Patent Publication Number: US-2022220469-A1

Title: Non-class i multi-component nucleic acid targeting systems

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims the benefit of and priority to co-pending U.S. Provisional Patent Application No. 62/850,494, filed on May 20, 2019 entitled “Non-Class I Multi-Component Nucleic Acid Targeting Systems,” the contents of which are incorporated by reference herein in their entirety. 
    
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH 
     This invention was made with government support under Grant No. HL141201 and HG009761 awarded by The National Institutes of Health. The government has certain rights in the invention. 
    
    
     SEQUENCE LISTING 
     This application contains a sequence listing filed in electronic form as an ASCII.txt file BROD-4280WP_ST25.txt, created on May 20, 2020, and having a size of 1,064,713 bytes. The content of the sequence listing is incorporated herein in its entirety. 
     TECHNICAL FIELD 
     The present invention generally relates to systems, methods and compositions related to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and components thereof. The present invention also generally relates to systems, methods, and compositions related to multi-component nucleic acid targeting systems. Additionally, the present invention relates to methods for developing or designing CRISPR-Cas system-based therapy or therapeutics. 
     BACKGROUND 
     Recent advances in genome sequencing techniques and analysis methods have significantly accelerated the ability to catalog and map genetic factors associated with a diverse range of biological functions and diseases. Precise genome targeting technologies are needed to enable systematic reverse engineering of causal genetic variations by allowing selective perturbation of individual genetic elements, as well as to advance synthetic biology, biotechnological, and medical applications. Although genome-editing techniques such as designer zinc fingers, transcription activator-like effectors (TALEs), or homing meganucleases are available for producing targeted genome perturbations, there remains a need for new genome engineering technologies that employ novel strategies and molecular mechanisms and are affordable, easy to set up, scalable, and amenable to targeting multiple positions within the eukaryotic genome. This would provide a major resource for new applications in genome engineering and biotechnology. 
     Gene editing via various base-editing systems, particularly the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas system, have revolutionized modern biotechnology and promise new avenues for clinical therapies. In addition to being faster, more economical, and more customizable, DNA and RNA guided systems offered a more precise mechanism for polynucleotide editing in comparison to other techniques. While the initial reports indicated that these systems, particularly the CRISPR-Cas system, were more precise than prior genetic modification techniques, they are not immune to producing unintended genomic modifications also referred to as “off-target” modifications. 
     With respect to DNA guided systems, there is a homeostatic level of cell replication and division that occurs and thus there is an inherent low level of off-target base editing that can occur as the DNA is unwound and opened during cell replication. This is one reason that DNA guided systems is not an ideal choice for editing dividing cells. RNA guided systems (e.g. CRISPR-Cas systems) are not immune to off-target base editing (see e.g. Kempton and Qi. Science (2019) 364:234-236; Wienert et al. Science (2019) 364:286-289; Zwo et al. Science (2019) 364:289-292; and Jin et al. Science (2019) 364:292-295). These off-target effects pose a significant barrier to translating these technologies into viable clinical therapies. Thus, there is a need for techniques for base editing that can have increased specificity and precision editing. 
     SUMMARY 
     Described in certain example embodiments herein are non-Class I engineered CRISPR-Cas polynucleotide targeting systems comprising two or more Cas proteins or one Cas protein and one or more non-Cas proteins. 
     In certain example embodiments, the non-Class I engineered CRISPR-Cas polynucleotide targeting systems further comprise a guide molecule capable of forming a complex with at least one of the two or more Cas proteins and directing site-specific binding to a target sequence of a target polynucleotide. 
     In certain example embodiments, the non-Class I engineered CRISPR-Cas polynucleotide targeting systems comprise at least two nuclease domains. 
     In certain example embodiments, the first nuclease domain is located on a first Cas protein and a second nuclease domain is located on a second Cas protein. 
     In certain example embodiments, the first nuclease domain is an HNH domain and the second nuclease domain is a RuvC domain. 
     In certain example embodiments, the first Cas protein further comprises an inactive RuvC domain, a bridge helix domain, or both. 
     In certain example embodiments, the system targets a dsDNA polynucleotide and wherein the first Cas protein acts as a nickase on a first strand of the dsDNA polynucleotide and the second Cas protein acts as a nickase on a second strand of the dsDNA polynucleotide. 
     In certain example embodiments, the first Cas protein and the second Cas protein allosterically interact upon target recognition to coordinate nicking of the first and second strands of the dsDNA polynucleotide. 
     In certain example embodiments, the first Cas and second Cas protein are modified to be catalytically inactive. 
     In certain example embodiments, the first Cas or second Cas protein further comprises a functional domain. 
     In certain example embodiments, the functional domain is activated upon allosteric interaction between the first and second Cas protein. 
     In certain example embodiments, the first Cas protein further comprises a first portion of a functional domain and the second Cas further comprises a second portion of a functional domain. 
     In certain example embodiments, the first and second portions form an active functional domain upon allosteric interaction between the first and second polypeptide. 
     In certain example embodiments, the functional domain comprises nucleotide deaminase activity, methylase activity, demethylase activity, translation activation activity, translation repression activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, nuclease activity, single-strand RNA cleavage activity, double-strand RNA cleavage activity, single-strand DNA cleavage activity, double-strand DNA cleavage activity and nucleic acid binding activity. 
     In certain example embodiments, the functional domain is a nucleotide deaminase. 
     In certain example embodiments, the first portion and second portion comprise a split fluorescent protein. 
     In certain example embodiments, the first portion and the second portion comprise a split apoptotic protein. 
     In certain example embodiments, the first portion and the second portion comprise a split transcription protein. 
     In certain example embodiments, the first Cas has at least 10-35% identity to IscB or at least 10-35% identity to a Cas9, preferably SpCas9. 
     In certain example embodiments, the second Cas has at least 10-35% identity to a Cas12a. 
     In certain example embodiments, the non Cas protein is a Cas-associated transposase. 
     In certain example embodiments, the Cas-associated transposase is a single strand DNA transposase. 
     In certain example embodiments, the single-strand DNA transposase is a TnpA. 
     Described in certain example embodiments herein are polynucleotide(s) that encode one or more components of a non-Class I engineered CRISPR-Cas polynucleotide targeting systems of paragraphs [0008]-[0030] and elsewhere herein. 
     In certain example embodiments, one or more regions of the polynucleotide is codon optimized for expression in a eukaryotic or plant cell. 
     Described in certain example embodiments herein are vectors that comprise a polynucleotide described in paragraphs [0031]-[0032] and elsewhere herein. 
     Described in certain example embodiments herein are vector systems that comprise a vector described in paragraph [0033] and elsewhere herein. 
     Described in certain example embodiments herein are cells that can comprise a polynucleotide described in paragraphs [0031]-[0032] and elsewhere herein, a vector described in paragraph [0033] and elsewhere herein, or a vector system described in paragraph [0034] and elsewhere herein. 
     In certain example embodiments, the cell is a eukaryotic cell or a prokaryotic cell. 
     Described in certain example embodiments herein are organisms comprising one or more cells described in paragraphs [0035]-[0036] and elsewhere herein. 
     In certain example embodiments, the modified organism is an animal. 
     In certain example embodiments, the modified organism is a non-human animal. 
     In certain example embodiments, the modified organism is a plant. 
     Described in certain example embodiments herein are methods of targeting and optionally modifying a polynucleotide, comprising contacting a sample that comprises the polynucleotide with the non-Class I engineered CRISPR-Cas polynucleotide targeting systems of paragraphs [0008]-[0030] and elsewhere herein. 
     In certain example embodiments, the method of targeting a polynucleotide can further comprise detecting binding of the complex to the polynucleotide. 
     In certain example embodiments, contacting results in modification of a gene product or modification of the amount or expression of a gene product. 
     In certain example embodiments, a target sequence of the polynucleotide is a disease-associated target sequence. 
     Described in certain example embodiments herein are methods of modifying an adenine or cytidine in a target DNA sequence, comprising delivering to said target DNA the system of paragraph [0022]. 
    
    
     DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTS 
     General Definitions 
     Overview 
     Disclosed herein are aspects of a non-Class I engineered multi-component nucleic acid targeting systems that can be used to specifically target a nucleic acid and allow for subsequent enzymatic and/or catalytic events and/or recruitment to occur at a target sequence of the targeted nucleic acid. It is noted the term “Class I” when used herein in reference to describe a CRISPR-Cas system or component thereof refers to any CRISPR-Cas system that would be classified as “Class I” as set forth in Makarova et al. 2018. The CRISPR J. 1(5): 325-336. Class I encompasses type I, type III, and type IV systems. As used herein, Class I is intended to be inclusive of all types and sub-types. Thus, where it is stated that a system or component thereof described herein is “not a Class I system” or a “non-Class I” system, this means that the system is not any of the Class I systems previously defined. 
     In certain embodiments, the multi-component nucleic acid targeting system described herein can provide increased specificity and control over catalytic events at and/or recruitment of various molecules to the specifically targeted nucleic acid. 
     In certain embodiments, the non-class I multi-component nucleic acid targeting system can include one or more Cas-like polypeptides that can allosterically interact with one or more polypeptides, such as another Cas-like polypeptide, to enzymatically act upon and/or specifically recognize a target polynucleotide. It is noted that the term “Cas-like” as used herein means that protein has similar, but not necessarily identical, features and/or functions as a Cas reference or wild-type protein. It will be appreciated that when this term is used to specifically call to a reference Cas protein (e.g. Cas9-like, Cas-12 like, Cas13-like, etc.) that the protein may be specifically labeled as Cas9-like, Cas12-like based on the reference Cas protein. Thus, the reference protein for a Cas9-like protein is a Cas9 protein. 
     In other aspects, the non-Class I multi-component systems may comprise a Cas-like polypeptide and a protein that is not Cas-like. For example, the system may comprise a Cas-like protein and a transposase. In certain embodiments, one or more of the Cas-like polypeptides can include an activatable functional domain. In some aspects, the activatable functional domain is inactive prior to allosteric interaction between one or more of the Cas-like polypeptides. Allosteric interaction can at least facilitate activation of one or more activatable functional domains present on one or more of the Cas-like polypeptides. In this way, the non-class I multi-component nucleic acid targeting system described herein can, in some aspects, allow for control over one or more catalytic or biological activities mediated by one or more of the activatable functional domains present on one or more of the Cas-like polypeptides. 
     Accordingly, some aspects relate to systems, compositions, methods for increasing the specificity and/or reducing off-target events of nucleic acid targeting systems (e.g. CRISPR-Cas systems), particularly for CRISPR-Cas based therapies. In a further aspect, the invention relates to methods for increasing safety of CRISPR-Cas systems, such as CRISPR-Cas system-based therapy or therapeutics. In a further aspect, the present invention relates to methods for increasing specificity, efficacy, and/or safety, preferably all, of CRISPR-Cas systems, such as CRISPR-Cas system-based therapy or therapeutics. 
     Aspects of methods of the present invention involve optimization of selected parameters or variables associated with the CRISPR-Cas system and/or its functionality, as described herein further elsewhere. Optimization of the CRISPR-Cas system in the methods as described herein may depend on the target(s), such as the therapeutic target or therapeutic targets, the mode or type of CRISPR-Cas system modulation, such as CRISPR-Cas system based therapeutic target(s) modulation, modification, or manipulation, as well as the delivery of the CRISPR-Cas system components. One or more targets may be selected, depending on the genotypic and/or phenotypic outcome. For instance, one or more therapeutic targets may be selected, depending on (genetic) disease etiology or the desired therapeutic outcome. The (therapeutic) target(s) may be a single gene, locus, or other genomic site, or may be multiple genes, loci or other genomic sites. As is known in the art, a single gene, locus, or other genomic site may be targeted more than once, such as by use of multiple gRNAs. 
     CRISPR-Cas system activity, such as CRISPR-Cas system design, may involve target disruption, such as target mutation, such as leading to gene knockout. CRISPR-Cas system activity, such as CRISPR-Cas system design may involve replacement of particular target sites, such as leading to target correction. CRISPR-Cas system design may involve removal of particular target sites, such as leading to target deletion. CRISPR-Cas system activity can involve modulation of target site functionality, such as target site activity or accessibility, leading for instance to (transcriptional and/or epigenetic) gene or genomic region activation or gene or genomic region silencing. The skilled person will understand that modulation of target site functionality may involve CRISPR effector mutation (such as, for instance, generation of a catalytically inactive CRISPR effector) and/or functionalization (such as, for instance, fusion of the CRISPR effector with a heterologous functional domain, such as a transcriptional activator or repressor), as described herein elsewhere. 
     In certain example embodiments, the non-Class I multi-component system may comprise a Cas-like and a non-Cas-like protein. In certain example embodiments, the Cas like protein is a Cas9 protein. Example Cas9 proteins are described below. In certain example embodiments, the non-Cas-like protein is a transposase. In certain example embodiment, the transposase is a single stranded DNA transposase. In certain example embodiments, the single stranded DNA transposase is TnpA. In certain example embodiments, the non-Class I multi-component system comprise a Cas9 associated transposase. In certain example embodiments, the transposase is a TnpA, or a functional fragment thereof. The Cas9 associated transposase systems may comprise a local architecture of Cas9-TnpA, Cas1-Cas2-CRISPR array. The Cas9 may or may not have a tracrRNA associated with it. The Cas9-associated systems may be coded on the same strand or be part of a larger operon. In certain embodiments, the Cas9 may confer target specificity, allowing the TnpA to move a polynucleotide cargo from other target sites in a sequence specific matter. In certain example embodiments, the Cas9-associated transposase are derived from  Flavobacterium granuli  strain DSM-19729 , Salinivirga cyanobacteriivorans  strain L21-Spi-D4 , Flavobacterium aciduliphilum  strain DSM 25663 , Flavobacterium glacii  strain DSM 19728 , Niabella soli  DSM 19437 , Salnivirga cyanobactriivorans  strain L21-Spi-D4 , Alkaliflexus imshenetskii  DSM 150055 strain Z-7010, or  Alkalitala saponilacus.    
     Non-Class I Engineered CRISPR-Cas Systems 
     General Overview 
     In general, CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats), also known as SPIDRs (SPacer Interspersed Direct Repeats), constitute a family of DNA loci that are usually specific to a particular bacterial species. It is noted that the terms “CRISPR-Cas system”, “CRISPR-Cas complex” “CRISPR complex” and “CRISPR system” are used interchangeably herein. Also, the terms “CRISPR enzyme”, “Cas enzyme”, or “CRISPR-Cas enzyme”, can be used interchangeably herein. The terms are inclusive of proteins and molecules in a CRISPR-Cas system, including those as described elsewhere herein that are Cas-like or CRISPR-Cas-like and the like. 
     The CRISPR locus comprises a distinct class of interspersed short sequence repeats (SSRs) that were recognized in  E. coli  (Ishino et al., J. Bacteriol., 169:5429-5433 [1987]; and Nakata et al., J. Bacteriol., 171:3553-3556 [1989]), and associated genes. Similar interspersed SSRs have been identified in  Haloferax mediterranei, Streptococcus pyogenes, Anabaena , and  Mycobacterium tuberculosis  (See, Groenen et al., Mol. Microbiol., 10:1057-1065 [1993]; Hoe et al., Emerg. Infect. Dis., 5:254-263 [1999]; Masepohl et al., Biochim. Biophys. Acta 1307:26-30 [1996]; and Mojica et al., Mol. Microbiol., 17:85-93 [1995]). The CRISPR loci typically differ from other SSRs by the structure of the repeats, which have been termed short regularly spaced repeats (SRSRs) (Janssen et al., OMICS J. Integ. Biol., 6:23-33 [2002]; and Mojica et al., Mol. Microbiol., 36:244-246 [2000]). In general, the repeats are short elements that occur in clusters that are regularly spaced by unique intervening sequences with a substantially constant length (Mojica et al., [2000], supra). Although the repeat sequences are highly conserved between strains, the number of interspersed repeats and the sequences of the spacer regions typically differ from strain to strain (van Embden et al., J. Bacteriol., 182:2393-2401 [2000]). CRISPR loci have been identified in more than 40 prokaryotes (See e.g., Jansen et al., Mol. Microbiol., 43:1565-1575 [2002]; and Mojica et al., [2005]) including, but not limited to  Aeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Halocarcula, Methanobacterium, Methanococcus, Methanosarcina, Methanopyrus, Pyrococcus, Picrophilus, Thermoplasma, Corynebacterium, Mycobacterium, Streptomyces, Aquifex, Porphyromonas, Chlorobium, Thermus, Bacillus, Listeria, Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma, Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomonas, Desulfovibrio, Geobacter, Myxococcus, Campylobacter, Wolinella, Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus, Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia, Treponema , and  Thermotoga.    
     Generally, CRISPR systems fall into two classes: Class I and Class II. Makarova et al. 2018. The CRISPR J. 1(5): 325-336. Class I encompasses CRISPR systems that involve effector complexes that are composed of multiple Cas protein subunits and have backbones composed of paralogous repeat-associated mysterious proteins (RAMPS), such as Cas7 and Cas5. This is in contrast to Class II CRISPR systems that have a much simpler organization, with a single effector molecule having a single large, multidomain and multifunctional protein (e.g. Cas9). 
     Described herein are engineered nucleic acid targeting systems (e.g. engineered nucleic acid CRISPR systems) that have structural and/or functional differences such that they are not a Class I system or Class II system as previously described. Such systems are also referred to herein as non-class I engineered CRISPR system. In certain embodiments, the non-class I engineered CRISPR systems described herein can have multiple Cas polypeptides or multiple Cas effector domains. In certain example embodiments, the systems may also be associated with or include non-Cas proteins such as transposases. In other example embodiments, the multiple Cas polypeptides may be capable of allosterically interacting to recognize, bind, and/or enzymatically act at a recognized target polynucleotide. Further aspects and advantages of the systems are described elsewhere herein. Further described herein are compositions, formulations, systems that can include, generate, and/or apply the non-Class I engineered CRISPR systems described herein. Also described elsewhere herein are methods of making and using the non-Class I engineered CRISPR systems and compositions, formulations and other systems thereof. 
     With respect to general information on CRISPR-Cas Systems, components thereof, and delivery of such components, including methods, materials, delivery vehicles, vectors, particles, AAV, and making and using thereof, including as to amounts and formulations, all useful in the practice of the instant invention, reference is made to: U.S. Pat. Nos. 8,697,359, 8,771,945, 8,795,965, 8,865,406, 8,871,445, 8,889,356, 8,889,418, 8,895,308, 8,906,616, 8,932,814, 8,945,839, 8,993,233 and 8,999,641; US Patent Publication Nos. US 2014-0310830 (U.S. application Ser. No. 14/105,031), US 2014-0287938 A1 (U.S. application Ser. No. 14/213,991), US 2014-0273234 A1 (U.S. application Ser. No. 14/293,674), US2014-0273232 A1 (U.S. application Ser. No. 14/290,575), US 2014-027323 A1 (U.S. application Ser. No. 14/259,420), US 2014-0256046 A1 (U.S. application Ser. No. 14/226,274), US 2014-0248702 A1 (U.S. application Ser. No. 14/258,458), US 2014-0242700 A1 (U.S. application Ser. No. 14/222,930), US 2014-0242699 A1 (U.S. application Ser. No. 14/183,512), US 2014-0242664 A1 (U.S. application Ser. No. 14/104,990), US 2014-0234972 A1 (U.S. application Ser. No. 14/183,471), US 2014-0227787 A1 (U.S. application Ser. No. 14/256,912), US 2014-0189896 A1 (U.S. application Ser. No. 14/105,035), US 2014-0186958 A1 (U.S. application Ser. No. 14/105,017), US 2014-0186919 A1 (U.S. application Ser. No. 14/104,977), US 2014-0186843 A1 (U.S. application Ser. No. 14/104,900), US 2014-0179770 A1 (U.S. application Ser. No. 14/104,837) and US 2014-0179006 A1 (U.S. application Ser. No. 14/183,486), US 2014-0170753 A1 (U.S. application Ser. No. 14/183,429); US 2015-0184139 A1 (U.S. application Ser. No. 14/324,960); U.S. application Ser. No. 14/054,414 European Patent Applications EP 2771468 (EP13818570.7), EP 2764103 (EP13824232.6), and EP 2784162 (EP14170383.5); and PCT Patent Publications WO 2014/093661 (PCT/US2013/074743), WO 2014/093694 (PCT/US2013/074790), WO 2014/093595 (PCT/US2013/074611), WO 2014/093718 (PCT/US2013/074825), WO 2014/093709 (PCT/US2013/074812), WO 2014/093622 (PCT/US2013/074667), WO 2014/093635 (PCT/US2013/074691), WO 2014/093655 (PCT/US2013/074736), WO 2014/093712 (PCT/US2013/074819), WO 2014/093701 (PCT/US2013/074800), WO 2014/018423 (PCT/US2013/051418), WO 2014/204723 (PCT/US2014/041790), WO 2014/204724 (PCT/US2014/041800), WO 2014/204725 (PCT/US2014/041803), WO 2014/204726 (PCT/US2014/041804), WO 2014/204727 (PCT/US2014/041806), WO 2014/204728 (PCT/US2014/041808), WO 2014/204729 (PCT/US2014/041809), WO 2015/089351 (PCT/US2014/069897), WO 2015/089354 (PCT/US2014/069902), WO 2015/089364 (PCT/US2014/069925), WO 2015/089427 (PCT/US2014/070068), WO 2015/089462 (PCT/US2014/070127), WO 2015/089419 (PCT/US2014/070057), WO 2015/089465 (PCT/US2014/070135), WO 2015/089486 (PCT/US2014/070175), PCT/US2015/051691, PCT/US2015/051830. Reference is also made to U.S. Provisional Application Nos. 61/758,468; 61/802,174; 61/806,375; 61/814,263; 61/819,803 and 61/828,130, filed on Jan. 30, 2013; Mar. 15, 2013; Mar. 28, 2013; Apr. 20, 2013; May 6, 2013 and May 28, 2013 respectively. Reference is also made to U.S. provisional patent application 61/836,123, filed on Jun. 17, 2013. Reference is additionally made to U.S. Provisional Application Nos. 61/835,931, 61/835,936, 61/835,973, 61/836,080, 61/836,101, and 61/836,127, each filed Jun. 17, 2013. Further reference is made to U.S. Provisional Application Nos. 61/862,468 and 61/862,355 filed on Aug. 5, 2013; 61/871,301 filed on Aug. 28, 2013; 61/960,777 filed on Sep. 25, 2013 and 61/961,980 filed on Oct. 28, 2013. Reference is yet further made to PCT/US2014/62558 filed Oct. 28, 2014, and U.S. Provisional Application Nos. 61/915,148, 61/915,150, 61/915,153, 61/915,203, 61/915,251, 61/915,301, 61/915,267, 61/915,260, and 61/915,397, each filed Dec. 12, 2013; 61/757,972 and 61/768,959, filed on Jan. 29, 2013 and Feb. 25, 2013; 62/010,888 and 62/010,879, both filed Jun. 11, 2014; 62/010,329, 62/010,439 and 62/010,441, each filed Jun. 10, 2014; 61/939,228 and 61/939,242, each filed Feb. 12, 2014; 61/980,012, filed Apr. 15, 2014; 62/038,358, filed Aug. 17, 2014; 62/055,484, 62/055,460 and 62/055,487, each filed Sep. 25, 2014; and 62/069,243, filed Oct. 27, 2014. Reference is made to International Patent Application No. PCT/US14/41806 designating, inter alia, the United States, filed Jun. 10, 2014. Reference is made to U.S. Provisional Application No. 61/930,214 filed on Jan. 22, 2014. Reference is made to International Patent Application No. PCT/US14/41806, designating, inter alia, the United States, filed Jun. 10, 2014. 
     Mention is also made of U.S. Provisional Application No. 62/180,709, filed 17 Jun. 2015, PROTECTED GUIDE RNAS (PGRNAS); U.S. Provisional Application No. 62/091,455, filed, 12 Dec. 2014, PROTECTED GUIDE RNAS (PGRNAS); U.S. Provisional Application No. 62/096,708, filed 24 Dec. 2014, PROTECTED GUIDE RNAS (PGRNAS); U.S. Provisional Application Nos. 62/091,462, filed 12 Dec. 2014, 62/096,324, filed 23 Dec. 2014, 62/180,681, filed 17 Jun. 2015, and 62/237,496, filed 5 Oct. 2015, DEAD GUIDES FOR CRISPR TRANSCRIPTION FACTORS; U.S. Provisional Application No. 62/091,456, filed 12 Dec. 2014 and 62/180,692, filed 17 Jun. 2015, ESCORTED AND FUNCTIONALIZED GUIDES FOR CRISPR-CAS SYSTEMS; U.S. Provisional Application No. 62/091,461, filed 12 Dec. 2014, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR GENOME EDITING AS TO HEMATOPOETIC STEM CELLS (HSCs); U.S. Provisional Application No. 62/094,903, filed 19 Dec. 2014, UNBIASED IDENTIFICATION OF DOUBLE-STRAND BREAKS AND GENOMIC REARRANGEMENT BY GENOME-WISE INSERT CAPTURE SEQUENCING; U.S. Provisional Application No. 62/096,761, filed 24-Dec-14, ENGINEERING OF SYSTEMS, METHODS AND OPTIMIZED ENZYME AND GUIDE SCAFFOLDS FOR SEQUENCE MANIPULATION; U.S. application Provisional Application No. 62/098,059, filed 30 Dec. 2014, 62/181,641, filed 18 Jun. 2015, and 62/181,667, filed 18 Jun. 2015, RNA-TARGETING SYSTEM; U.S. Provisional Application No. 62/096,656, filed 24 Dec. 2014 and 62/181,151, filed 17 Jun. 2015, CRISPR HAVING OR ASSOCIATED WITH DESTABILIZATION DOMAINS; U.S. Provisional Application No. 62/096,697, filed 24 Dec. 2014, CRISPR HAVING OR ASSOCIATED WITH AAV; U.S. application 62/098,158, filed 30 Dec. 2014, ENGINEERED CRISPR COMPLEX INSERTIONAL TARGETING SYSTEMS; U.S. Provisional Application No. 62/151,052, 22-Apr-15, CELLULAR TARGETING FOR EXTRACELLULAR EXOSOMAL REPORTING; U.S. Provisional Application No. 62/054,490, filed 24 Sep. 2014, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR TARGETING DISORDERS AND DISEASES USING PARTICLE DELIVERY COMPONENTS; U.S. Provisional Application No. 61/939,154, filed 12 Feb. 2014, SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S. Provisional Application No. 62/055,484, filed 25 Sep. 2014, SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S. Provisional Application No. 62/087,537, filed 4 Dec. 2014, SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S. Provisional Application No. 62/054,651, filed 24 Sep. 2014, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR MODELING COMPETITION OF MULTIPLE CANCER MUTATIONS IN VIVO; U.S. Provisional Application No. 62/067,886, filed 23 Oct. 2014, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR MODELING COMPETITION OF MULTIPLE CANCER MUTATIONS IN VIVO; U.S. Provisional Application Nos. 62/054,675, filed 24 Sep. 2014 and 62/181,002, filed 17 Jun. 2015, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS IN NEURONAL CELLS/TISSUES; U.S. Provisional Application No. 62/054,528, filed 24 Sep. 2014, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS IN IMMUNE DISEASES OR DISORDERS; U.S. Provisional Application No. 62/055,454, 25 Sep. 2014, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR TARGETING DISORDERS AND DISEASES USING CELL PENETRATION PEPTIDES (CPP); U.S. Provisional Application No. 62/055,460, filed 25 Sep. 2014, MULTIFUNCTIONAL-CRISPR COMPLEXES AND/OR OPTIMIZED ENZYME LINKED FUNCTIONAL-CRISPR COMPLEXES; U.S. Provisional Application No. 62/087,475, filed 4 Dec. 2014 and 62/181,690, filed 18 Jun. 2015, FUNCTIONAL SCREENING WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S. Provisional Application No. 62/055,487, filed 25 Sep. 2014, FUNCTIONAL SCREENING WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S. Provisional Application No. 62/087,546, filed 4 Dec. 2014 and 62/181,687, filed 18 Jun. 2015, MULTIFUNCTIONAL CRISPR COMPLEXES AND/OR OPTIMIZED ENZYME LINKED FUNCTIONAL-CRISPR COMPLEXES; and U.S. Provisional Application No. 62/098,285, filed 30 Dec. 2014, CRISPR MEDIATED IN VIVO MODELING AND GENETIC SCREENING OF TUMOR GROWTH AND METASTASIS. 
     Mention is made of U.S. Provisional Application Nos. 62/181,659, filed 18 Jun. 2015 and 62/207,318, filed 19 Aug. 2015, ENGINEERING AND OPTIMIZATION OF SYSTEMS, METHODS, ENZYME AND GUIDE SCAFFOLDS OF CAS9 ORTHOLOGS AND VARIANTS FOR SEQUENCE MANIPULATION. Mention is made of U.S. Provisional Application Nos. 62/181,663, filed 18 Jun. 2015 and 62/245,264, filed 22 Oct. 2015, NOVEL CRISPR ENZYMES AND SYSTEMS, U.S. Provisional Application Nos. 62/181,675, filed 18 Jun. 2015, 62/285,349, filed 22 Oct. 2015, 62/296,522, filed 17 Feb. 2016, and 62/320,231, filed 8 Apr. 2016, NOVEL CRISPR ENZYMES AND SYSTEMS, U.S. Provisional Application No. 62/232,067, filed 24 Sep. 2015, U.S. application Ser. No. 14/975,085, filed 18 Dec. 2015, European application No. 16150428.7, U.S. Provisional Application No. 62/205,733, filed 16 Aug. 2015, U.S. Provisional Application No. 62/201,542, filed 5 Aug. 2015, U.S. Provisional Application No. 62/193,507, filed 16 Jul. 2015, and U.S. Provisional Application No. 62/181,739, filed 18 Jun. 2015, each entitled NOVEL CRISPR ENZYMES AND SYSTEMS and of U.S. Provisional Application No. 62/245,270, filed 22 Oct. 2015, NOVEL CRISPR ENZYMES AND SYSTEMS. Mention is also made of U.S. Provisional Application No. 61/939,256, filed 12 Feb. 2014, and International Patent Publication No. WO 2015/089473 (PCT/US2014/070152), filed 12 Dec. 2014, each entitled ENGINEERING OF SYSTEMS, METHODS AND OPTIMIZED GUIDE COMPOSITIONS WITH NEW ARCHITECTURES FOR SEQUENCE MANIPULATION. Mention is also made of International Patent Application No. PCT/US2015/045504, filed 15 Aug. 2015, U.S. Provisional Application No. 62/180,699, filed 17 Jun. 2015, and U.S. Provisional Application No. 62/038,358, filed 17 Aug. 2014, each entitled GENOME EDITING USING CAS9 NICKASES. 
     In addition, mention is made of PCT application PCT/US14/70057, entitled “DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR TARGETING DISORDERS AND DISEASES USING PARTICLE DELIVERY COMPONENTS (claiming priority from one or more or all of U.S. Provisional Application No. 62/054,490, filed Sep. 24, 2014; 62/010,441, filed Jun. 10, 2014; and 61/915,118, 61/915,215 and 61/915,148, each filed on Dec. 12, 2013) (“the Particle Delivery PCT”), incorporated herein by reference, and of PCT application PCT/US14/70127, Attorney Reference 47627.99.2091 and BI-2013/101 entitled “DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR GENOME EDITING” (claiming priority from one or more or all of U.S. provisional patent application 61/915,176; 61/915,192; 61/915,215; 61/915,107, 61/915,145; 61/915,148; and 61/915,153 each filed Dec. 12, 2013) (“the Eye PCT”), incorporated herein by reference. 
     Cas-Like Effector Proteins and Domains 
     General Discussion 
     As generally discussed, the non-Class I engineered nucleic acid targeting system described herein can have multiple effector proteins. In certain embodiments, the non-Class I nucleic acid targeting system can be a non-Class I engineered CRISPR-Cas polynucleotide targeting system comprising two or more Cas proteins. In certain embodiments, non-Class I engineered CRISPR-Cas polynucleotide targeting system can further comprise a guide molecule capable of forming a complex with at least one of the two or more Cas proteins and directing site-specific binding to a target sequence of a target polynucleotide. In certain embodiments, the system comprises at least two nuclease domains. In other embodiments, a first nuclease domain is located on a first Cas protein and a second nuclease domain is located on a second Cas protein. In certain example embodiments, the first nuclease domain is an HNH domain and the second nuclease domain is a RuvC domain. In certain example embodiments, the first Cas protein further comprises an inactive RuvC domain, a bridge helix, or both. In certain embodiments, the system targets a dsDNA polynucleotide and wherein the first Cas protein acts as a nickase on a first strand of the dsDNA polynucleotide and the second Cas protein acts as a nickase on a second strand of the dsDNA polynucleotide. In other example embodiments, the first Cas protein and the second Cas protein allosterically interact upon target recognition to coordinate nicking of the first and second strands of the dsDNA polynucleotide. In other embodiments, the first Cas protein, the second Cas protein, or both are modified to be catalytically inactive. 
     In certain embodiments, the first Cas or second Cas protein further comprises a functional domain. In certain embodiments, the functional domain is activated upon allosteric interaction between the first and second Cas protein. In certain embodiments, the first Cas protein further comprises a first portion of a functional domain and the second Cas further comprises a second portion of a functional domain. In certain embodiments, the first and second portions form an active functional domain upon allosteric interaction between the first and second polypeptide. In certain embodiments, the functional domain comprises nucleotide deaminase activity, methylase activity, demethylase activity, translation activation activity, translation repression activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, nuclease activity, single-strand RNA cleavage activity, double-strand RNA cleavage activity, single-strand DNA cleavage activity, double-strand DNA cleavage activity, transposase activity, reverse transcriptase activity, and nucleic acid binding activity. In certain embodiments, the functional domain is a nucleotide deaminase. In certain example embodiments, the functional domain is a reverse transcriptase. In certain embodiments, the first portion and second portion comprise a split fluorescent protein. In certain embodiments, the first portion and the second portion comprise a split apoptotic protein. In certain embodiments, the first portion and the second portion comprise a split transcription protein. 
     In certain embodiments, the first Cas has at least 10-35%, 10-30%, 10-25%, 10-20%, 15-35%, 15-30%, 15-25%, 15-20%, 20-35%, 25-35%, or 30-35% identity to IscB. In another embodiment, the first Cas has at least 10-35%, 10-30%, 10-25%, 10-20%, 15-35%, 15-30%, 15-25%, 15-20%, 20-35%, 25-35%, or 30-35% identity with SpCas9. In certain example embodiments the second Cas has at least 10-35%, 10-30%, 10-25%, 10-20%, 15-35%, 15-30%, 15-25%, 15-20%, 20-35%, 25-35%, or 30-35% identity to a Cas12. 
     Also provided herein are polynucleotides encoding the one or more components of the non-class I engineered CRISPR-Cas nucleic acid targeting system. In certain embodiments, one or more regions of the polynucleotide is codon optimized for expression in a eukaryotic or plant cell. 
     Also provided herein are vectors and systems thereof that can include a polynucleotide described herein. 
     Also provided herein are cells comprising a polynucleotide described herein, a vector described herein, and/or a vector system described herein. In certain embodiments, the cell is a eukaryotic cell, a prokaryotic cell, or a plant cell. 
     Also provided herein is a plant or a non-human animal comprising one or more cells described herein. 
     Also provided herein is a method of targeting a polynucleotide that comprises contacting a sample that comprises the polynucleotide with a non-class I engineered CRISPR-Cas nucleic acid targeting system described herein. In certain embodiments, the method can further comprise detecting binding of the complex to the polynucleotide. In certain embodiments, contacting results in modification of a gene product or modification of the amount or expression of a gene product. In certain embodiments, a target sequence of the polynucleotide is a disease-associated target sequence. 
     Also provided herein is a method of modifying an adenine or cytidine in a target DNA sequence, comprising delivering to said target DNA using a non-class I engineered CRISPR-Cas nucleic acid targeting system described herein. 
     In certain embodiments, two or more of the effector proteins can allosterically interact within the CRISPR-Cas system. In certain embodiments, the allosteric interaction is not akin to any allosteric interaction of a known Class I CRISPR-Cas system. The allosteric interaction between two or more of the effector proteins can result in target polynucleotide recognition, binding, recruitment of other effectors and/or accessory molecules, activation of a functional domain, and combinations thereof. The effector proteins that allosterically interact in this way are also referred to herein as Cas-like effectors, Cas-like polypeptides, or Cas-like proteins. In some aspects, at least one of the Cas-like proteins is a Cas9-like (or IscB-like) protein. In some aspects, at least one of the Cas-like proteins is a Cas12-like protein. In some aspects, the non-class I nucleic acid targeting system described herein can include at least one Cas9-like protein and at least one Cas12-like protein. In certain embodiments, the Cas9-like and the Cas12-like proteins can allosterically interact within the system. Allosteric interaction between the Cas9-like and the Cas12-like proteins can result in, among other things, target polynucleotide recognition, binding, recruitment of other effectors and/or accessory molecules, activation of a functional domain, and combinations thereof. Additional features of the Cas-like proteins are further described elsewhere herein. The non-Class I nucleic acid targeting system described herein can also optionally include other effector, accessory molecules, and/or adaptor molecules. 
     Cas9-Like Effectors 
     General Features of the Cas9-like Effectors 
     In certain embodiments, the Cas9-like protein can include a polypeptide that contains an HNH domain and optionally includes an inactive RuvC domain. In certain embodiments, the Cas9-like polypeptide can have 10-35%, 10-30%, 10-25%, 10-20%, 15-35%, 15-30%, 15-25%, 15-20%, 20-35%, 25-35%, or 30-35% identity to a reference or wild-type Cas9 polypeptide, which are discussed elsewhere herein. In certain embodiments, the Cas9-like polypeptide can have 10-35%, 10-30%, 10-25%, 10-20%, 15-35%, 15-30%, 15-25%, 15-20%, 20-35%, 25-35%, or 30-35% identity to a reference or wild-type IscB polypeptide, which are discussed elsewhere herein. 
     In certain embodiments, the Cas9-like polypeptide can have 80, 85, 90, 95 and 100% identity to a polypeptide encoded by one or more of the polynucleotides of SEQ ID NOs: 57-100 and/or one or more regions therein (see also e.g. Tables 14-23 in the Working Example(s) herein), which are incorporated by reference herein as if expressed in their entireties. In aspects, the Cas12-like polypeptide can have 80-100% identity to a polypeptide encoded by one or more of the polynucleotides provided in SEQ ID NOs: 57-87 and/or one or more regions therein (See also, e.g. Tables 14-23 in the Working Example(s) herein). The Cas9-like protein can contain other domains as described elsewhere herein that can give the Cas9-like polypeptide other functionalities that can or are not dependent on allosteric interaction between the Cas9-like protein and another Cas (e.g. Cas12-like) protein. 
     Description of Cas9 Reference Protein 
     As previously described, the Cas9-like polypeptide can have a varying sequence identity (e.g. not 100% sequence identity) to a reference or wild-type Cas9. It will be appreciated that any of the following Cas9 polypeptides described herein can serve as a reference or wild-type sequence to a Cas9-like polypeptide as discussed elsewhere herein. 
     The Cas9 gene is found in several diverse bacterial genomes, typically in the same locus with cas1, cas2, and cas4 genes and a CRISPR cassette. Furthermore, the Cas9 protein contains a readily identifiable C-terminal region that is homologous to the transposon ORF-B and includes an active RuvC-like nuclease, an arginine-rich region. 
     In particular embodiments, Cas9 is from an organism from a genus comprising  Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium , or Corynebacte. 
     In particular embodiments, the Cas9 is from an organism from a genus comprising  Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae, Clostridiaridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes, Helcococcus, Letospira, Desulfovibrio, Desulfonatronum, Opitutaceae, Tuberibacillus, Bacillus, Brevibacilus, Methylobacterium  or  Acidaminococcus.    
     In further particular embodiments, the Cas9 protein is from an organism selected from  S. mutans, S. agalactiae, S. equisimilis, S. sanguinis, S. pneumonia; C. jejuni, C. coli; N. salsuginis, N. tergarcus; S. auricularis, S. carnosus; N. meningitides, N. gonorrhoeae; L. monocytogenes, L. ivanovii; C. botulinum, C. difficile, C. tetani, C. sordellii . In particular embodiments, the effector protein is a Cas9 effector protein from an organism from  Streptococcus pyogenes  (SpCas9),  Staphylococcus aureus  (SaCas9),  Streptococcus canis  (ScCas9),  Streptococcus macacae  (SmCas9as, or  Streptococcus thermophilus  (StCas9) Cas9. 
     In an embodiment, the Cas9 is derived from a bacterial species selected from  Streptococcus pyogenes, Staphylococcus aureus , or  Streptococcus thermophilus  Cas9. 
     In certain embodiments, the Cas9 is derived from a bacterial species selected from  Francisella tularensis  1 , Prevotella albensis , Lachnospiraceae bacterium MC2017 1 , Butyrivibrio proteoclasticus , Peregrinibacteria bacterium GW2011_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44_17 , Smithella  sp. SCADC,  Acidaminococcus  sp. BV3L6, Lachnospiraceae bacterium MA2020 , Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi  237 , Leptospira inadai, Lachnospiraceae bacterium  ND2006 , Porphyromonas crevioricanis  3 , Prevotella disiens  and  Porphyromonas macacae . In certain embodiments, the Cas9p is derived from a bacterial species selected from  Acidaminococcus  sp. BV3L6 or  Lachnospiraceae bacterium  MA2020. In certain embodiments, the effector protein is derived from a subspecies of  Francisella tularensis  1, including but not limited to  Francisella tularensis  subsp.  Novicida.    
     In certain embodiments, the wild-type or reference Cas9 is an ortholog or homolog of a Cas9 protein described herein. The terms “orthologue” (also referred to as “ortholog” herein) and “homologue” (also referred to as “homolog” herein) are well known in the art. By means of further guidance, a “homologue” of a protein as used herein is a protein of the same species which performs the same or a similar function as the protein it is a homologue of Homologous proteins may but need not be structurally related, or are only partially structurally related. An “orthologue” of a protein as used herein is a protein of a different species which performs the same or a similar function as the protein it is an orthologue of Orthologous proteins may but need not be structurally related, or are only partially structurally related. Homologs and orthologs may be identified by homology modelling (see, e.g., Greer, Science vol. 228 (1985) 1055, and Blundell et al. Eur J Biochem vol 172 (1988), 513) or “structural BLAST” (Dey F, Cliff Zhang Q, Petrey D, Honig B. Toward a “structural BLAST”: using structural relationships to infer function. Protein Sci. 2013 April; 22(4):359-66. doi: 10.1002/pro.2225.). See also Shmakov et al. (2015) for application in the field of CRISPR-Cas loci. Homologous proteins may but need not be structurally related, or are only partially structurally related. 
     Sequence homologies may be generated by any of a number of computer programs known in the art, for example BLAST or FASTA, etc. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A; Devereux et al., 1984, Nucleic Acids Research 12:387). Examples of other software than may perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 ibid—Chapter 18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60). However, it is preferred to use the GCG Bestfit program. Percentage (%) sequence homology may be calculated over contiguous sequences, i.e., one sequence is aligned with the other sequence and each amino acid or nucleotide in one sequence is directly compared with the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called an “ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues. Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion may cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without unduly penalizing the overall homology or identity score. This is achieved by inserting “gaps” in the sequence alignment to try to maximize local homology or identity. However, these more complex methods assign “gap penalties” to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible—reflecting higher relatedness between the two compared sequences—may achieve a higher score than one with many gaps. “Affinity gap costs” are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties may, of course, produce optimized alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example, when using the GCG Wisconsin Bestfit package the default gap penalty for amino acid sequences is −12 for a gap and −4 for each extension. Calculation of maximum % homology therefore first requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (Devereux et al., 1984  Nuc. Acids Research  12 p 387). Examples of other software than may perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 Short Protocols in Molecular Biology, 4 th  Ed.—Chapter 18), FASTA (Altschul et al., 1990  J. Mol. Biol.  403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al., 1999 , Short Protocols in Molecular Biology , pages 7-58 to 7-60). However, for some applications, it is preferred to use the GCG Bestfit program. A new tool, called BLAST 2 Sequences is also available for comparing protein and nucleotide sequences (see  FEMS Microbiol Lett.  1999 174(2): 247-50 ; FEMS Microbiol Lett.  1999 177(1): 187-8 and the website of the National Center for Biotechnology information at the website of the National Institutes for Health). Although the final % homology may be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pair-wise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix—the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table, if supplied (see user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62. Alternatively, percentage homologies may be calculated using the multiple alignment feature in DNASIS™ (Hitachi Software), based on an algorithm, analogous to CLUSTAL (Higgins D G &amp; Sharp P M (1988),  Gene  73(1), 237-244). Once the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result. The sequences may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance. Deliberate amino acid substitutions may be made on the basis of similarity in amino acid properties (such as polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues) and it is therefore useful to group amino acids together in functional groups. Amino acids may be grouped together based on the properties of their side chains alone. However, it is more useful to include mutation data as well. The sets of amino acids thus derived are likely to be conserved for structural reasons. These sets may be described in the form of a Venn diagram (Livingstone C. D. and Barton G. J. (1993) “Protein sequence alignments: a strategy for the hierarchical analysis of residue conservation”  Comput. Appl. Biosci.  9: 745-756) (Taylor W. R. (1986) “The classification of amino acid conservation”  J. Theor. Biol.  119; 205-218). Conservative substitutions may be made, for example according to the Table 1, which describes a generally accepted Venn diagram grouping of amino acids. 
     
       
         
           
               
               
               
             
               
                 TABLE 1 
               
               
                   
               
               
                   
                 Set 
                 Sub-set 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 Hydrophobic 
                 F W Y H 
                 Aromatic 
                 F W Y H 
               
               
                   
                   
                 K M I L 
                   
                 SEQ ID 
               
               
                   
                   
                 V A G C 
                   
                 NO: 4) 
               
               
                   
                   
                 (SEQ 
                 Aliphatic 
                 I L V 
               
               
                   
                   
                 ID 
                   
                   
               
               
                   
                   
                 NO: 
                   
                   
               
               
                   
                   
                 1) 
                   
                   
               
               
                   
               
               
                   
                 Polar 
                 W Y H K 
                 Charged 
                 H K R E D 
               
               
                   
                   
                 R E D C 
                   
                 SEQ ID 
               
               
                   
                   
                 S T N Q 
                   
                 NO: 5) 
               
               
                   
                   
                 SEQ 
                 Positively 
                 H K R 
               
               
                   
                   
                 ID 
                 charged 
                   
               
               
                   
                   
                 NO: 
                 Negatively 
                 E D 
               
               
                   
                   
                 2) 
                 charged 
                   
               
               
                   
               
               
                   
                 Small 
                 V C A G 
                 Tiny 
                 A G S 
               
               
                   
                   
                 S P T N 
                   
                   
               
               
                   
                   
                 D 
                   
                   
               
               
                   
                   
                 SEQ 
                   
                   
               
               
                   
                   
                 ID 
                   
                   
               
               
                   
                   
                 NO: 
                   
                   
               
               
                   
                   
                 3) 
               
               
                   
               
            
           
         
       
     
     In certain embodiments, the Cas9 is an ortholog or homologue of Cas9 and can have a sequence homology or identity of at least 80%, more preferably at least 85%, even more preferably at least 90%, such as for instance at least 95% with Cas9. In further embodiments, the homologue or orthologue of Cas9 as referred to herein has a sequence identity of at least 80%, more preferably at least 85%, even more preferably at least 90%, such as for instance at least 95% with the wild type Cas9. Where the Cas9 has one or more mutations (mutated), the homologue or orthologue of said Cas9 as referred to herein has a sequence identity of at least 80%, more preferably at least 85%, even more preferably at least 90%, such as for instance at least 95% with the mutated Cas9. 
     In an embodiment, the Cas9 protein may be an ortholog of an organism of a genus which includes, but is not limited to  Streptococcus  sp. or  Staphylococcus  sp.; in particular embodiments, Cas9 protein may be an ortholog of an organism of a species which includes, but is not limited to SpCas9, SaCas9, ScCas9, SmCas9, or StCas9. In particular embodiments, the homologue or orthologue of Cas9p as referred to herein has a sequence homology or identity of at least 80%, more preferably at least 85%, even more preferably at least 90%, such as for instance at least 95% with one or more of the Cas9 sequences disclosed herein. In further embodiments, the homologue or orthologue of Cas9 as referred to herein has a sequence identity of at least 80%, more preferably at least 85%, even more preferably at least 90%, such as for instance at least 95% with the wild type SpCas9, SaCas9, ScCas9, SmCas9, or StCas9. 
     In particular embodiments, the Cas9 has a sequence homology or identity of at least 60%, more particularly at least 70%, such as at least 80%, more preferably at least 85%, even more preferably at least 90%, such as for instance at least 95% with SpCas9, SaCas9, ScCas9, SmCas9, or StCas9. In further embodiments, the Cas9 protein as referred to herein has a sequence identity of at least 60%, such as at least 70%, more particularly at least 80%, more preferably at least 85%, even more preferably at least 90%, such as for instance at least 95% with the wild type SpCas9, SaCas9, ScCas9, SmCas9, or StCas9. The skilled person will understand that this includes truncated forms of the Cas9 protein whereby the sequence identity is determined over the length of the truncated form. 
     IscB Reference Protein 
     As previously described, the Cas9-like polypeptide can have a varying sequence identity (e.g. not 100% sequence identity) to a reference or wild-type IscB. In some aspects the Cas9-like Effector can have structural and/or functional similarity to the IscB. It will be appreciated that any of the following IscB polypeptides described herein can serve as a reference or wild-type sequence to a Cas9-like polypeptide as discussed elsewhere herein. 
     Bacterial genomes encode numerous homologues of Cas9, the effector protein of the type II CRISPR-Cas systems. The homology region includes the arginine-rich helix and the HNH nuclease domain that is inserted into the RuvC-like nuclease domain. These genes, however, are not linked to cas genes or CRISPR. A group of Cas9 homologous represent a distinct group of nonautonomous transposons denoted ISC (Insertion Sequences Cas-9 related/like) (Kapitonov et al. J. Bacteriol. 2016 Mar. 1; 198(5): 797-807). Based on their structural features and the predicted target site specificity, the ISC elements form a distinct group within the IS605/IS200 superfamily of bacterial and archaeal transposons that are mobilized by the Y1 tyrosine transposase. Kapitonov et al. suggests that the Cas9 evolved via immobilization of an ISC transposon and that the ISC transposon-encoded two nuclease domain-containing proteins are the likely ancestors of the CRISPR-associated Cas9. 
     IscB is part of the ISC family of transposons that share domain architectures with Cas9, in which an HNH endonuclease domain is inserted into the RuvC-like domain (see e.g. Chylinski K, et al., 2014. Classification and evolution of type II CRISPR-Cas systems. Nucleic Acids Res. 42(10):6091-6105 and Shmakov et al. 2015. Discovery and functional characterization of diverse class 2 CRISPR-Cas systems. Mol Cell 60(3):385-397). 
     In some aspects, the reference or wild-type IscB can be an IscB Ktendonobacter racemifer DSM 44963 , Geitlerinema  sp. PCC7105 , Salipiger mucosus  DSM 16094, Youngiibacter fragilis 232.1, Coleofasciculus chthonoplastes PCC 7420. 
     Cas9-Like Effector Domains 
     As described elsewhere herein the Cas9-like polypeptide can include an HNH domain and optionally an inactive RuvC domain. In certain embodiments the Cas9-like polypeptide can include a bridge-helix domain. In certain embodiments, the Cas9-like polypeptide can include or be modified to include one or more other domains, such as, a nucleic acid interaction domain, a PAM interacting domain. These are discussed in greater detail elsewhere herein. The Cas9-like polypeptide can be further mutated or modified. Exemplary mutant Cas9-like polypeptides are discussed in greater detail elsewhere herein. 
     HNH Domain 
     As previously discussed, the Cas9-like polypeptide can have an HNH domain. HNH here refers to its structural motif bearing the conserved amino acid sequence of H—N—H. Proteins that harbor the HNH motif usually have a consensus sequence of approximately 30 amino acids including two pairs of conserved histidines and one asparagine that forms a zinc-finger domain. Proteins that contain the HNH motif fall into the HNH superfamily. Mechanistically, the HNH motif interacts mostly with the minor groove of the DNA where it is capable of inducing a strand break. The HNH domain can be similar or the same as a Cas9 HNH domain. In certain embodiments, the HNH domain can have nuclease activity or nickase activity. In some aspects the HNH domain of the Cas9-like polypeptide has 10-35%, 10-30%, 10-25%, 10-20%, 15-35%, 15-30%, 15-25%, 15-20%, 20-35%, 25-35%, or 30-35% identity to a reference or wild type Cas9 HNH domain. In certain embodiments, the nuclease activity at a target polynucleotide by the HNH domain can be absent until conformational transition of the Cas-like polypeptide as a result of allosteric interaction with another Cas effector (e.g. Cas12-like protein). In certain embodiments, conformational change of the Cas9 like effector can result in a positional change in the HNH domain such that it can be brought into effective proximity of a target polynucleotide. Thus, aspects, the nuclease and/or nickase activity of the Cas9-like protein can be dependent on the allosteric interaction between the Cas19like protein or domain and another Cas protein or domain (e.g. a Cas12-like protein or domain). 
     RuvC Domain 
     As previously discussed, the Cas9-like polypeptide can have a RuvC domain. The RuvC domain may be a RuvI, RuvII, or RuvIII domain. In certain example embodiments, the RuvC may be an inactive RuvC domain. The inactive RuvC domain can be similar to a Cas9 RuvC domain. In the context of e.g. Cas9, RuvC is active and cleaves the non-target DNA strand. The inactive RuvC domain of the Cas9-like polypeptide does not have nuclease activity. 
     Cas12-Like Effector Protein 
     General Features 
     In certain embodiments, the Cas12-like protein or domain can include a polypeptide that contains a RuvC or RuvC-like domain. In certain embodiments, the Cas12-like polypeptide can have 10-35%, 10-30%, 10-25%, 10-20%, 15-35%, 15-30%, 15-25%, 15-20%, 20-35%, 25-35%, or 30-35% identity to a reference or wild-type Cas12 polypeptide, which are discussed elsewhere herein. In certain embodiments, the Cas12-like polypeptide can have 80-100% identity to a polypeptide encoded by one or more of the polynucleotides provided in SEQ ID NOs: 57-100 and/or one or more regions therein (See also, e.g. Tables 14-23 in the Working Example(s) herein), which are incorporated by reference herein as if expressed in their entireties. In certain embodiments, the Cas12-like polypeptide can have 80-100% identity to a polypeptide encoded by one or more of the polynucleotides provided in SEQ ID NOs: 57-87 and/or one or more regions therein (See also, e.g. Tables 14-23 in the Working Example(s) herein). In aspects, the RuvC or RuvC-like domain can have nuclease and/or nickase activity. In certain embodiments, the Cas12-like protein or domain can be capable of allosterically interacting with another Cas polypeptide (e.g. a Cas9-like protein) and eliciting an enzymatic or other biological effect. The Cas12-like protein can contain other domains as described elsewhere herein that can give the Cas12-like polypeptide other functionalities that can or are not dependent on allosteric interaction between the Cas12-like protein and another Cas (e.g. Cas9-like) protein. 
     Description of Cas12 (Cpfl) Reference Proteins 
     Cas12a (or Cpfl) is a Class II, Type V CRISPR-Cas system. The reference or wild-type Cas12a can be a Cas12a from  Prevotella  or  Francisella . Generally, Cas 12 is a smaller endonuclease than Cas9 and contains about 1300 amino acids, depending on variant. In some embodiments, the reference or wild-type Cas12 can be Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY) or Cas12e(CasX) (see e.g. Burstein et al., Nature 542, 237-241(2107); Liu et al., Nature 566, 218-223 (2019); Shmakov et al., Molecular Cell. 60:385-397(2015); and International Publication No. WO 2016/205749). 
     The Cpfl locus contains a mixed alpha-beta domain, a RuvC-1 followed by a helical region, a RuvC-II and a zinc finger-like domain. Zetsche et al. (2015) Cell 163(3):759-771. The Cpfl protein has a RuvC-like nuclease domain that is similar to the RuvC domain of Cas9. Further, Cpfl lacks an HNH domain, and the N-terminal does not have the alpha-helical recognition lobe of Cas9. Makarova et al. Nature Rev. Microbiol. (2015) “An updated evolutionary classification of CRISPR-Cas systems.” 
     The Cpfl does not require a tracrRNA and therefore only a crRNA is required. The Cpfl-crRNA complex cleaves target DNA and RNA by identification of a PAM (5′-YTN-3′), where Y is a pyrimidine and N is any nucleobase. This is in contrast to the G-rich PAM targeted by Cas9. After identification of PAM, Cpfl can introduce a sticky-end-like double stranded break of about 4-5 nucleotides overhang. 
     As previously discussed, the Cas12-like protein can be similar, but not identical, in structure and/or function to a wild-type or reference Cas12 protein. Suitable reference Cas12 reference or wild-type proteins are discussed herein. 
     In some aspects, the reference or wild-type Cas12 is that as discussed in Zetsche et al. (2015), which reported characterization of Cpfl, a class 2 CRISPR nuclease from  Francisella novicida  U112 having features distinct from Cas9. Cpfl is a single RNA-guided endonuclease lacking tracrRNA, utilizes a T-rich protospacer-adjacent motif, and cleaves DNA via a staggered DNA double-stranded break. 
     In some aspects, the reference or wild-type Cas12 is that as discussed Shmakov et al. (2015), which reported three distinct Class 2 CRISPR-Cas systems. Two system CRISPR enzymes (C2c1 and C2c3) contain RuvC-like endonuclease domains distantly related to Cpfl. Unlike Cpfl, C2c1 depends on both crRNA and tracrRNA for DNA cleavage. The third enzyme (C2c2) contains two predicted HEPN RNase domains and is tracrRNA independent. 
     In some aspects, the reference or wild-type Cas12 is that as discussed Gao et al, “Engineered Cpfl Enzymes with Altered PAM Specificities,” bioRxiv 091611; doi: http://dx.doi.org/10.1101/091611 (Dec. 4, 2016). 
     Cas12-Like Effector Domains 
     As previously discussed, the Cas12-like protein or domain contains a RuvC or RuvC-like domain. In certain embodiments, the RuvC or RuvC-like domain can have nuclease and/or nickase activity. In certain embodiments, the nuclease and/or nickase activity of the Cas12-like protein can be dependent on the allosteric interaction between the Cas12-like protein or domain and another Cas protein or domain (e.g. a Cas9-like protein or domain). In certain embodiments, the Cas12-like polypeptide can include or be modified to include one or more other domains, such as, a nucleic acid interaction domain, a PAM interacting domain. These are discussed in greater detail elsewhere herein. The Cas12-like polypeptide can be further mutated or modified. Exemplary mutant Cas12-like polypeptides are discussed in greater detail elsewhere herein. 
     RuvC/RuvC-Like Domain 
     The RuvC or RuvC-like domain can be similar or the same as a Cas12 RuvC or RuvC-like domain. In certain embodiments, the RuvC or RuvC-like domain can have nuclease activity or nickase activity. In certain embodiments, the nuclease or nickase activity at a target polynucleotide by the RuvC domain can be absent until conformational transition of the Cas12-like polypeptide as a result of allosteric interaction with another Cas effector (e.g. Cas9-like protein). In certain embodiments, conformational change of the Cas12-like protein can result in a positional change in the RuvC domain such that it can be brought into effective proximity of a target polynucleotide. 
     General Features of Cas-Like Effectors 
     Activatable Functional Domains 
     In addition to the domains previously discussed, the Cas-like effectors can have other optional domains. In some aspects the Cas-like effectors the Cas-like effectors can have one or more activatable functional domains. As used in this context herein “activatable functional domain” refers to a functional domain that can interact with another activatable functional domain to induce one or both of the activatable functional domains to activate, associate, interact, and/or fuse to form a new single active functional domain to elicit an enzymatic or other biological activity to affect a target with the attributed function. A pair of activatable functional domains that matched such that their association, interaction, or fusion elicits an enzymatic or other biological activity is referred to herein as a “matched pair of activatable functional domains”. In certain embodiments, association, interaction, and/or fusion of matched pair of activatable functional domains occurs after allosteric interaction between two or more of the same or different Cas-like proteins. In certain embodiments, the enzymatic or other biological activity is elicited at the target after association, interaction, and/or fusion of matched pair of activatable functional domains. In certain embodiments, the enzymatic or other biological activity is elicited at the target after allosteric interaction of two or more of the same or different Cas-like proteins. 
     In some aspects, a Cas-like protein described herein can change conformation upon allosteric interaction that results in exposure of an active site in a functional domain such that it can interact with a substrate. In some aspects, a Cas-like protein described herein or domain thereof can change in spatial position within the system upon allosteric interaction that results in exposure or accessibility of an active site in a functional domain such to a substrate (e.g. a target substrate). In some aspects, a functional domain of a Cas-like protein can be in an inactive state prior to allosteric interaction due to the presence of a protector molecule or group. In these aspects, an inactive functional domain of a first Cas-like protein can interact with a functional domain on a second Cas-like protein upon or after direct or indirect allosteric interaction between the two Cas-like proteins such that the second functional domain alters the protection group on the first functional domain and thus activates the functional domain on the first Cas-like protein. In some aspects, allosteric interaction two Cas-like proteins can bring an inactive functional domain on one Cas-like protein into effective proximity of a domain (e.g. another functional domain) on another protein in the CRISPR-Cas system (e.g. another Cas-like protein) such that the first functional domain is activated. Such examples include fluorescent proteins that can be activated (or in activated) based on resonant energy transfer. It will be appreciated that the system can be configured in some aspects as a “switched-off” system, meaning that the functional group can be active until allosteric interaction between two Cas-like proteins. One example of this may be a system where the first functional domain is optically active until allosteric interaction between the two Cas-like proteins. It will be appreciated that the system can be configured as a “switched-on” system, meaning that the functional group can be inactive until allosteric interaction between two Cas-like proteins occurs. 
     One or both of the activatable functional domains in a matched activatable functional domain pair can have activity selected from the group comprising, consisting essentially of, or consisting of methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, deaminase activity, reverse-transcriptase, transposase, optical activity (e.g. emits a wavelength of light), molecular switch activity (e.g., light inducible), base excision repair inhibiting activity and combinations thereof. 
     In some embodiments, one or more of the activatable functional domains comprise a transcriptional activator, repressor, a recombinase, a transposase, a histone remodeler, a demethylase, a DNA methyltransferase, a cryptochrome, a light inducible/controllable domain, a chemically inducible/controllable domain, an optically active protein domain, a deaminase, base excision repair inhibiting domain. an epigenetic modifying domain, or a combination thereof. The functional domain can include an activator, repressor or nuclease. 
     In general, the positioning of the one or more activatable functional domain on the Cas-like enzyme is one which allows for correct spatial orientation for the activatable functional domain to affect the target with the attributed functional effect upon or after allosteric interaction with another Cas-like protein described herein. For example, if the functional domain is a transcription activator (e.g., VP64 or p65), the transcription activator is placed in a spatial orientation which allows it to affect the transcription of the target. Likewise, a transcription repressor will be advantageously positioned to affect the transcription of the target, and a nuclease (e.g., Fok1) will be advantageously positioned to cleave or partially cleave the target. This may include positions other than the N-/C-terminus of the CRISPR enzyme. 
     A split protein approach may be used with respect to the activatable functional domain. The so-called ‘split protein’ approach allows for the following. The protein (e.g. complete active functional domain) is split into two pieces and each of these are fused to one half of a dimer or each to a different Cas-like polypeptide or different Cas-like domain on a single polypeptide. Upon dimerization and/or other allosteric interaction between the two Cas-like proteins, the two parts of the split protein (or split functional domain) are brought together and the reconstituted protein and/or functional domain becomes functional. It will be appreciated that in the context of an AAV or other viral delivery system (described in greater detail herein), one Cas-like protein with one part of the split protein or split functional domain can be associated with one VP domain (e.g. VP2) and the second Cas-like protein with another part of the split protein or split functional domain can be on another or different VP (e.g. VP2) domain. The two VP domains (e.g. VP2 domains) may be in the same or different capsid. In other words, the split parts of the split protein or split functional domain can be on the same virus particle or on different virus particles. Likewise, one Cas-like protein can be on the same virus particle or on different virus particles. The split protein or split functional domain can be derived or generated from or be based on any other functional protein or functional domain described herein. 
     In some embodiments, one or more functional domains may be associated with or tethered to one or more CRISPR-Cas enzymes and/or may be associated with or tethered to nucleic acid components (e.g. modified guides) via adaptor proteins. These can be used irrespective of the fact that the CRISPR enzyme may also be tethered to a virus outer protein or capsid or envelope, such as a VP2 domain or a capsid, via modified guides with aptamer RNA sequences that recognize correspond adaptor proteins. 
     As previously discussed the activatable functional domains can include or form functional domains that are not necessarily base-editors as discussed above. This can provide alternative or additional functionalities and/or control to the CRISPR-Cas systems described herein other than or in addition to base editing. In embodiments, or both of the activatable functional domains in a matched activatable functional domain pair can have activity selected from the group comprising, consisting essentially of, or consisting of methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, optical activity (e.g. emits a wavelength of light), molecular switch activity (e.g., light inducible), and combinations thereof. 
     In some embodiments, one or more of the activatable functional domains comprise a transcriptional activator, repressor, a recombinase, a transposase, a histone remodeler, a demethylase, a DNA methyltransferase, a cryptochrome, a light inducible/controllable domain, a chemically inducible/controllable domain, an optically active protein domain, an epigenetic modifying domain, or a combination thereof. The functional domain can include an activator, repressor or nuclease. 
     Examples of activators include P65, a tetramer of the herpes simplex activation domain VP16, termed VP64, optimized use of VP64 for activation through modification of both the sgRNA design and addition of additional helper molecules, MS2, P65 and HSF1 in the system called the synergistic activation mediator (SAM) (Konermann et al, “Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex,” Nature 517(7536):583-8 (2015)); and examples of repressors include the KRAB (Kruppel-associated box) domain of Kox1 or SID domain (e.g. SID4X); and an example of a nuclease or nuclease domain suitable for a functional domain comprises Fok1. 
     Example of optically active molecules include, dyes (e.g. fluorescent dyes, infrared, near-IR, and UV dyes) chemiluminescent molecules, and quantum dots. Examples of optically active proteins include, but are not limited to fluorescent proteins and bioluminescent proteins (e.g. luciferase). Fluorescent proteins can be engineered to fluoresce at a variety of wavelengths to yield proteins that fluoresce in different colors or in UV. Blue and UV fluorescent proteins include, but are not limited to, BFP, tagBFP, mTagBFB2, Azurite, EBFP2, mKalama1, Sirius, Sapphire, and T-Sapphire. Cyan fluorescent proteins include, but are not limited to, ECFP, Cerulean, SCFP3A, mTurquoise, mTurquoise2, monomeric Midoriishi-Cyan, TagCFP, and mTFP1. Green fluorescent proteins include, but are not limited to, GFP, EGFP, Emerald, Superfolder GFP, Monomeric Azami Green, TagGFP2, mUKG, mWasabi, Clover, mNeonGreen. Yellow fluorescent proteins include, but are not limited to, YFP, EYFP, Citrine, Venus, SYFP2, TagYFP. Orange fluorescent proteins include, but are not limited to, Monomeric Kusabira-Orange, mKOk, mKO2, mOrange, and mOrange2. Red fluorescent proteins include, but are not limited to RFP, mRaspberry, mCherry, mStrwberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, mRuby, and mRuby2. Far-Red proteins include, but are not limited to mPlum, HcRed-tandem, mKate2, mNeptune, and NirFP. Near-IR proteins include, but are not limited to, IFP1.4 and iRFP. Long Stokes Shift proteins include, but are not limited to mKeimaRed, LSS-mKatel, LSS-mKate2, and mBeRFP. 
     Examples of photoactivatable proteins include, but are not limited to, Kaede (green), Kaede (red), KikGR1 (green), KikGR1 (red), PS-CFP2, mEos2 (green), mEos3.2 (green), mEos3.2 (red), PSmOrange. 
     Examples of photoswitchable proteins include, but are not limited to Dronapa. 
     Attachment of a functional domain or fusion protein can be via a linker, e.g., a flexible glycine-serine (GlyGlyGlySer) (SEQ ID NO: 6), GGGGS (SEQ ID NO: 7) or (GGGS) 3  (SEQ ID NO: 8) or a rigid alpha-helical linker such as (Ala(GluAlaAlaAlaLys)Ala) (SEQ ID NO: 9). Linkers such as (GGGGS) 3  (SEQ ID NO: 10) are preferably used herein to separate protein or peptide domains. (GGGGS) 3  (SEQ ID NO: 10) is preferable because it is a relatively long linker (15 amino acids). The glycine residues are the most flexible and the serine residues enhance the chance that the linker is on the outside of the protein. (GGGGS) 6  (SEQ ID NO: 11), (GGGGS) 9  (SEQ ID NO: 12) or (GGGGS) 12  (SEQ ID NO: 13) may preferably be used as alternatives. Other preferred alternatives are (GGGGS) 1  (SEQ ID NO: 7), (GGGGS) 2  (SEQ ID NO: 14), (GGGGS) 4  (SEQ ID NO: 15), (GGGGS) 5  (SEQ ID NO: 16), (GGGGS) 7  (SEQ ID NO: 17), (GGGGS) 8  (SEQ ID NO: 18), (GGGGS) 10  (SEQ ID NO: 19), or (GGGGS) 11  (SEQ ID NO: 20). Alternative linkers are available, but highly flexible linkers are thought to work best to allow for maximum opportunity for the 2 parts of the Cas protein to come together and thus reconstitute Cas protein activity. One alternative is that the NLS of nucleoplasmin can be used as a linker. For example, a linker can also be used between the Cas protein and any functional domain. Again, a (GGGGS) 3  (SEQ ID NO: 10) linker may be used here (or the 6, 9, or 12 repeat versions therefore) or the NLS of nucleoplasmin can be used as a linker between a Cas protein and the functional domain. Other linkers are described herein and/or will be instantly appreciated by those of ordinary skill in the art in view of the disclosure herein. 
     Additional Functional Domains 
     The Cas-like polypeptides can have additional domains including, but not limited to a nucleic acid interaction domain, a PAM interacting domain, a HEPN domain and combinations thereof. In some aspects, one or more of the Cas-like proteins comprise at least one nucleic acid interacting domain, including but not limited to nucleic acid interaction domains described herein, nucleic acid interaction domains known in the art, and domains recognized to be nucleic acid interaction by comparison to consensus sequences and motifs. In some aspects, one or more of the Cas-like proteins comprise at least one PAM interacting domain, including but not limited to PAM interacting domains described herein, PAM interacting domains known in the art, and domains recognized to be PAM interacting domains by comparison to consensus sequences and motifs. In some aspects, one or more of the Cas-like proteins comprise at least one HEPN domain, including but not limited to HEPN domains described herein, HEPN domains known in the art, and domains recognized to be HEPN domains by comparison to consensus sequences and motifs. 
     Nucleic Acid Interaction Domain 
     One or more of the Cas-like proteins can be capable of directing sequence-specific binding. In certain embodiments, sequence-specific binding can be facilitated by a nucleic acid interaction domain. In some aspects, one or more of the Cas-like proteins can include a nucleic interaction domain. The nucleic acid interaction domain can be a domain that is capable of complexing with a nucleic acid component. The nucleic acid component can specifically hybridize with a target sequence as is discussed in greater detail elsewhere herein. In some aspects, one or more of the Cas-like proteins is complexed to a nucleic acid component. Nucleic acid components are discussed elsewhere herein. 
     Modified Cas-Like Effectors 
     The Cas-like polypeptides described herein can be mutated or otherwise modified. 
     In particular embodiments, it is of interest to make use of an engineered Cas-like protein as defined herein, wherein the protein complexes with a nucleic acid molecule comprising RNA to form a CRISPR complex, wherein when in the CRISPR complex, the nucleic acid molecule targets one or more target polynucleotide loci, the protein comprises at least one modification compared to unmodified Cas-like protein, and wherein the CRISPR complex comprising the modified protein has altered activity as compared to the complex comprising the unmodified Cas-like protein. 
     In one embodiment, a modified Cas or Cas-like protein comprises at least one modification that alters editing preference as composed to wild type. In certain embodiments, the editing preference is for a specific insert or deletion within the target region. In certain example embodiments, the at least one modification increases formation of one or more specific indels. In one example embodiment, the at least on modification is in the binding region including the targeting region and/or a PAM interacting region. In another example embodiment, the at least one modification is not in the binding region including the targeting region and/or the PAM interacting region. In one example embodiment, the one or more modifications are located in or proximate to an active or inactive RuvC domain. In another example embodiment, the one or more modifications are located in or proximate to an HNH domain or Nuc lobe. In another example embodiment, the one or more modifications are in or proximate to a bridge helix. In another example embodiment, the one or more modifications are in or proximate to a recognition (REC) lobe. In another example embodiment, the at least one modification is present or proximate to a D10 active site residue. In another example embodiment, the at least one modification is present in or proximate to a linker region. The linker region may form a linker from an optional active or inactive RuCv domain to the bridge helix. In certain example embodiments, the one or more modifications are located at residues 6-19, 51-60, 690-696, 698-700, 725-734, 764-786, 802-811, 837-871, 902-929, 976-982, 998-1007, or a combination thereof, of SpCas9 or a residue in an ortholog corresponding or functionally equivalent to a Cas9-like protein described herein. 
     In certain example embodiments, the at least one modification increases formation of one or more specific insertions. In certain example embodiments, the at least one modification results in an insertion of an A adjacent to an A, T, G, or C in the target region. In another example embodiment, the at least one modification results in insertion of a T adjacent to an A, T, G, or C in the target region. In another example embodiment, the at least one modification results in insertion of a G adjacent to an A, T, G, or C in the target region. In another example embodiment, the at least one modification results in insertion of a C adjacent to an A, T, C, or G in the target region. The insertion may be 5′ or 3′ to the adjacent nucleotide. In one example embodiment, the one or more modification direct insertion of a T adjacent to an existing T. In certain example embodiments, the existing T corresponds to the 4 th  position in the binding region of a guide sequence. In certain example embodiments, the one or more modifications result in an enzyme which ensures more precise one-base insertions or deletions, such as those described above. More particularly, the one or more modifications may reduce the formations of other types of indels by the enzyme. The ability to generate one-base insertions or deletions can be of interest in a number of applications, such as correction of genetic mutants in diseases caused by small deletions, more particularly where HDR is not possible. For example, correction of the F508del mutation in CFTR via delivery of three sRNA directing insertion of three T&#39;s, which is the most common genotype of cystic fibrosis, or correction of Alia Jafar&#39;s single nucleotide deletion in CDKL5 in the brain. As the editing method only requires NHEJ, the editing would be possible in post-mitotic cells such as the brain. The ability to generate one base pair insertions/deletions may also be useful in genome-wide CRISPR-Cas negative selection screens. In certain example embodiments, the at least one modification is a mutation. In certain other example embodiment, the one or more modification may be combined with one or more additional modifications or mutations described below including modifications to increase binding specificity, decrease off-target effects, modify allosteric interaction one or more other polypeptides, e.g. a Cas12-like polypeptide, Cas9-like, and combinations thereof. 
     In certain example embodiments, the Cas polypeptide comprising at least one modification that alters editing preference as compared to wild type Cas polypeptide may further comprise one or more additional modifications that alters the binding property as to a nucleic acid component, nucleic acid molecule comprising RNA and/or the target polypeptide loci, altering binding kinetics as to the nucleic acid molecule or target molecule or target polynucleotide, alters binding specificity as to a polynucleotide such as a nucleic acid component and/or a target sequence, and/or alters the allosteric interaction capability described herein of the Cas polypeptide. Example of such modifications are summarized in the following paragraph. 
     Suitable polypeptide modifications which enhance specificity in particular by reducing off-target effects, are described for instance in International Patent Application No. PCT/US2016/038034, which is incorporated herein by reference in its entirety. In particular embodiments, a reduction of off-target cleavage is ensured by destabilizing strand separation, more particularly by introducing mutations in the Cas enzyme decreasing the positive charge in the DNA interacting regions (as described herein and further exemplified for Cas9 by Slaymaker et al. 2016 (Science, 1; 351(6268):84-8). In further embodiments, a reduction of off-target cleavage is ensured by introducing mutations into one or more Cas enzyme which affect the interaction between the target strand and the guide RNA sequence, more particularly disrupting interactions between a Cas protein and the phosphate backbone of the target DNA strand in such a way as to retain target specific activity but reduce off-target activity (as described for Cas9 by Kleinstiver et al. 2016, Nature, 28; 529(7587):490-5). In particular embodiments, the off-target activity is reduced by way of a modified Cas wherein both interaction with target strand and non-target strand are modified compared to wild-type Cas. 
     The methods and mutations which can be employed in various combinations to increase or decrease activity and/or specificity of on-target vs. off-target activity, or increase or decrease binding and/or specificity of on-target vs. off-target binding, can be used to compensate or enhance mutations or modifications made to promote other effects. Such mutations or modifications made to promote other effects include mutations or modification to the Cas effector protein and or mutation or modification made to a guide RNA. 
     With a similar strategy used to improve Cas specificity (Slaymaker et al. 2015 “Rationally engineered Cas9 nucleases with improved specificity”), specificity of Cas-like polypeptide can be further improved by mutating residues that stabilize the non-targeted DNA strand. This may be accomplished without a crystal structure by using linear structure alignments to predict 1) which domain of Cas polypeptide binds to which strand of DNA and 2) which residues within these domains contact DNA. It may be desirable to probe the function of all likely DNA interacting amino acids (lysine, histidine and arginine) of the Cas polypeptide (e.g. a Cas-like (e.g. Cas9-like or Cas12-like protein) described herein. 
     Without being bound by theory, in an aspect, the methods and mutations described can enhance conformational rearrangement of Cas domains or proteins to positions that results in cleavage at on-target sites and avoidance of those conformational states at off-target sites. In certain embodiments, the confirmation rearrangement of the Cas domains or proteins occurs upon allosteric interaction of two or more Cas polypeptides. 
     In certain embodiments, a Cas cleaves target DNA in a series of coordinated steps. First, the PAM-interacting domain recognizes the PAM sequence 5′ of the target DNA. After PAM binding, the first 10-12 nucleotides of the target sequence (seed sequence) are sampled for sgRNA:DNA complementarity, a process dependent on DNA duplex separation. If the seed sequence nucleotides complement the sgRNA, the remainder of DNA is unwound and the full length of sgRNA hybridizes with the target DNA strand. The nt-groove between the RuvC and HNH domains stabilizes the non-targeted DNA strand and facilitates unwinding through nonspecific interactions with positive charges of the DNA phosphate backbone. RNA:cDNA and Cas:ncDNA interactions drive DNA unwinding in competition against cDNA:ncDNA rehybridization. Other Cas9 and/or Cas12 domains can affect the conformation of nuclease domains as well, for example linkers connecting HNH with RuvCII and RuvCIII, RuvC-like, RuvC (inactive or active). 
     The methods and mutations described herein encompass, without limitation, RuvCI, RuvCIII, RuvCIII and HNH domains and linkers. Conformational changes in Cas and/or Cas-like protein brought about by allosteric interaction with other Cas and/or Cas-like proteins, target DNA binding, including seed sequence interaction, and interactions with the target and non-target DNA strand determine whether the domains are positioned to trigger nickase, nuclease, and/or other enzymatic activity. Thus, the Cas and Cas-like protein mutations and methods provided herein demonstrate and enable modifications that go beyond PAM recognition and RNA-DNA base pairing. In an aspect, the invention provides Cas-like proteins that comprise an improved equilibrium towards conformations associated with cleavage activity when involved in on-target interactions and/or improved equilibrium away from conformations associated with cleavage activity when involved in off-target interactions. In one aspect, the invention provides Cas-like proteins with or improved proof-reading function, i.e. a Cas or Cas-like nickase or nuclease which adopts a conformation comprising nickase or nuclease activity at an on-target site, and which conformation has increased unfavorability at an off-target site. Sternberg et al., Nature 527(7576):110-3, doi: 10.1038/nature15544, published online 28 Oct. 2015. Epub 2015 Oct. 28, used Förster resonance energy transfer FRET) experiments to detect relative orientations of the Cas9 catalytic domains when associated with on- and off-target DNA. Similar assays can be used to detect the relative orientations of the Cas-like effector (e.g. Cas9-like or Cas12-like) domains described herein. 
     Where the Cas polypeptide has nuclease activity, the Cas polypeptide can be modified to have diminished nuclease activity e.g., nuclease inactivation of at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% as compared with the wild type enzyme; or to put in another way, a Cas enzyme having advantageously about 0% of the nuclease activity of the non-mutated or wild type Cas enzyme or reference Cas CRISPR enzyme, or no more than about 3% or about 5% or about 10% of the nuclease activity of the non-mutated or wild type Cas-like or Cas enzyme. This is possible by introducing mutations into the nuclease domains of the Cas-polypeptide and orthologs thereof. In certain embodiments, the Cas enzyme is engineered and can comprise one or more mutations that reduce or eliminate a nuclease activity. When the enzyme is not SpCas9 (e.g. is a Cas-like protein (e.g. Cas9-like or Cas12-like)), mutations may be made at any or all residues corresponding to positions 10, 762, 840, 854, 863 and/or 986 of SpCas9 (which may be ascertained for instance by standard sequence comparison tools). In particular, any or all of the following mutations are preferred in SpCas9 or SpCas9-like: D10, E762, H840, N854, N863, or D986; as well as conservative substitution for any of the replacement amino acids is also envisaged. The point mutations to be generated to substantially reduce nuclease activity include but are not limited to D10A, E762A, H840A, N854A, N863A and/or D986A. In an aspect, the invention provides a herein-discussed composition, wherein the Cas polypeptide comprises two or more mutations, wherein the two or more mutations are two or more of D10, E762, H840, N854, N863, or D986 according or corresponding to the SpCas9 or SpCas9-like protein or any corresponding to N580 according or corresponding to the SaCas9 or SaCas9-like protein ortholog are mutated, or the Cas polypeptide comprises at least one mutation wherein at least H840 is mutated. In an aspect, the invention provides a herein-discussed composition wherein the Cas polypeptide comprises two or more mutations comprising D10A, E762A, H840A, N854A, N863A or D986A according or corresponding to SpCas9 or SpCas9-likeprotein or any corresponding ortholog, or N580A according or corresponding to SaCas9 or SaCas9-like protein, or at least one mutation comprising H840A, or, optionally wherein the Cas polypeptide comprises: N580A according or corresponding to SaCas9 or SaCas-like protein or any corresponding ortholog; or D10A according or corresponding to SpCas9 or SpCas9 protein, or any corresponding ortholog, and N580A according to or corresponding to SaCas9 or SaCas-like protein. In an aspect, the invention provides a herein-discussed composition, wherein the Cas polypeptide comprises H840A, or D10A and H840A, or D10A and N863A, according or corresponding to SpCas9 or SpCas9-like protein or any corresponding ortholog. 
     Mutations can also be made at neighboring residues, e.g., at amino acids near those indicated above that participate in the nuclease activity. In some embodiments, the RuvC domain is inactivated, and in other embodiments, another putative nuclease domain is inactivated, wherein the effector protein complex functions as a nickase and cleaves only one DNA strand as discussed elsewhere herein. In a preferred embodiment, the other putative nuclease domain is a HincII-like endonuclease domain. In some embodiments, two Cas or Cas-like variants (each a different nickase) are used to increase specificity, two nickase variants are used to cleave DNA at a target (where both nickases cleave a DNA strand, while minimizing or eliminating off-target modifications where only one DNA strand is cleaved and subsequently repaired). In a preferred embodiment, a homodimer may comprise two Cas or Cas-like effector protein molecules comprising a different mutation in their respective RuvC domains. 
     In certain embodiments, the modification or mutation comprises a mutation in a RuvCI, RuvCIII, RuvCIII or HNH domain. In certain embodiments, the modification or mutation comprises an amino acid substitution at one or more of positions corresponding to positions 12, 13, 63, 415, 610, 775, 779, 780, 810, 832, 848, 855, 861, 862, 866, 961, 968, 974, 976, 982, 983, 1000, 1003, 1014, 1047, 1060, 1107, 1108, 1109, 1114, 1129, 1240, 1289, 1296, 1297, 1300, 1311, and 1325; preferably 855; 810, 1003, and 1060; or 848, 1003 with reference to amino acid position numbering of SpCas9. Corresponding locations can be identified in a Cas polypeptide as described elsewhere herein. In certain embodiments, the modification or mutation corresponding to position(s) 63, 415, 775, 779, 780, 810, 832, 848, 855, 861, 862, 866, 961, 968, 974, 976, 982, 983, 1000, 1003, 1014, 1047, 1060, 1107, 1108, 1109, 1114, 1129, 1240, 1289, 1296, 1297, 1300, 1311, or 1325; preferably 855; 810, 1003, and 1060; 848, 1003, and 1060; or 497, 661, 695, and 926 comprises an alanine substitution with corresponding reference to amino acid position numbering of SpCas9. Corresponding locations can be identified in a Cas polypeptide as described elsewhere herein. In certain embodiments, the modification comprises K855A; K810A, K1003A, and R1060A; or K848A, K1003A (with reference to SpCas9), and R1060A. in certain embodiments, in certain embodiments, the modification comprises N497A, R661A, Q695A, and Q926A, with reference to amino acid position numbering of SpCas9. Corresponding locations can be identified in a Cas polypeptide as described elsewhere herein. 
     Other mutations may include N692A, M694A, Q695A, H698A or combinations thereof and as otherwise described in Kleinstiver et al. “High-fidelity CRISP-Cas9 nucleases with no detectable genome-wide off-target effects” Nature 529, 590-607 (2016). Where the mutations are made in reference to a non-Cas-like protein, corresponding locations can be identified in a Cas-like polypeptide as described elsewhere herein. In addition, mutations and or modifications within a REC3 domain (with reference to SpCas9-HF1 and eSpCas9(1.1)) may also be targeted for increased target specify and as further described in Chen et al. “Enhanced proofreading governs CRISPR-Cas9 targeting accuracy” bioRxv Jul. 6, 2017 doi: http://dx.doi.org/10.1101/160036. Other mutations may be located in an HNH nuclease domain as further described in Sternberg et al. Nature 2015 doi:10.1038/nature15544. Where the mutations are made in reference to a non-Cas-like protein, corresponding locations can be identified in a Cas-like polypeptide as described elsewhere herein. 
     Where the Cas protein has nuclease activity, the Cas protein may be modified to have diminished nuclease activity e.g., nuclease inactivation of at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% as compared with the wild type enzyme; or to put in another way, a Cas enzyme having advantageously about 0% of the nuclease activity of the non-mutated or wild type Cas enzyme or CRISPR enzyme, or no more than about 3% or about 5% or about 10% of the nuclease activity of the non-mutated or wild type Cas enzyme. In some embodiments, a nucleic acid-targeting effector protein may be considered to substantially lack all RNA cleavage activity when the RNA cleavage activity of the mutated enzyme is about no more than 25%, 10%, 5%, 1%, 0.1%, 0.01%, or less of the nucleic acid cleavage activity of the non-mutated form of the enzyme; an example can be when the nucleic acid cleavage activity of the mutated form is nil or negligible as compared with the non-mutated form. This is possible by introducing mutations into the nuclease domains of the Cas and orthologs thereof. 
     Embodiments of the invention include sequences (both polynucleotide or polypeptide) which may comprise homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue or nucleotide, with an alternative residue or nucleotide) that may occur i.e., like-for-like substitution in the case of amino acids such as basic for basic, acidic for acidic, polar for polar, etc. Non-homologous substitution may also occur i.e., from one class of residue to another or alternatively involving the inclusion of unnatural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O), pyriylalanine, thienylalanine, naphthylalanine and phenylglycine. Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence including alkyl groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or β-alanine residues. A further form of variation, which involves the presence of one or more amino acid residues in peptoid form, may be well understood by those skilled in the art. For the avoidance of doubt, “the peptoid form” is used to refer to variant amino acid residues wherein the α-carbon substituent group is on the residue&#39;s nitrogen atom rather than the α-carbon. Processes for preparing peptides in the peptoid form are known in the art, for example Simon R J et al., PNAS (1992) 89(20), 9367-9371 and Horwell D C, Trends Biotechnol. (1995) 13(4), 132-134. 
     Homology modelling: Corresponding residues in other Cas orthologs can be identified by the methods of Zhang et al., 2012 (Nature; 490(7421): 556-60) and Chen et al., 2015 (PLoS Comput Biol; 11(5): e1004248)—a computational protein-protein interaction (PPI) method to predict interactions mediated by domain-motif interfaces. PrePPI (Predicting PPI), a structure-based PPI prediction method, combines structural evidence with non-structural evidence using a Bayesian statistical framework. The method involves taking a pair of query proteins and using structural alignment to identify structural representatives that correspond to either their experimentally determined structures or homology models. Structural alignment is further used to identify both close and remote structural neighbours by considering global and local geometric relationships. Whenever two neighbours of the structural representatives form a complex reported in the Protein Data Bank, this defines a template for modelling the interaction between the two query proteins. Models of the complex are created by superimposing the representative structures on their corresponding structural neighbour in the template. This approach is further described in Dey et al., 2013 (Prot Sci; 22: 359-66). 
     For purpose of this invention, amplification means any method employing a primer and a polymerase capable of replicating a target sequence with reasonable fidelity. Amplification may be carried out by natural or recombinant DNA polymerases such as TaqGold™, T7 DNA polymerase, Klenow fragment of  E. coli  DNA polymerase, and reverse transcriptase. A preferred amplification method is PCR. 
     In some embodiments the Cas effector (e.g. a Cas-like effector or other Cas effector described herein that is part of the non-class I engineered CRISPR-Cas system described herein) can have one or more nuclear localization sequences (NLSs) such as about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. More particularly, vector comprises one or more NLSs not naturally present in the Cas effector protein. Most particularly, the NLS is present in the vector 5′ and/or 3′ of the Cas effector protein sequence In some embodiments, the RNA-targeting effector protein comprises about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the amino-terminus, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the carboxy-terminus, or a combination of these (e.g., zero or at least one or more NLS at the amino-terminus and zero or at one or more NLS at the carboxy terminus). When more than one NLS is present, each may be selected independently of the others, such that a single NLS may be present in more than one copy and/or in combination with one or more other NLSs present in one or more copies. In some embodiments, an NLS is considered near the N- or C-terminus when the nearest amino acid of the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptide chain from the N- or C-terminus. Non-limiting examples of NLSs include an NLS sequence derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO: 21); the NLS from nucleoplasmin (e.g., the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 22)); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 23) or RQRRNELKRSP (SEQ ID NO: 24); the hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 25); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 26) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 27) and PPKKARED (SEQ ID NO: 28) of the myoma T protein; the sequence PQPKKKPL (SEQ ID NO: 29) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO: 30) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 31) and PKQKKRK (SEQ ID NO: 32) of the influenza virus NS1; the sequence RKLKKKIKKL (SEQ ID NO: 33) of the Hepatitis virus delta antigen; the sequence REKKKFLKRR (SEQ ID NO: 34) of the mouse Mx1 protein; the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 35) of the human poly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 36) of the steroid hormone receptors (human) glucocorticoid. In general, the one or more NLSs are of sufficient strength to drive accumulation of the DNA/RNA-targeting Cas protein in a detectable amount in the nucleus of a eukaryotic cell. In general, strength of nuclear localization activity may derive from the number of NLSs in the nucleic acid-targeting effector protein, the particular NLS(s) used, or a combination of these factors. Detection of accumulation in the nucleus may be performed by any suitable technique. For example, a detectable marker may be fused to the nucleic acid-targeting protein, such that location within a cell may be visualized, such as in combination with a means for detecting the location of the nucleus (e.g., a stain specific for the nucleus such as DAPI). Cell nuclei may also be isolated from cells, the contents of which may then be analyzed by any suitable process for detecting protein, such as immunohistochemistry, Western blot, or enzyme activity assay. Accumulation in the nucleus may also be determined indirectly, such as by an assay for the effect of nucleic acid-targeting complex formation (e.g., assay for DNA or RNA cleavage or mutation at the target sequence, or assay for altered gene expression activity affected by DNA or RNA-targeting complex formation and/or DNA or RNA-targeting Cas protein activity), as compared to a control not exposed to the nucleic acid-targeting Cas protein or nucleic acid-targeting complex, or exposed to a nucleic acid-targeting Cas protein lacking the one or more NLSs. In preferred embodiments of the herein described Cas effector proteins, complexes thereof and systems thereof, the codon optimized Cas effector proteins comprise an NLS attached to the C-terminal of the protein. In certain embodiments, other localization tags may be fused to the Cas protein, such as without limitation for localizing the Cas to particular sites in a cell, such as organelles, such mitochondria, plastids, chloroplast, vesicles, Golgi, (nuclear or cellular) membranes, ribosomes, nucleolus, ER, cytoskeleton, vacuoles, centrosome, nucleosome, granules, centrioles, etc. These and other targeting/localization/retention signals are discussed elsewhere herein and can be used to modify the Cas effectors(s). 
     Nucleic Acid Components 
     General Discussion 
     The nucleic acid interaction domain can interact with, associate, and/or bind to one or more nucleic acid components. The term “nucleic acid components” is inclusive of crRNA, guide RNA, single guide RNA and variants thereof described herein. As used herein, the term “crRNA” or “guide RNA” or “single guide RNA” or “sgRNA” or “one or more nucleic acid components” of a CRISPR-Cas effector protein described herein comprises any polynucleotide sequence having sufficient complementarity with a target nucleic acid sequence to hybridize with the target nucleic acid sequence and direct sequence-specific binding of a nucleic acid-targeting complex to the target nucleic acid sequence. In some embodiments, the degree of complementarity, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at www.novocraft.com), ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). The ability of a guide sequence (within a nucleic acid-targeting guide RNA) to direct sequence-specific binding of a nucleic acid-targeting complex to a target nucleic acid sequence may be assessed by any suitable assay. For example, the components of a nucleic acid-targeting CRISPR system sufficient to form a nucleic acid-targeting complex, including the guide sequence to be tested, may be provided to a host cell having the corresponding target nucleic acid sequence, such as by transfection with vectors encoding the components of the nucleic acid-targeting complex, followed by an assessment of preferential targeting (e.g., cleavage) within the target nucleic acid sequence, such as by Surveyor assay as described herein. Similarly, cleavage of a target nucleic acid sequence may be evaluated in a test tube by providing the target nucleic acid sequence, components of a nucleic acid-targeting complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions. Other assays are possible, and will occur to those skilled in the art. A guide sequence, and hence a nucleic acid-targeting guide may be selected to target any target nucleic acid sequence. The target sequence may be DNA. The target sequence may be any RNA sequence. In some embodiments, the target sequence may be a sequence within an RNA molecule selected from the group consisting of messenger RNA (mRNA), pre-mRNA, ribosomal RNA (rRNA), transfer RNA (tRNA), micro-RNA (miRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), double stranded RNA (dsRNA), non-coding RNA (ncRNA), long non-coding RNA (lncRNA), and small cytoplasmatic RNA (scRNA). In some preferred embodiments, the target sequence may be a sequence within a RNA molecule selected from the group consisting of mRNA, pre-mRNA, and rRNA. In some preferred embodiments, the target sequence may be a sequence within an RNA molecule selected from the group consisting of ncRNA, and lncRNA. In some more preferred embodiments, the target sequence may be a sequence within an mRNA molecule or a pre-mRNA molecule. 
     In some embodiments, a nucleic acid-targeting guide is selected to reduce the degree secondary structure within the nucleic acid-targeting guide. In some embodiments, about or less than about 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or fewer of the nucleotides of the nucleic acid-targeting guide participate in self-complementary base pairing when optimally folded. Optimal folding may be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimal Gibbs free energy. An example of one such algorithm is mFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example folding algorithm is the online webserver RNAfold, developed at Institute for Theoretical Chemistry at the University of Vienna, using the centroid structure prediction algorithm (see e.g., A. R. Gruber et al., 2008, Cell 106(1): 23-24; and P A Carr and G M Church, 2009, Nature Biotechnology 27(12): 1151-62). 
     In certain embodiments, a guide RNA or crRNA may comprise, consist essentially of, or consist of a direct repeat (DR) sequence and a guide sequence or spacer sequence. In certain embodiments, the guide RNA or crRNA may comprise, consist essentially of, or consist of a direct repeat sequence fused or linked to a guide sequence or spacer sequence. In certain embodiments, the direct repeat sequence may be located upstream (i.e., 5′) from the guide sequence or spacer sequence. In other embodiments, the direct repeat sequence may be located downstream (i.e., 3′) from the guide sequence or spacer sequence. 
     In certain embodiments, the crRNA comprises a stem loop, preferably a single stem loop. In certain embodiments, the direct repeat sequence forms a stem loop, preferably a single stem loop. 
     In certain embodiments, the spacer length of the guide RNA is from 15 to 35 nt. In certain embodiments, the spacer length of the guide RNA is at least 15 nucleotides. In certain embodiments, the spacer length is from 15 to 17 nt, e.g., 15, 16, or 17 nt, from 17 to 20 nt, e.g., 17, 18, 19, or 20 nt, from 20 to 24 nt, e.g., 20, 21, 22, 23, or 24 nt, from 23 to 25 nt, e.g., 23, 24, or 25 nt, from 24 to 27 nt, e.g., 24, 25, 26, or 27 nt, from 27 to 30 nt, e.g., 27, 28, 29, or 30 nt, from 3 to 35 nt, e.g., 30, 31, 32, 33, 34, or 35 nt, or 35 nt or longer. 
     The “tracrRNA” sequence or analogous terms includes any polynucleotide sequence that has sufficient complementarity with a crRNA sequence to hybridize. In some embodiments, the degree of complementarity between the tracrRNA sequence and crRNA sequence along the length of the shorter of the two when optimally aligned is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher. In some embodiments, the tracr sequence is about or more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides in length. In some embodiments, the tracr sequence and crRNA sequence are contained within a single transcript, such that hybridization between the two produces a transcript having a secondary structure, such as a hairpin. In an embodiment of the invention, the transcript or transcribed polynucleotide sequence has at least two or more hairpins. In preferred embodiments, the transcript has two, three, four or five hairpins. In a further embodiment of the invention, the transcript has at most five hairpins. In a hairpin structure the portion of the sequence 5′ of the final “N” and upstream of the loop corresponds to the tracr mate sequence, and the portion of the sequence 3′ of the loop corresponds to the tracr sequence. 
     In general, degree of complementarity is with reference to the optimal alignment of the sca sequence and tracr sequence, along the length of the shorter of the two sequences. Optimal alignment may be determined by any suitable alignment algorithm, and may further account for secondary structures, such as self-complementarity within either the sca sequence or tracr sequence. In some embodiments, the degree of complementarity between the tracr sequence and sca sequence along the length of the shorter of the two when optimally aligned is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher. 
     In general, the CRISPR-Cas, CRISPR-Cas-like or CRISPR system may be as used in the foregoing documents, such as International Patent Publication No. WO 2014/093622 (PCT/US2013/074667) and refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, in particular a Cas9-like or cas12-like gene in the case of CRISPR-Cas9-like or CRISPR-Cas12-like, a tracr (trans-activating CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), or “RNA(s)” as that term is herein used (e.g., RNA(s) to guide Cas9-like and/or Cas12-like, e.g. CRISPR RNA and transactivating (tracr) RNA or a single guide RNA (sgRNA) (chimeric RNA)) or other sequences and transcripts from a CRISPR locus. In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system). In the context of formation of a CRISPR complex, “target sequence” refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. The section of the guide sequence through which complementarity to the target sequence is important for cleavage activity is referred to herein as the seed sequence. A target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides. In some embodiments, a target sequence is located in the nucleus or cytoplasm of a cell, and may include nucleic acids in or from mitochondrial, organelles, vesicles, liposomes or particles present within the cell. In some embodiments, especially for non-nuclear uses, NLSs are not preferred. In some embodiments, a CRISPR system comprises one or more nuclear exports signals (NESs). In some embodiments, a CRISPR system comprises one or more NLSs and one or more NESs. In some embodiments, direct repeats may be identified in silico by searching for repetitive motifs that fulfill any or all of the following criteria: 1. found in a 2 Kb window of genomic sequence flanking the type II CRISPR locus; 2. span from 20 to 50 bp; and 3. interspaced by 20 to 50 bp. In some embodiments, 2 of these criteria may be used, for instance 1 and 2, 2 and 3, or 1 and 3. In some embodiments, all 3 criteria may be used. 
     In embodiments of the invention the terms guide sequence and guide RNA, i.e. RNA capable of guiding Cas to a target genomic locus, are used interchangeably as in foregoing cited documents such as WO 2014/093622 (PCT/US2013/074667). In general, a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences as is described elsewhere herein. In particular embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is greater than 94.5% or 95% or 95.5% or 96% or 96.5% or 97% or 97.5% or 98% or 98.5% or 99% or 99.5% or 99.9%, or 100%. Off target is less than 100% or 99.9% or 99.5% or 99% or 99% or 98.5% or 98% or 97.5% or 97% or 96.5% or 96% or 95.5% or 95% or 94.5% or 94% or 93% or 92% or 91% or 90% or 89% or 88% or 87% or 86% or 85% or 84% or 83% or 82% or 81% or 80% complementarity between the sequence and the guide, with it advantageous that off target is 100% or 99.9% or 99.5% or 99% or 99% or 98.5% or 98% or 97.5% or 97% or 96.5% or 96% or 95.5% or 95% or 94.5% complementarity between the sequence and the guide. 
     In some embodiments, a guide sequence is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. Preferably the guide sequence is 10 30 nucleotides long. The ability of a guide sequence to direct sequence-specific binding of a CRISPR complex to a target sequence may be assessed by any suitable assay. For example, the components of a CRISPR system sufficient to form a CRISPR complex, including the guide sequence to be tested, may be provided to a host cell having the corresponding target sequence, such as by transfection with vectors encoding the components of the CRISPR sequence, followed by an assessment of preferential cleavage within the target sequence, such as by Surveyor assay as described herein. Similarly, cleavage of a target polynucleotide sequence may be evaluated in a test tube by providing the target sequence, components of a CRISPR complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions. Other assays are possible, and will occur to those skilled in the art. 
     In some embodiments of CRISPR-Cas systems, the degree of complementarity between a guide sequence and its corresponding target sequence can be about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or 100%; a guide or RNA or sgRNA can be about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length; or guide or RNA or sgRNA can be less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length; and advantageously tracr RNA is 30 or 50 nucleotides in length. However, an aspect of the invention is to reduce off-target interactions, e.g., reduce the guide interacting with a target sequence having low complementarity. Indeed, in the examples, it is shown that the invention involves mutations that result in the CRISPR-Cas system being able to distinguish between target and off-target sequences that have greater than 80% to about 95% complementarity, e.g., 83%-84% or 88-89% or 94-95% complementarity (for instance, distinguishing between a target having 18 nucleotides from an off-target of 18 nucleotides having 1, 2 or 3 mismatches). Accordingly, in the context of the present invention the degree of complementarity between a guide sequence and its corresponding target sequence is greater than 94.5% or 95% or 95.5% or 96% or 96.5% or 97% or 97.5% or 98% or 98.5% or 99% or 99.5% or 99.9%, or 100%. Off target is less than 100% or 99.9% or 99.5% or 99% or 99% or 98.5% or 98% or 97.5% or 97% or 96.5% or 96% or 95.5% or 95% or 94.5% or 94% or 93% or 92% or 91% or 90% or 89% or 88% or 87% or 86% or 85% or 84% or 83% or 82% or 81% or 80% complementarity between the sequence and the guide, with it advantageous that off target is 100% or 99.9% or 99.5% or 99% or 99% or 98.5% or 98% or 97.5% or 97% or 96.5% or 96% or 95.5% or 95% or 94.5% complementarity between the sequence and the guide. 
     In particularly preferred embodiments according to the invention, the guide RNA (capable of guiding Cas to a target locus) may comprise (1) a guide sequence capable of hybridizing to a genomic target locus in the eukaryotic cell; (2) a tracr sequence; and (3) a tracr mate sequence. All (1) to (3) may reside in a single RNA, i.e. an sgRNA (arranged in a 5′ to 3′ orientation), or the tracr RNA may be a different RNA than the RNA containing the guide and tracr sequence. The tracr hybridizes to the tracr mate sequence and directs the CRISPR/Cas complex to the target sequence. Where the tracr RNA is on a different RNA than the RNA containing the guide and tracr sequence, the length of each RNA may be optimized to be shortened from their respective native lengths, and each may be independently chemically modified to protect from degradation by cellular RNase or otherwise increase stability. 
     The methods according to the invention as described herein comprehend inducing one or more mutations in a eukaryotic cell (in vitro, i.e. in an isolated eukaryotic cell) as herein discussed comprising delivering to cell a vector as herein discussed. The mutation(s) can include the introduction, deletion, or substitution of one or more nucleotides at each target sequence of cell(s) via the guide(s) RNA(s) or sgRNA(s). The mutations can include the introduction, deletion, or substitution of 1-75 nucleotides at each target sequence of said cell(s) via the guide(s) RNA(s) or sgRNA(s). The mutations can include the introduction, deletion, or substitution of 1, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides at each target sequence of said cell(s) via the guide(s) RNA(s) or sgRNA(s). The mutations can include the introduction, deletion, or substitution of 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides at each target sequence of said cell(s) via the guide(s) RNA(s) or sgRNA(s). The mutations include the introduction, deletion, or substitution of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides at each target sequence of said cell(s) via the guide(s) RNA(s) or sgRNA(s). The mutations can include the introduction, deletion, or substitution of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides at each target sequence of said cell(s) via the guide(s) RNA(s) or sgRNA(s). The mutations can include the introduction, deletion, or substitution of 40, 45, 50, 75, 100, 200, 300, 400 or 500 nucleotides at each target sequence of said cell(s) via the guide(s) RNA(s) or sgRNA(s). 
     For minimization of toxicity and off-target effect, it may be important to control the concentration of Cas mRNA and guide RNA delivered. Optimal concentrations of Cas mRNA and guide RNA can be determined by testing different concentrations in a cellular or non-human eukaryote animal model and using deep sequencing the analyze the extent of modification at potential off-target genomic loci. Alternatively, to minimize the level of toxicity and off-target effect, Cas nickase mRNA (for example  S. pyogenes  Cas9-like with the D10A mutation) can be delivered with a pair of guide RNAs targeting a site of interest. Guide sequences and strategies to minimize toxicity and off-target effects can be as in International Patent Publication No. WO 2014/093622 (PCT/US2013/074667); or, via mutation as herein. 
     Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence. Without wishing to be bound by theory, the tracr sequence, which may comprise or consist of all or a portion of a wild-type tracr sequence (e.g. about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of a wild-type tracr sequence), may also form part of a CRISPR complex, such as by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence that is operably linked to the guide sequence. 
     In some embodiments, the Cas effector and/or CRISPR-Cas system can be modified such that it is and/or includes a double nickase. Alternatively, to minimize the level of toxicity and off-target effect, a Cas-like (e.g. Cas9-like and/or Cas12-like) nickase can be used with a pair of guide RNAs targeting a site of interest. Guide sequences and strategies to minimize toxicity and off-target effects can be as in WO 2014/093622 (PCT/US2013/074667); or, via mutation as described herein. The invention thus contemplates methods of using two or more nickases, in particular a dual or double nickase approach. In some aspects and embodiments, a single type nickase may be delivered, for example a modified nickase as described herein. This results in the target DNA being bound by two nickases. In addition, it is also envisaged that different orthologs may be used, e.g., a nickase on one strand (e.g., the coding strand) of the DNA and an ortholog on the non-coding or opposite DNA strand. The ortholog can be, but is not limited to, a Cas-like (e.g. Cas9-like and/or Cas12-like) nickase such as a SaCas9-like nickase or a SpCas9-like nickase or a StCas9-like. It may be advantageous to use two different orthologs that require different PAMs and may also have different guide requirements, thus allowing a greater deal of control for the user. In certain embodiments, DNA cleavage will involve at least four types of nickases, wherein each type is guided to a different sequence of target DNA, wherein each pair introduces a first nick into one DNA strand and the second introduces a nick into the second DNA strand. In such methods, at least two pairs of single stranded breaks are introduced into the target DNA wherein upon introduction of first and second pairs of single-strand breaks, target sequences between the first and second pairs of single-strand breaks are excised. In certain embodiments, one or both of the orthologs is controllable, i.e. inducible. 
     Nucleic Acid Component Modifications 
     In certain embodiments, guides of the invention comprise non-naturally occurring nucleic acids and/or non-naturally occurring nucleotides and/or nucleotide analogs, and/or chemically modifications. Non-naturally occurring nucleic acids can include, for example, mixtures of naturally and non-naturally occurring nucleotides. Non-naturally occurring nucleotides and/or nucleotide analogs may be modified at the ribose, phosphate, and/or base moiety. In an embodiment of the invention, a guide nucleic acid comprises ribonucleotides and non-ribonucleotides. In one such embodiment, a guide comprises one or more ribonucleotides and one or more deoxyribonucleotides. In an embodiment of the invention, the guide comprises one or more non-naturally occurring nucleotide or nucleotide analog such as a nucleotide with phosphorothioate linkage, boranophosphate linkage, a locked nucleic acid (LNA) nucleotides comprising a methylene bridge between the 2′ and 4′ carbons of the ribose ring, peptide nucleic acids (PNA), or bridged nucleic acids (BNA). Other examples of modified nucleotides include 2′-O-methyl analogs, 2′-deoxy analogs, 2-thiouridine analogs, N6-methyladenosine analogs, or 2′-fluoro analogs. Further examples of modified nucleotides include linkage of chemical moieties at the 2′ position, including but not limited to peptides, nuclear localization sequence (NLS), peptide nucleic acid (PNA), polyethylene glycol (PEG), triethylene glycol, or tetraethyleneglycol (TEG). Further examples of modified bases include, but are not limited to, 2-aminopurine, 5-bromo-uridine, pseudouridine (Ψ), N 1 -methylpseudouridine (me 1 Ψ), 5-methoxyuridine (5moU), inosine, 7-methylguanosine. Examples of guide RNA chemical modifications include, without limitation, incorporation of 2′-O-methyl (M), 2′-O-methyl-3′-phosphorothioate (MS), phosphorothioate (PS), S-constrained ethyl(cEt), 2′-O-methyl-3′-thioPACE (MSP), or 2′-O-methyl-3′-phosphonoacetate (MP) at one or more terminal nucleotides. Such chemically modified guides can comprise increased stability and increased activity as compared to unmodified guides, though on-target vs. off-target specificity is not predictable. (See, Hendel, 2015, Nat Biotechnol. 33(9):985-9, doi: 10.1038/nbt.3290, published online 29 Jun. 2015; Ragdarm et al., 0215 , PNAS , E7110-E7111; Allerson et al.,  J. Med. Chem.  2005, 48:901-904; Bramsen et al.,  Front. Genet.,  2012, 3:154; Deng et al.,  PNAS,  2015, 112:11870-11875; Sharma et al.,  MedChemComm.,  2014, 5:1454-1471; Hendel et al.,  Nat. Biotechnol . (2015) 33(9): 985-989; Li et al.,  Nature Biomedical Engineering,  2017, 1, 0066 DOI:10.1038/s41551-017-0066; Ryan et al.,  Nucleic Acids Res . (2018) 46(2): 792-803). In some embodiments, the 5′ and/or 3′ end of a guide RNA is modified by a variety of functional moieties including fluorescent dyes, polyethylene glycol, cholesterol, proteins, or detection tags. (See Kelly et al., 2016 , J. Biotech.  233:74-83). In certain embodiments, a guide comprises ribonucleotides in a region that binds to a target DNA and one or more deoxyribonucleotides and/or nucleotide analogs in a region that binds to Cas, Cas-like (e.g. Cas9-like and/or Cas12-like), Cas9, Cpfl, or C2c1. In an embodiment of the invention, deoxyribonucleotides and/or nucleotide analogs are incorporated in engineered guide structures, such as, without limitation, 5′ and/or 3′ end, stem-loop regions, and the seed region. In certain embodiments, the modification is not in the 5′-handle of the stem-loop regions. Chemical modification in the 5′-handle of the stem-loop region of a guide may abolish its function (see Li, et al.,  Nature Biomedical Engineering,  2017, 1:0066). In certain embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides of a guide is chemically modified. In some embodiments, 3-5 nucleotides at either the 3′ or the 5′ end of a guide is chemically modified. In some embodiments, only minor modifications are introduced in the seed region, such as 2′-F modifications. In some embodiments, 2′-F modification is introduced at the 3′ end of a guide. In certain embodiments, three to five nucleotides at the 5′ and/or the 3′ end of the guide are chemically modified with 2′-O-methyl (M), 2′-O-methyl-3′-phosphorothioate (MS), S-constrained ethyl(cEt), 2′-O-methyl-3′-thioPACE (MSP), or 2′-O-methyl-3′-phosphonoacetate (MP). Such modification can enhance genome editing efficiency (see Hendel et al.,  Nat. Biotechnol . (2015) 33(9): 985-989; Ryan et al.,  Nucleic Acids Res . (2018) 46(2): 792-803). In certain embodiments, all of the phosphodiester bonds of a guide are substituted with phosphorothioates (PS) for enhancing levels of gene disruption. In certain embodiments, more than five nucleotides at the 5′ and/or the 3′ end of the guide are chemically modified with 2′-O-Me, 2′-F or S-constrained ethyl(cEt). Such chemically modified guide can mediate enhanced levels of gene disruption (see Ragdarm et al., 0215 , PNAS , E7110-E7111). In an embodiment of the invention, a guide is modified to comprise a chemical moiety at its 3′ and/or 5′ end. Such moieties include, but are not limited to amine, azide, alkyne, thio, dibenzocyclooctyne (DBCO), Rhodamine, peptides, nuclear localization sequence (NLS), peptide nucleic acid (PNA), polyethylene glycol (PEG), triethylene glycol, or tetraethyleneglycol (TEG). In certain embodiment, the chemical moiety is conjugated to the guide by a linker, such as an alkyl chain. In certain embodiments, the chemical moiety of the modified guide can be used to attach the guide to another molecule, such as DNA, RNA, protein, or nanoparticles. Such chemically modified guide can be used to identify or enrich cells generically edited by a CRISPR system (see Lee et al.,  eLife,  2017, 6:e25312, DOI:10.7554). In some embodiments, 3 nucleotides at each of the 3′ and 5′ ends are chemically modified. In a specific embodiment, the modifications comprise 2′-O-methyl or phosphorothioate analogs. In a specific embodiment, 12 nucleotides in the tetraloop and 16 nucleotides in the stem-loop region are replaced with 2′-O-methyl analogs. Such chemical modifications improve in vivo editing and stability (see Finn et al.,  Cell Reports  (2018), 22: 2227-2235). In some embodiments, more than 60 or 70 nucleotides of the guide are chemically modified. In some embodiments, this modification comprises replacement of nucleotides with 2′-O-methyl or 2′-fluoro nucleotide analogs or phosphorothioate (PS) modification of phosphodiester bonds. In some embodiments, the chemical modification comprises 2′-O-methyl or 2′-fluoro modification of guide nucleotides extending outside of the nuclease protein when the CRISPR complex is formed or PS modification of 20 to 30 or more nucleotides of the 3′-terminus of the guide. In a particular embodiment, the chemical modification further comprises 2′-O-methyl analogs at the 5′ end of the guide or 2′-fluoro analogs in the seed and tail regions. Such chemical modifications improve stability to nuclease degradation and maintain or enhance genome-editing activity or efficiency, but modification of all nucleotides may abolish the function of the guide (see Yin et al.,  Nat. Biotech . (2018), 35(12): 1179-1187). Such chemical modifications may be guided by knowledge of the structure of the CRISPR complex, including knowledge of the limited number of nuclease and RNA 2′-OH interactions (see Yin et al.,  Nat. Biotech . (2018), 35(12): 1179-1187). In some embodiments, one or more guide RNA nucleotides may be replaced with DNA nucleotides. In some embodiments, up to 2, 4, 6, 8, 10, or 12 RNA nucleotides of the 5′-end tail/seed guide region are replaced with DNA nucleotides. In certain embodiments, the majority of guide RNA nucleotides at the 3′ end are replaced with DNA nucleotides. In particular embodiments, 16 guide RNA nucleotides at the 3′ end are replaced with DNA nucleotides. In particular embodiments, 8 guide RNA nucleotides of the 5′-end tail/seed region and 16 RNA nucleotides at the 3′ end are replaced with DNA nucleotides. In particular embodiments, guide RNA nucleotides that extend outside of the nuclease protein when the CRISPR complex is formed are replaced with DNA nucleotides. Such replacement of multiple RNA nucleotides with DNA nucleotides leads to decreased off-target activity but similar on-target activity compared to an unmodified guide; however, replacement of all RNA nucleotides at the 3′ end may abolish the function of the guide (see Yin et al.,  Nat. Chem. Biol . (2018) 14, 311-316). Such modifications may be guided by knowledge of the structure of the CRISPR complex, including knowledge of the limited number of nuclease and RNA 2′-OH interactions (see Yin et al.,  Nat. Chem. Biol . (2018) 14, 311-316). 
     In one aspect of the invention, the guide comprises a modified crRNA for Cpfl or a guide similarly modified to a crRNA for Cpfl, having a 5′-handle and a guide segment further comprising a seed region and a 3′-terminus. In some embodiments, the modified guide can be used with a Cpfl of any one of  Acidaminococcus  sp. BV3L6 Cpfl (AsCpfl);  Francisella tularensis  subsp.  Novicida  U112 Cpfl (FnCpfl);  L. bacterium  MC2017 Cpfl (Lb3Cpfl);  Butyrivibrio proteoclasticus  Cpfl (BpCpfl);  Parcubacteria bacterium  GWC2011_GWC2_44_17 Cpfl (PbCpfl); Peregrinibacteria bacterium GW2011_GWA_33_10 Cpfl (PeCpfl);  Leptospira inadai  Cpfl (LiCpfl);  Smithella  sp. SC_K08D17 Cpfl (SsCpfl);  L. bacterium  MA2020 Cpfl (Lb2Cpfl);  Porphyromonas crevioricanis  Cpfl (PcCpfl);  Porphyromonas macacae  Cpfl (PmCpfl);  Candidatus Methanoplasma termitum  Cpfl (CMtCpfl);  Eubacterium eligens  Cpfl (EeCpfl);  Moraxella bovoculi  237 Cpfl (MbCpfl);  Prevotella disiens  Cpfl (PdCpfl); or  L. bacterium  ND2006 Cpfl (LbCpfl). 
     In some embodiments, the modification to the guide is a chemical modification, an insertion, a deletion or a split. In some embodiments, the chemical modification includes, but is not limited to, incorporation of 2′-O-methyl (M) analogs, 2′-deoxy analogs, 2-thiouridine analogs, N6-methyladenosine analogs, 2′-fluoro analogs, 2-aminopurine, 5-bromo-uridine, pseudouridine (T), N 1 -methylpseudouridine (me 1 Ψ), 5-methoxyuridine (5moU), inosine, 7-methylguanosine, 2′-O-methyl-3′-phosphorothioate (MS), S-constrained ethyl(cEt), phosphorothioate (PS), 2′-O-methyl-3′-thioPACE (MSP), or 2′-O-methyl-3′-phosphonoacetate (MP). In some embodiments, the guide comprises one or more of phosphorothioate modifications. In certain embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 25 nucleotides of the guide are chemically modified. In some embodiments, all nucleotides are chemically modified. In certain embodiments, one or more nucleotides in the seed region are chemically modified. In certain embodiments, one or more nucleotides in the 3′-terminus are chemically modified. In certain embodiments, none of the nucleotides in the 5′-handle is chemically modified. In some embodiments, the chemical modification in the seed region is a minor modification, such as incorporation of a 2′-fluoro analog. In a specific embodiment, one nucleotide of the seed region is replaced with a 2′-fluoro analog. In some embodiments, 5 or 10 nucleotides in the 3′-terminus are chemically modified. Such chemical modifications at the 3′-terminus of the Cpfl crRNA improve gene cutting efficiency (see Li, et al.,  Nature Biomedical Engineering,  2017, 1:0066). In a specific embodiment, 5 nucleotides in the 3′-terminus are replaced with 2′-fluoro analogues. In a specific embodiment, 10 nucleotides in the 3′-terminus are replaced with 2′-fluoro analogues. In a specific embodiment, 5 nucleotides in the 3′-terminus are replaced with 2′-O-methyl (M) analogs. In some embodiments, 3 nucleotides at each of the 3′ and 5′ ends are chemically modified. In a specific embodiment, the modifications comprise 2′-O-methyl or phosphorothioate analogs. In a specific embodiment, 12 nucleotides in the tetraloop and 16 nucleotides in the stem-loop region are replaced with 2′-O-methyl analogs. Such chemical modifications improve in vivo editing and stability (see Finn et al.,  Cell Reports  (2018), 22: 2227-2235). 
     In some embodiments, the loop of the 5′-handle of the guide is modified. In some embodiments, the loop of the 5′-handle of the guide is modified to have a deletion, an insertion, a split, or chemical modifications. In certain embodiments, the loop comprises 3, 4, or 5 nucleotides. In certain embodiments, the loop comprises the sequence of UCUU, UUUU, UAUU, or UGUU. In some embodiments, the guide molecule forms a stemloop with a separate non-covalently linked sequence, which can be DNA or RNA. 
     Synthetically Linked Guides 
     In one aspect, the guide comprises a tracr sequence and a tracr mate sequence that are chemically linked or conjugated via a non-phosphodiester bond. In one aspect, the guide comprises a tracr sequence and a tracr mate sequence that are chemically linked or conjugated via a non-nucleotide loop. In some embodiments, the tracr and tracr mate sequences are joined via a non-phosphodiester covalent linker. Examples of the covalent linker include but are not limited to a chemical moiety selected from the group consisting of carbamates, ethers, esters, amides, imines, amidines, aminotrizines, hydrozone, disulfides, thioethers, thioesters, phosphorothioates, phosphorodithioates, sulfonamides, sulfonates, fulfones, sulfoxides, ureas, thioureas, hydrazide, oxime, triazole, photolabile linkages, C—C bond forming groups such as Diels-Alder cyclo-addition pairs or ring-closing metathesis pairs, and Michael reaction pairs. 
     In some embodiments, the tracr and tracr mate sequences are first synthesized using the standard phosphoramidite synthetic protocol (Herdewijn, P., ed., Methods in Molecular Biology Col 288, Oligonucleotide Synthesis: Methods and Applications, Humana Press, New Jersey (2012)). In some embodiments, the tracr or tracr mate sequences can be functionalized to contain an appropriate functional group for ligation using the standard protocol known in the art (Hermanson, G. T., Bioconjugate Techniques, Academic Press (2013)). Examples of functional groups include, but are not limited to, hydroxyl, amine, carboxylic acid, carboxylic acid halide, carboxylic acid active ester, aldehyde, carbonyl, chlorocarbonyl, imidazolylcarbonyl, hydrozide, semicarbazide, thio semicarbazide, thiol, maleimide, haloalkyl, sulfonyl, ally, propargyl, diene, alkyne, and azide. Once the tracr and the tracr mate sequences are functionalized, a covalent chemical bond or linkage can be formed between the two oligonucleotides. Examples of chemical bonds include, but are not limited to, those based on carbamates, ethers, esters, amides, imines, amidines, aminotrizines, hydrozone, disulfides, thioethers, thioesters, phosphorothioates, phosphorodithioates, sulfonamides, sulfonates, fulfones, sulfoxides, ureas, thioureas, hydrazide, oxime, triazole, photolabile linkages, C—C bond forming groups such as Diels-Alder cyclo-addition pairs or ring-closing metathesis pairs, and Michael reaction pairs. 
     In some embodiments, the tracr and tracr mate sequences can be chemically synthesized. In some embodiments, the chemical synthesis uses automated, solid-phase oligonucleotide synthesis machines with 2′-acetoxyethyl orthoester (2′-ACE) (Scaringe et al., J. Am. Chem. Soc. (1998) 120: 11820-11821; Scaringe, Methods Enzymol. (2000) 317: 3-18) or 2′-thionocarbamate (2′-TC) chemistry (Dellinger et al., J. Am. Chem. Soc. (2011) 133: 11540-11546; Hendel et al., Nat. Biotechnol. (2015) 33:985-989). 
     In some embodiments, the tracr and tracr mate sequences can be covalently linked using various bioconjugation reactions, loops, bridges, and non-nucleotide links via modifications of sugar, internucleotide phosphodiester bonds, purine and pyrimidine residues. Sletten et al., Angew. Chem. Int. Ed. (2009) 48:6974-6998; Manoharan, M. Curr. Opin. Chem. Biol. (2004) 8: 570-9; Behlke et al., Oligonucleotides (2008) 18: 305-19; Watts, et al., Drug. Discov. Today (2008) 13: 842-55; Shukla, et al., ChemMedChem (2010) 5: 328-49. 
     In some embodiments, the tracr and tracr mate sequences can be covalently linked using click chemistry. In some embodiments, the tracr and tracr mate sequences can be covalently linked using a triazole linker. In some embodiments, the tracr and tracr mate sequences can be covalently linked using Huisgen 1,3-dipolar cycloaddition reaction involving an alkyne and azide to yield a highly stable triazole linker (He et al., ChemBioChem (2015) 17: 1809-1812; WO 2016/186745). In some embodiments, the tracr and tracr mate sequences are covalently linked by ligating a 5′-hexyne tracrRNA and a 3′-azide crRNA. In some embodiments, either or both of the 5′-hexyne tracrRNA and a 3′-azide crRNA can be protected with 2′-acetoxyethl orthoester (2′-ACE) group, which can be subsequently removed using Dharmacon protocol (Scaringe et al., J. Am. Chem. Soc. (1998) 120: 11820-11821; Scaringe, Methods Enzymol. (2000) 317: 3-18). 
     In some embodiments, the tracr and tracr mate sequences can be covalently linked via a linker (e.g., a non-nucleotide loop) that comprises a moiety such as spacers, attachments, bioconjugates, chromophores, reporter groups, dye labeled RNAs, and non-naturally occurring nucleotide analogues. More specifically, suitable spacers for purposes of this invention include, but are not limited to, polyethers (e.g., polyethylene glycols, polyalcohols, polypropylene glycol or mixtures of ethylene and propylene glycols), polyamines group (e.g., spennine, spermidine and polymeric derivatives thereof), polyesters (e.g., poly(ethyl acrylate)), polyphosphodiesters, alkylenes, and combinations thereof. Suitable attachments include any moiety that can be added to the linker to add additional properties to the linker, such as but not limited to, fluorescent labels. Suitable bioconjugates include, but are not limited to, peptides, glycosides, lipids, cholesterol, phospholipids, diacyl glycerols and dialkyl glycerols, fatty acids, hydrocarbons, enzyme substrates, steroids, biotin, digoxigenin, carbohydrates, polysaccharides. Suitable chromophores, reporter groups, and dye-labeled RNAs include, but are not limited to, fluorescent dyes such as fluorescein and rhodamine, chemiluminescent, electrochemiluminescent, and bioluminescent marker compounds. The design of example linkers conjugating two RNA components are also described in WO 2004/015075. 
     The linker (e.g., a non-nucleotide loop) can be of any length. In some embodiments, the linker has a length equivalent to about 0-16 nucleotides. In some embodiments, the linker has a length equivalent to about 0-8 nucleotides. In some embodiments, the linker has a length equivalent to about 0-4 nucleotides. In some embodiments, the linker has a length equivalent to about 2 nucleotides. Example linker design is also described in International Patent Publication No. WO2011/008730. 
     A typical Type II Cas9 sgRNA comprises (in 5′ to 3′ direction): a guide sequence, a poly U tract, a first complimentary stretch (the “repeat”), a loop (tetraloop), a second complimentary stretch (the “anti-repeat” being complimentary to the repeat), a stem, and further stem loops and stems and a poly A (often poly U in RNA) tail (terminator). In certain embodiments of guide architecture are retained or similar to that of a Cas9 sgRNA, certain aspect of guide architecture cam be modified, for example by addition, subtraction, or substitution of features, whereas certain other aspects of guide architecture are maintained. Preferred locations for engineered sgRNA modifications, including but not limited to insertions, deletions, and substitutions include guide termini and regions of the sgRNA that are exposed when complexed with CRISPR protein and/or target, for example the tetraloop and/or loop2. 
     In certain embodiments, guides of the invention comprise specific binding sites (e.g. aptamers) for adapter proteins, which may comprise one or more functional domains (e.g. via fusion protein). When such a guides forms a CRISPR complex (i.e. CRISPR enzyme binding to guide and target) the adapter proteins bind and, the functional domain associated with the adapter protein is positioned in a spatial orientation which is advantageous for the attributed function to be effective. For example, if the functional domain is a transcription activator (e.g. VP64 or p65), the transcription activator is placed in a spatial orientation which allows it to affect the transcription of the target. Likewise, a transcription repressor will be advantageously positioned to affect the transcription of the target and a nuclease (e.g. Fok1) will be advantageously positioned to cleave or partially cleave the target. 
     The skilled person will understand that modifications to the guide which allow for binding of the adapter+functional domain but not proper positioning of the adapter+functional domain (e.g. due to steric hindrance within the three-dimensional structure of the CRISPR complex) are modifications which are not intended. The one or more modified guide may be modified at the tetra loop, the stem loop 1, stem loop 2, or stem loop 3, as described herein, preferably at either the tetra loop or stem loop 2, and most preferably at both the tetra loop and stem loop 2. 
     The repeat:anti repeat duplex will be apparent from the secondary structure of the sgRNA. It may be typically a first complimentary stretch after (in 5′ to 3′ direction) the poly U tract and before the tetraloop; and a second complimentary stretch after (in 5′ to 3′ direction) the tetraloop and before the poly A tract. The first complimentary stretch (the “repeat”) is complimentary to the second complimentary stretch (the “anti-repeat”). As such, they Watson-Crick base pair to form a duplex of dsRNA when folded back on one another. As such, the anti-repeat sequence is the complimentary sequence of the repeat and in terms to A-U or C-G base pairing, but also in terms of the fact that the anti-repeat is in the reverse orientation due to the tetraloop. 
     In an embodiment of the invention, modification of guide architecture comprises replacing bases in stemloop 2. For example, in some embodiments, “actt” (“acuu” in RNA) and “aagt” (“aagu” in RNA) bases in stemloop2 are replaced with “cgcc” and “gcgg”. In some embodiments, “actt” and “aagt” bases in stemloop2 are replaced with complimentary GC-rich regions of 4 nucleotides. In some embodiments, the complimentary GC-rich regions of 4 nucleotides are “cgcc” and “gcgg” (both in 5′ to 3′ direction). In some embodiments, the complimentary GC-rich regions of 4 nucleotides are “gcgg” and “cgcc” (both in 5′ to 3′ direction). Other combination of C and G in the complimentary GC-rich regions of 4 nucleotides will be apparent including CCCC and GGGG. 
     In one aspect, the stemloop 2, e.g., “ACTTgtttAAGT” can be replaced by any “XXXXgtttYYYY”, e.g., where XXXX and YYYY represent any complementary sets of nucleotides that together will base pair to each other to create a stem. 
     In one aspect, the stem comprises at least about 4 bp comprising complementary X and Y sequences, although stems of more, e.g., 5, 6, 7, 8, 9, 10, 11 or 12 or fewer, e.g., 3, 2, base pairs are also contemplated. Thus, for example X2-12 and Y2-12 (wherein X and Y represent any complementary set of nucleotides) may be contemplated. In one aspect, the stem made of the X and Y nucleotides, together with the “gttt,” will form a complete hairpin in the overall secondary structure; and, this may be advantageous and the amount of base pairs can be any amount that forms a complete hairpin. In one aspect, any complementary X:Y basepairing sequence (e.g., as to length) is tolerated, so long as the secondary structure of the entire sgRNA is preserved. In one aspect, the stem can be a form of X:Y basepairing that does not disrupt the secondary structure of the whole sgRNA in that it has a DR:tracr duplex, and 3 stemloops. In one aspect, the “gttt” tetraloop that connects ACTT and AAGT (or any alternative stem made of X:Y basepairs) can be any sequence of the same length (e.g., 4 basepair) or longer that does not interrupt the overall secondary structure of the sgRNA. In one aspect, the stemloop can be something that further lengthens stemloop2, e.g. can be MS2 aptamer. In one aspect, the stemloop3 “GGCACCGagtCGGTGC” (SEQ ID NO: 37) can likewise take on a “XXXXXXXagtYYYYYYY” form, e.g., wherein X7 and Y7 represent any complementary sets of nucleotides that together will base pair to each other to create a stem. In one aspect, the stem comprises about 7 bp comprising complementary X and Y sequences, although stems of more or fewer basepairs are also contemplated. In one aspect, the stem made of the X and Y nucleotides, together with the “agt”, will form a complete hairpin in the overall secondary structure. In one aspect, any complementary X:Y basepairing sequence is tolerated, so long as the secondary structure of the entire sgRNA is preserved. In one aspect, the stem can be a form of X:Y basepairing that doesn&#39;t disrupt the secondary structure of the whole sgRNA in that it has a DR:tracr duplex, and 3 stemloops. In one aspect, the “agt” sequence of the stemloop 3 can be extended or be replaced by an aptamer, e.g., a MS2 aptamer or sequence that otherwise generally preserves the architecture of stemloop3. In one aspect for alternative Stemloops 2 and/or 3, each X and Y pair can refer to any basepair. In one aspect, non-Watson Crick basepairing is contemplated, where such pairing otherwise generally preserves the architecture of the stemloop at that position. 
     In one aspect, the DR:tracrRNA duplex can be replaced with the form: gYYYYag(N)NNNNxxxxNNNN(AAN)uuRRRRu (using standard IUPAC nomenclature for nucleotides), wherein (N) and (AAN) represent part of the bulge in the duplex, and “xxxx” represents a linker sequence. NNNN on the direct repeat can be anything so long as it basepairs with the corresponding NNNN portion of the tracrRNA. In one aspect, the DR:tracrRNA duplex can be connected by a linker of any length (xxxx . . . ), any base composition, as long as it doesn&#39;t alter the overall structure. 
     In one aspect, the sgRNA structural requirement is to have a duplex and 3 stemloops. In most aspects, the actual sequence requirement for many of the particular base requirements are lax, in that the architecture of the DR:tracrRNA duplex should be preserved, but the sequence that creates the architecture, i.e., the stems, loops, bulges, etc., may be altered. 
     In certain embodiments, the sgRNA are modified in a manner that provides specific binding sites (e.g., aptamers) for adapter proteins comprising one or more functional domains (e.g., via fusion protein) to bind to. The modified sgRNA can be modified such that once the sgRNA forms a AAV-CRISPR complex (i.e. AAV-CRISPR enzyme binding to sgRNA and target) the adapter proteins bind and, the functional domain on the adapter protein is positioned in a spatial orientation which is advantageous for the attributed function to be effective. For example, if the functional domain comprise, consist essentially of a transcription activator (e.g., VP64 or p65), the transcription activator is placed in a spatial orientation which allows it to affect the transcription of the target. Likewise, a transcription repressor will be advantageously positioned to affect the transcription of the target and a nuclease (e.g., Fok1) will be advantageously positioned to cleave or partially cleave the target. 
     Aptamers 
     One guide with a first aptamer/RNA-binding protein pair can be linked or fused to an activator, whilst a second guide with a second aptamer/RNA-binding protein pair can be linked or fused to a repressor. The guides are for different targets (loci), so this allows one gene to be activated and one repressed. For example, the following schematic shows such an approach. 
     Guide 1—MS2 aptamer-------MS2 RNA-binding protein-------VP64 activator; and 
     Guide 2—PP7 aptamer-------PP7 RNA-binding protein------SID4x repressor 
     The present invention also relates to orthogonal PP7/MS2 gene targeting. In this example, sgRNA targeting different loci are modified with distinct RNA loops in order to recruit MS2-VP64 or PP7-SID4X, which activate and repress their target loci, respectively. PP7 is the RNA-binding coat protein of the bacteriophage  Pseudomonas . Like MS2, it binds a specific RNA sequence and secondary structure. The PP7 RNA-recognition motif is distinct from that of MS2. Consequently, PP7 and MS2 can be multiplexed to mediate distinct effects at different genomic loci simultaneously. For example, an sgRNA targeting locus A can be modified with MS2 loops, recruiting MS2-VP64 activators, while another sgRNA targeting locus B can be modified with PP7 loops, recruiting PP7-SID4X repressor domains. In the same cell, dCas9-like or dCas12-like can thus mediate orthogonal, locus-specific modifications. This principle can be extended to incorporate other orthogonal RNA-binding proteins such as Q-beta. 
     An alternative option for orthogonal repression includes incorporating non-coding RNA loops with transactive repressive function into the guide (either at similar positions to the MS2/PP7 loops integrated into the guide or at the 3′ terminus of the guide). For instance, guides were designed with non-coding (but known to be repressive) RNA loops (e.g. using the Alu repressor (in RNA) that interferes with RNA polymerase II in mammalian cells). The Alu RNA sequence was located: in place of the MS2 RNA sequences as used herein (e.g. at tetraloop and/or stem loop 2); and/or at 3′ terminus of the guide. This gives possible combinations of MS2, PP7 or Alu at the tetraloop and/or stemloop 2 positions, as well as, optionally, addition of Alu at the 3′ end of the guide (with or without a linker). 
     The use of two different aptamers (distinct RNA) allows an activator-adaptor protein fusion and a repressor-adaptor protein fusion to be used, with different guides, to activate expression of one gene, whilst repressing another. They, along with their different guides can be administered together, or substantially together, in a multiplexed approach. A large number of such modified guides can be used all at the same time, for example 10 or 20 or 30 and so forth, whilst only one (or at least a minimal number) of Cas and or Cas-like (e.g. Cas9-like or Cas12-like) proteins to be delivered, as a comparatively small number of Cas and or Cas-like (e.g. Cas9-like or Cas12-like) proteins can be used with a large-number modified guides. The adaptor protein may be associated (preferably linked or fused to) one or more activators or one or more repressors. For example, the adaptor protein may be associated with a first activator and a second activator. The first and second activators may be the same, but they are preferably different activators. For example, one might be VP64, whilst the other might be p65, although these are just examples and other transcriptional activators are envisaged. Three or more or even four or more activators (or repressors) may be used, but package size may limit the number being higher than 5 different functional domains. Linkers are preferably used, over a direct fusion to the adaptor protein, where two or more functional domains are associated with the adaptor protein. Suitable linkers might include the GlySer linker. Other linkers are described elsewhere herein. 
     It is also envisaged that the enzyme-guide complex as a whole may be associated with two or more functional domains. For example, there may be two or more functional domains associated with the enzyme, or there may be two or more functional domains associated with the guide (via one or more adaptor proteins), or there may be one or more functional domains associated with the enzyme and one or more functional domains associated with the guide (via one or more adaptor proteins). 
     The fusion between the adaptor protein and the activator or repressor may include a linker. For example, GlySer linkers GGGS (SEQ ID NO: 6) can be used. They can be used in repeats of 3 ((GGGGS) 3 ) (SEQ ID NO: 10) or 6, 9 or even 12 or more (see e.g. SEQ ID NOS: 6-20), to provide suitable lengths, as required. Linkers can be used between the RNA-binding protein and the functional domain (activator or repressor), or between the CRISPR Enzyme (Cas-like (e.g. Cas9-like or Cas12-like)) and the functional domain (activator or repressor). The linkers can be used to engineer appropriate amounts of “mechanical flexibility”. 
     Dead Guides 
     Guide RNAs comprising a dead guide sequence may be used in the present invention. In one aspect, the invention provides guide sequences which are modified in a manner which allows for formation of the CRISPR complex and successful binding to the target, while at the same time, not allowing for successful nuclease activity (i.e. without nuclease activity/without indel activity). For matters of explanation such modified guide sequences are referred to as “dead guides” or “dead guide sequences”. These dead guides or dead guide sequences can be thought of as catalytically inactive or conformationally inactive with regard to nuclease activity. Nuclease activity may be measured using surveyor analysis or deep sequencing as commonly used in the art, preferably surveyor analysis. Similarly, dead guide sequences may not sufficiently engage in productive base pairing with respect to the ability to promote catalytic activity or to distinguish on-target and off-target binding activity. Briefly, the surveyor assay involves purifying and amplifying a CRISPR target site for a gene and forming heteroduplexes with primers amplifying the CRISPR target site. After re-anneal, the products are treated with SURVEYOR nuclease and SURVEYOR enhancer S (Transgenomics) following the manufacturer&#39;s recommended protocols, analyzed on gels, and quantified based upon relative band intensities. 
     Hence, in a related aspect, the invention provides a non-naturally occurring or engineered composition CRISPR-Cas system comprising a Cas-like protein as described herein, and guide RNA (gRNA) wherein the gRNA comprises a dead guide sequence whereby the gRNA is capable of hybridizing to a target sequence such that the CRISPR-Cas system is directed to a genomic locus of interest in a cell without detectable indel activity resultant from nuclease activity of a non-mutant Cas enzyme of the system as detected by a SURVEYOR assay. For shorthand purposes, a gRNA comprising a dead guide sequence whereby the gRNA is capable of hybridizing to a target sequence such that the CRISPR-Cas system is directed to a genomic locus of interest in a cell without detectable indel activity resultant from nuclease activity of a non-mutant Cas enzyme of the system as detected by a SURVEYOR assay is herein termed a “dead gRNA”. It is to be understood that any of the gRNAs according to the invention as described herein elsewhere may be used as dead gRNAs/gRNAs comprising a dead guide sequence as described herein below. Any of the methods, products, compositions and uses as described herein elsewhere is equally applicable with the dead gRNAs/gRNAs comprising a dead guide sequence as further detailed below. By means of further guidance, the following particular aspects and embodiments are provided. 
     The ability of a dead guide sequence to direct sequence-specific binding of a CRISPR complex to a target sequence may be assessed by any suitable assay. For example, the components of a CRISPR system sufficient to form a CRISPR complex, including the dead guide sequence to be tested, may be provided to a host cell having the corresponding target sequence, such as by transfection with vectors encoding the components of the CRISPR sequence, followed by an assessment of preferential cleavage within the target sequence, such as by Surveyor assay as described herein. Similarly, cleavage of a target polynucleotide sequence may be evaluated in a test tube by providing the target sequence, components of a CRISPR complex, including the dead guide sequence to be tested and a control guide sequence different from the test dead guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions. Other assays are possible, and will occur to those skilled in the art. A dead guide sequence may be selected to target any target sequence. In some embodiments, the target sequence is a sequence within a genome of a cell. 
     As explained further herein, several structural parameters allow for a proper framework to arrive at such dead guides. Dead guide sequences are shorter than respective guide sequences which result in active Cas-specific indel formation. Dead guides are 5%, 10%, 20%, 30%, 40%, 50%, shorter than respective guides directed to the same Cas leading to active Cas-specific indel formation. 
     As explained below and known in the art, one aspect of gRNA—Cas specificity is the direct repeat sequence, which is to be appropriately linked to such guides. In particular, this implies that the direct repeat sequences are designed dependent on the origin of the Cas. Thus, structural data available for validated dead guide sequences may be used for designing Cas specific equivalents. Structural similarity between, e.g., the orthologous nuclease domains RuvC of two or more Cas effector proteins may be used to transfer design equivalent dead guides. Thus, the dead guide herein may be appropriately modified in length and sequence to reflect such Cas specific equivalents, allowing for formation of the CRISPR complex and successful binding to the target, while at the same time, not allowing for successful nuclease activity. 
     The use of dead guides in the context herein as well as the state of the art provides a surprising and unexpected platform for network biology and/or systems biology in both in vitro, ex vivo, and in vivo applications, allowing for multiplex gene targeting, and in particular bidirectional multiplex gene targeting. Prior to the use of dead guides, addressing multiple targets, for example for activation, repression and/or silencing of gene activity, has been challenging and in some cases not possible. With the use of dead guides, multiple targets, and thus multiple activities, may be addressed, for example, in the same cell, in the same animal, or in the same patient. Such multiplexing may occur at the same time or staggered for a desired timeframe. 
     For example, the dead guides now allow for the first time to use gRNA as a means for gene targeting, without the consequence of nuclease activity, while at the same time providing directed means for activation or repression. Guide RNA comprising a dead guide may be modified to further include elements in a manner which allow for activation or repression of gene activity, in particular protein adaptors (e.g. aptamers) as described herein elsewhere allowing for functional placement of gene effectors (e.g. activators or repressors of gene activity). One example is the incorporation of aptamers, as explained herein and in the state of the art. By engineering the gRNA comprising a dead guide to incorporate protein-interacting aptamers (Konermann et al., “Genome-scale transcription activation by an engineered CRISPR-Cas9 complex,” doi:10.1038/nature14136, incorporated herein by reference), one may assemble a synthetic transcription activation complex consisting of multiple distinct effector domains. Such may be modeled after natural transcription activation processes. For example, an aptamer, which selectively binds an effector (e.g. an activator or repressor; dimerized MS2 bacteriophage coat proteins as fusion proteins with an activator or repressor), or a protein which itself binds an effector (e.g. activator or repressor) may be appended to a dead gRNA tetraloop and/or a stem-loop 2. In the case of MS2, the fusion protein MS2-VP64 binds to the tetraloop and/or stem-loop 2 and in turn mediates transcriptional up-regulation, for example for Neurog2. Other transcriptional activators are, for example, VP64. P65, HSF1, and MyoD1. By mere example of this concept, replacement of the MS2 stem-loops with PP7-interacting stem-loops may be used to recruit repressive elements. 
     Thus, one aspect is a gRNA of the invention which comprises a dead guide, wherein the gRNA further comprises modifications which provide for gene activation or repression, as described herein. The dead gRNA may comprise one or more aptamers. The aptamers may be specific to gene effectors, gene activators or gene repressors. Alternatively, the aptamers may be specific to a protein which in turn is specific to and recruits/binds a specific gene effector, gene activator or gene repressor. If there are multiple sites for activator or repressor recruitment, it is preferred that the sites are specific to either activators or repressors. If there are multiple sites for activator or repressor binding, the sites may be specific to the same activators or same repressors. The sites may also be specific to different activators or different repressors. The gene effectors, gene activators, gene repressors may be present in the form of fusion proteins. 
     In an embodiment, the dead gRNA as described herein or the CRISPR-Cas complex as described herein includes a non-naturally occurring or engineered composition comprising two or more adaptor proteins, wherein each protein is associated with one or more functional domains and wherein the adaptor protein binds to the distinct RNA sequence(s) inserted into the at least one loop of the dead gRNA. 
     Hence, an aspect provides a non-naturally occurring or engineered composition comprising a guide RNA (gRNA) comprising a dead guide sequence capable of hybridizing to a target sequence in a genomic locus of interest in a cell, wherein the dead guide sequence is as defined herein, a Cas comprising at least one or more nuclear localization sequences, wherein the Cas optionally comprises at least one mutation wherein at least one loop of the dead gRNA is modified by the insertion of distinct RNA sequence(s) that bind to one or more adaptor proteins, and wherein the adaptor protein is associated with one or more functional domains; or, wherein the dead gRNA is modified to have at least one non-coding functional loop, and wherein the composition comprises two or more adaptor proteins, wherein the each protein is associated with one or more functional domains. 
     In certain embodiments, the adaptor protein is a fusion protein comprising the functional domain, the fusion protein optionally comprising a linker between the adaptor protein and the functional domain, the linker optionally including a GlySer linker (e.g., SEQ ID NOS: 6-20). 
     In certain embodiments, the at least one loop of the dead gRNA is not modified by the insertion of distinct RNA sequence(s) that bind to the two or more adaptor proteins. 
     In certain embodiments, the one or more functional domains associated with the adaptor protein is a transcriptional activation domain. 
     In certain embodiments, the one or more functional domains associated with the adaptor protein is a transcriptional activation domain comprising VP64, p65, MyoD1, HSF1, RTA or SETT/9. 
     In certain embodiments, the one or more functional domains associated with the adaptor protein is a transcriptional repressor domain. 
     In certain embodiments, the transcriptional repressor domain is a KRAB domain. 
     In certain embodiments, the transcriptional repressor domain is a NuE domain, NcoR domain, SID domain or a SID4X domain. 
     In certain embodiments, at least one of the one or more functional domains associated with the adaptor protein have one or more activities comprising methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, DNA integration activity RNA cleavage activity, DNA cleavage activity or nucleic acid binding activity. 
     In certain embodiments, the DNA cleavage activity is due to a Fok1 nuclease. 
     In certain embodiments, the dead gRNA is modified so that, after dead gRNA binds the adaptor protein and further binds to the Cas and target, the functional domain is in a spatial orientation allowing for the functional domain to function in its attributed function. 
     In certain embodiments, the at least one loop of the dead gRNA is tetra loop and/or loop2. In certain embodiments, the tetra loop and loop 2 of the dead gRNA are modified by the insertion of the distinct RNA sequence(s). 
     In certain embodiments, the insertion of distinct RNA sequence(s) that bind to one or more adaptor proteins is an aptamer sequence. In certain embodiments, the aptamer sequence is two or more aptamer sequences specific to the same adaptor protein. In certain embodiments, the aptamer sequence is two or more aptamer sequences specific to different adaptor protein. 
     In certain embodiments, the adaptor protein comprises MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, 7s, PRR1. 
     In certain embodiments, the cell is a eukaryotic cell. In certain embodiments, the eukaryotic cell is a mammalian cell, optionally a mouse cell. In certain embodiments, the mammalian cell is a human cell. 
     In certain embodiments, a first adaptor protein is associated with a p65 domain and a second adaptor protein is associated with a HSF1 domain. 
     In certain embodiments, the composition comprises a Cas CRISPR-Cas complex having at least three functional domains, at least one of which is associated with the Cas and at least two of which are associated with dead gRNA. 
     In certain embodiments, the composition further comprises a second gRNA, wherein the second gRNA is a live gRNA capable of hybridizing to a second target sequence such that a second Cas CRISPR-Cas system is directed to a second genomic locus of interest in a cell with detectable indel activity at the second genomic locus resultant from nuclease activity of the Cas enzyme of the system. 
     In certain embodiments, the composition further comprises a plurality of dead gRNAs and/or a plurality of live gRNAs. 
     One aspect of the invention is to take advantage of the modularity and customizability of the gRNA scaffold to establish a series of gRNA scaffolds with different binding sites (in particular aptamers) for recruiting distinct types of effectors in an orthogonal manner. Again, for matters of example and illustration of the broader concept, replacement of the MS2 stem-loops with PP7-interacting stem-loops may be used to bind/recruit repressive elements, enabling multiplexed bidirectional transcriptional control. Thus, in general, gRNA comprising a dead guide may be employed to provide for multiplex transcriptional control and preferred bidirectional transcriptional control. This transcriptional control is most preferred of genes. For example, one or more gRNA comprising dead guide(s) may be employed in targeting the activation of one or more target genes. At the same time, one or more gRNA comprising dead guide(s) may be employed in targeting the repression of one or more target genes. Such a sequence may be applied in a variety of different combinations, for example the target genes are first repressed and then at an appropriate period other targets are activated, or select genes are repressed at the same time as select genes are activated, followed by further activation and/or repression. As a result, multiple components of one or more biological systems may advantageously be addressed together. 
     In an aspect, the invention provides nucleic acid molecule(s) encoding dead gRNA or the Cas CRISPR-Cas complex or the composition as described herein. 
     In an aspect, the invention provides a vector system comprising: a nucleic acid molecule encoding dead guide RNA as defined herein. In certain embodiments, the vector system further comprises a nucleic acid molecule(s) encoding Cas. In certain embodiments, the vector system further comprises a nucleic acid molecule(s) encoding (live) gRNA. In certain embodiments, the nucleic acid molecule or the vector further comprises regulatory element(s) operable in a eukaryotic cell operably linked to the nucleic acid molecule encoding the guide sequence (gRNA) and/or the nucleic acid molecule encoding Cas and/or the optional nuclear localization sequence(s). 
     In another aspect, structural analysis may also be used to study interactions between the dead guide and the active Cas nuclease that enable DNA binding, but no DNA cutting. In this way amino acids important for nuclease activity of Cas are determined. Modification of such amino acids allows for improved Cas enzymes used for gene editing. 
     A further aspect is combining the use of dead guides as explained herein with other applications of CRISPR, as explained herein as well as known in the art. For example, gRNA comprising dead guide(s) for targeted multiplex gene activation or repression or targeted multiplex bidirectional gene activation/repression may be combined with gRNA comprising guides which maintain nuclease activity, as explained herein. Such gRNA comprising guides which maintain nuclease activity may or may not further include modifications which allow for repression of gene activity (e.g. aptamers). Such gRNA comprising guides which maintain nuclease activity may or may not further include modifications which allow for activation of gene activity (e.g. aptamers). In such a manner, a further means for multiplex gene control is introduced (e.g. multiplex gene targeted activation without nuclease activity/without indel activity may be provided at the same time or in combination with gene targeted repression with nuclease activity). 
     For example, 1) using one or more gRNA (e.g. 1-50, 1-40, 1-30, 1-20, preferably 1-10, more preferably 1-5) comprising dead guide(s) targeted to one or more genes and further modified with appropriate aptamers for the recruitment of gene activators; 2) may be combined with one or more gRNA (e.g. 1-50, 1-40, 1-30, 1-20, preferably 1-10, more preferably 1-5) comprising dead guide(s) targeted to one or more genes and further modified with appropriate aptamers for the recruitment of gene repressors. 1) and/or 2) may then be combined with 3) one or more gRNA (e.g. 1-50, 1-40, 1-30, 1-20, preferably 1-10, more preferably 1-5) targeted to one or more genes. This combination can then be carried out in turn with 1)+2)+3) with 4) one or more gRNA (e.g. 1-50, 1-40, 1-30, 1-20, preferably 1-10, more preferably 1-5) targeted to one or more genes and further modified with appropriate aptamers for the recruitment of gene activators. This combination can then be carried in turn with 1)+2)+3)+4) with 5) one or more gRNA (e.g. 1-50, 1-40, 1-30, 1-20, preferably 1-10, more preferably 1-5) targeted to one or more genes and further modified with appropriate aptamers for the recruitment of gene repressors. As a result various uses and combinations are included in the invention. For example, combination 1)+2); combination 1)+3); combination 2)+3); combination 1)+2)+3); combination 1)+2)+3)+4); combination 1)+3)+4); combination 2)+3)+4); combination 1)+2)+4); combination 1)+2)+3)+4)+5); combination 1)+3)+4)+5); combination 2)+3)+4)+5); combination 1)+2)+4)+5); combination 1)+2)+3)+5); combination 1)+3)+5); combination 2)+3)+5); combination 1)+2)+5). 
     In an aspect, the invention provides an algorithm for designing, evaluating, or selecting a dead guide RNA targeting sequence (dead guide sequence) for guiding a Cas CRISPR-Cas system to a target gene locus. In particular, it has been determined that dead guide RNA specificity relates to and can be optimized by varying i) GC content and ii) targeting sequence length. In an aspect, the invention provides an algorithm for designing or evaluating a dead guide RNA targeting sequence that minimizes off-target binding or interaction of the dead guide RNA. In an embodiment of the invention, the algorithm for selecting a dead guide RNA targeting sequence for directing a CRISPR system to a gene locus in an organism comprises a) locating one or more CRISPR motifs in the gene locus, analyzing the 20 nt sequence downstream of each CRISPR motif by i) determining the GC content of the sequence; and ii) determining whether there are off-target matches of the 15 downstream nucleotides nearest to the CRISPR motif in the genome of the organism, and c) selecting the 15 nucleotide sequence for use in a dead guide RNA if the GC content of the sequence is 70% or less and no off-target matches are identified. In an embodiment, the sequence is selected for a targeting sequence if the GC content is 60% or less. In certain embodiments, the sequence is selected for a targeting sequence if the GC content is 55% or less, 50% or less, 45% or less, 40% or less, 35% or less or 30% or less. In an embodiment, two or more sequences of the gene locus are analyzed and the sequence having the lowest GC content, or the next lowest GC content, or the next lowest GC content is selected. In an embodiment, the sequence is selected for a targeting sequence if no off-target matches are identified in the genome of the organism. In an embodiment, the targeting sequence is selected if no off-target matches are identified in regulatory sequences of the genome. 
     In an aspect, the invention provides a method of selecting a dead guide RNA targeting sequence for directing a functionalized CRISPR system to a gene locus in an organism, which comprises: a) locating one or more CRISPR motifs in the gene locus; b) analyzing the 20 nt sequence downstream of each CRISPR motif by: i) determining the GC content of the sequence; and ii) determining whether there are off-target matches of the first 15 nt of the sequence in the genome of the organism; c) selecting the sequence for use in a guide RNA if the GC content of the sequence is 70% or less and no off-target matches are identified. In an embodiment, the sequence is selected if the GC content is 50% or less. In an embodiment, the sequence is selected if the GC content is 40% or less. In an embodiment, the sequence is selected if the GC content is 30% or less. In an embodiment, two or more sequences are analyzed and the sequence having the lowest GC content is selected. In an embodiment, off-target matches are determined in regulatory sequences of the organism. In an embodiment, the gene locus is a regulatory region. An aspect provides a dead guide RNA comprising the targeting sequence selected according to the aforementioned methods. 
     In an aspect, the invention provides a dead guide RNA for targeting a functionalized CRISPR system to a gene locus in an organism. In an embodiment of the invention, the dead guide RNA comprises a targeting sequence wherein the CG content of the target sequence is 70% or less, and the first 15 nt of the targeting sequence does not match an off-target sequence downstream from a CRISPR motif in the regulatory sequence of another gene locus in the organism. In certain embodiments, the GC content of the targeting sequence 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less or 30% or less. In certain embodiments, the GC content of the targeting sequence is from 70% to 60% or from 60% to 50% or from 50% to 40% or from 40% to 30%. In an embodiment, the targeting sequence has the lowest CG content among potential targeting sequences of the locus. 
     In an embodiment of the invention, the first 15 nt of the dead guide match the target sequence. In another embodiment, first 14 nt of the dead guide match the target sequence. In another embodiment, the first 13 nt of the dead guide match the target sequence. In another embodiment first 12 nt of the dead guide match the target sequence. In another embodiment, first 11 nt of the dead guide match the target sequence. In another embodiment, the first 10 nt of the dead guide match the target sequence. In an embodiment of the invention the first 15 nt of the dead guide does not match an off-target sequence downstream from a CRISPR motif in the regulatory region of another gene locus. In other embodiments, the first 14 nt, or the first 13 nt of the dead guide, or the first 12 nt of the guide, or the first 11 nt of the dead guide, or the first 10 nt of the dead guide, does not match an off-target sequence downstream from a CRISPR motif in the regulatory region of another gene locus. In other embodiments, the first 15 nt, or 14 nt, or 13 nt, or 12 nt, or 11 nt of the dead guide do not match an off-target sequence downstream from a CRISPR motif in the genome. 
     In certain embodiments, the dead guide RNA includes additional nucleotides at the 3′-end that do not match the target sequence. Thus, a dead guide RNA that includes the first 15 nt, or 14 nt, or 13 nt, or 12 nt, or 11 nt downstream of a CRISPR motif can be extended in length at the 3′ end to 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, or longer. 
     The invention provides a method for directing a CRISPR-Cas system, including but not limited to a dead Cas-like, Cas (dCas) or functionalized Cas system (which may comprise a functionalized Cas or functionalized guide) to a gene locus. In an aspect, the invention provides a method for selecting a dead guide RNA targeting sequence and directing a functionalized CRISPR system to a gene locus in an organism. In an aspect, the invention provides a method for selecting a dead guide RNA targeting sequence and effecting gene regulation of a target gene locus by a functionalized Cas CRISPR-Cas system. In certain embodiments, the method is used to effect target gene regulation while minimizing off-target effects. In an aspect, the invention provides a method for selecting two or more dead guide RNA targeting sequences and effecting gene regulation of two or more target gene loci by a functionalized Cas CRISPR-Cas system. In certain embodiments, the method is used to effect regulation of two or more target gene loci while minimizing off-target effects. 
     In an aspect, the invention provides a method of selecting a dead guide RNA targeting sequence for directing a functionalized Cas to a gene locus in an organism, which comprises: a) locating one or more CRISPR motifs in the gene locus; b) analyzing the sequence downstream of each CRISPR motif by: i) selecting 10 to 15 nt adjacent to the CRISPR motif, ii) determining the GC content of the sequence; and c) selecting the 10 to 15 nt sequence as a targeting sequence for use in a guide RNA if the GC content of the sequence is 40% or more. In an embodiment, the sequence is selected if the GC content is 50% or more. In an embodiment, the sequence is selected if the GC content is 60% or more. In an embodiment, the sequence is selected if the GC content is 70% or more. In an embodiment, two or more sequences are analyzed and the sequence having the highest GC content is selected. In an embodiment, the method further comprises adding nucleotides to the 3′ end of the selected sequence which do not match the sequence downstream of the CRISPR motif. An aspect provides a dead guide RNA comprising the targeting sequence selected according to the aforementioned methods. 
     In an aspect, the invention provides a dead guide RNA for directing a functionalized CRISPR system to a gene locus in an organism wherein the targeting sequence of the dead guide RNA consists of 10 to 15 nucleotides adjacent to the CRISPR motif of the gene locus, wherein the CG content of the target sequence is 50% or more. In certain embodiments, the dead guide RNA further comprises nucleotides added to the 3′ end of the targeting sequence which do not match the sequence downstream of the CRISPR motif of the gene locus. 
     In an aspect, the invention provides for a single effector to be directed to one or more, or two or more gene loci. In certain embodiments, the effector is associated with a Cas, and one or more, or two or more selected dead guide RNAs are used to direct the Cas-associated effector to one or more, or two or more selected target gene loci. In certain embodiments, the effector is associated with one or more, or two or more selected dead guide RNAs, each selected dead guide RNA, when complexed with a Cas enzyme, causing its associated effector to localize to the dead guide RNA target. One non-limiting example of such CRISPR systems modulates activity of one or more, or two or more gene loci subject to regulation by the same transcription factor. 
     In an aspect, the invention provides for two or more effectors to be directed to one or more gene loci. In certain embodiments, two or more dead guide RNAs are employed, each of the two or more effectors being associated with a selected dead guide RNA, with each of the two or more effectors being localized to the selected target of its dead guide RNA. One non-limiting example of such CRISPR systems modulates activity of one or more, or two or more gene loci subject to regulation by different transcription factors. Thus, in one non-limiting embodiment, two or more transcription factors are localized to different regulatory sequences of a single gene. In another non-limiting embodiment, two or more transcription factors are localized to different regulatory sequences of different genes. In certain embodiments, one transcription factor is an activator. In certain embodiments, one transcription factor is an inhibitor. In certain embodiments, one transcription factor is an activator and another transcription factor is an inhibitor. In certain embodiments, gene loci expressing different components of the same regulatory pathway are regulated. In certain embodiments, gene loci expressing components of different regulatory pathways are regulated. 
     In an aspect, the invention also provides a method and algorithm for designing and selecting dead guide RNAs that are specific for target DNA cleavage or target binding and gene regulation mediated by an active CRISPR-Cas system. In certain embodiments, the CRISPR-Cas system provides orthogonal gene control using an active Cas which cleaves target DNA at one gene locus while at the same time binds to and promotes regulation of another gene locus. 
     In an aspect, the invention provides an method of selecting a dead guide RNA targeting sequence for directing a functionalized Cas to a gene locus in an organism, without cleavage, which comprises a) locating one or more CRISPR motifs in the gene locus; b) analyzing the sequence downstream of each CRISPR motif by i) selecting 10 to 15 nt adjacent to the CRISPR motif, ii) determining the GC content of the sequence, and c) selecting the 10 to 15 nt sequence as a targeting sequence for use in a dead guide RNA if the GC content of the sequence is 30% more, 40% or more. In certain embodiments, the GC content of the targeting sequence is 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, or 70% or more. In certain embodiments, the GC content of the targeting sequence is from 30% to 40% or from 40% to 50% or from 50% to 60% or from 60% to 70%. In an embodiment of the invention, two or more sequences in a gene locus are analyzed and the sequence having the highest GC content is selected. 
     In an embodiment of the invention, the portion of the targeting sequence in which GC content is evaluated is 10 to 15 contiguous nucleotides of the 15 target nucleotides nearest to the PAM. In an embodiment of the invention, the portion of the guide in which GC content is considered is the 10 to 11 nucleotides or 11 to 12 nucleotides or 12 to 13 nucleotides or 13, or 14, or 15 contiguous nucleotides of the 15 nucleotides nearest to the PAM. 
     In an aspect, the invention further provides an algorithm for identifying dead guide RNAs which promote CRISPR system gene locus cleavage while avoiding functional activation or inhibition. It is observed that increased GC content in dead guide RNAs of 16 to 20 nucleotides coincides with increased DNA cleavage and reduced functional activation. 
     It is also demonstrated herein that efficiency of functionalized Cas can be increased by addition of nucleotides to the 3′ end of a guide RNA which do not match a target sequence downstream of the CRISPR motif. For example, of dead guide RNA 11 to 15 nt in length, shorter guides may be less likely to promote target cleavage, but are also less efficient at promoting CRISPR system binding and functional control. In certain embodiments, addition of nucleotides that don&#39;t match the target sequence to the 3′ end of the dead guide RNA increase activation efficiency while not increasing undesired target cleavage. In an aspect, the invention also provides a method and algorithm for identifying improved dead guide RNAs that effectively promote CRISPR system function in DNA binding and gene regulation while not promoting DNA cleavage. Thus, in certain embodiments, the invention provides a dead guide RNA that includes the first 15 nt, or 14 nt, or 13 nt, or 12 nt, or 11 nt downstream of a CRISPR motif and is extended in length at the 3′ end by nucleotides that mismatch the target to 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, or longer. 
     In an aspect, the invention provides a method for effecting selective orthogonal gene control. As will be appreciated from the disclosure herein, dead guide selection according to the invention, taking into account guide length and GC content, provides effective and selective transcription control by a functional CRISPR-Cas system, for example to regulate transcription of a gene locus by activation or inhibition and minimize off-target effects. Accordingly, by providing effective regulation of individual target loci, the invention also provides effective orthogonal regulation of two or more target loci. 
     In certain embodiments, orthogonal gene control is by activation or inhibition of two or more target loci. In certain embodiments, orthogonal gene control is by activation or inhibition of one or more target locus and cleavage of one or more target locus. 
     In one aspect, the invention provides a cell comprising a non-naturally occurring CRISPR-Cas system comprising one or more dead guide RNAs disclosed or made according to a method or algorithm described herein wherein the expression of one or more gene products has been altered. In an embodiment of the invention, the expression in the cell of two or more gene products has been altered. The invention also provides a cell line from such a cell. 
     In one aspect, the invention provides a multicellular organism comprising one or more cells comprising a non-naturally occurring CRISPR-Cas system comprising one or more dead guide RNAs disclosed or made according to a method or algorithm described herein. In one aspect, the invention provides a product from a cell, cell line, or multicellular organism comprising a non-naturally occurring CRISPR-Cas system comprising one or more dead guide RNAs disclosed or made according to a method or algorithm described herein. 
     A further aspect of this invention is the use of gRNA comprising dead guide(s) as described herein, optionally in combination with gRNA comprising guide(s) as described herein or in the state of the art, in combination with systems e.g. cells, transgenic animals, transgenic mice, inducible transgenic animals, inducible transgenic mice) which are engineered for either overexpression of Cas or preferably knock in Cas. As a result, a single system (e.g. transgenic animal, cell) can serve as a basis for multiplex gene modifications in systems/network biology. On account of the dead guides, this is now possible in both in vitro, ex vivo, and in vivo. 
     For example, once the Cas is provided for, one or more dead gRNAs may be provided to direct multiplex gene regulation, and preferably multiplex bidirectional gene regulation. The one or more dead gRNAs may be provided in a spatially and temporally appropriate manner if necessary or desired (for example tissue specific induction of Cas expression). Because the transgenic/inducible Cas is provided for (e.g. expressed) in the cell, tissue, animal of interest, both gRNAs comprising dead guides or gRNAs comprising guides are equally effective. In the same manner, a further aspect of this invention is the use of gRNA comprising dead guide(s) as described herein, optionally in combination with gRNA comprising guide(s) as described herein or in the state of the art, in combination with systems (e.g. cells, transgenic animals, transgenic mice, inducible transgenic animals, inducible transgenic mice) which are engineered for knockout Cas CRISPR-Cas. 
     As a result, the combination of dead guides as described herein with CRISPR applications described herein and CRISPR applications known in the art results in a highly efficient and accurate means for multiplex screening of systems (e.g. network biology). Such screening allows, for example, identification of specific combinations of gene activities for identifying genes responsible for diseases (e.g. on/off combinations), in particular gene related diseases. A preferred application of such screening is cancer. In the same manner, screening for treatment for such diseases is included in the invention. Cells or animals may be exposed to aberrant conditions resulting in disease or disease like effects. Candidate compositions may be provided and screened for an effect in the desired multiplex environment. For example, a patient&#39;s cancer cells may be screened for which gene combinations will cause them to die, and then use this information to establish appropriate therapies. 
     In one aspect, the invention provides a kit comprising one or more of the components described herein. The kit may include dead guides as described herein with or without guides as described herein. 
     The structural information provided herein allows for interrogation of dead gRNA interaction with the target DNA and the Cas permitting engineering or alteration of dead gRNA structure to optimize functionality of the entire CRISPR-Cas system. For example, loops of the dead gRNA may be extended, without colliding with the Cas protein by the insertion of adaptor proteins that can bind to RNA. These adaptor proteins can further recruit effector proteins or fusions which comprise one or more functional domains. 
     In some preferred embodiments, the functional domain is a transcriptional activation domain, preferably VP64. In some embodiments, the functional domain is a transcription repression domain, preferably KRAB. In some embodiments, the transcription repression domain is SID, or concatemers of SID (e.g. SID4X). In some embodiments, the functional domain is an epigenetic modifying domain, such that an epigenetic modifying enzyme is provided. In some embodiments, the functional domain is an activation domain, which may be the P65 activation domain. 
     An aspect of the invention is that the above elements are comprised in a single composition or comprised in individual compositions. These compositions may advantageously be applied to a host to elicit a functional effect on the genomic level. 
     In general, the dead gRNA are modified in a manner that provides specific binding sites (e.g. aptamers) for adapter proteins comprising one or more functional domains (e.g. via fusion protein) to bind to. The modified dead gRNA are modified such that once the dead gRNA forms a CRISPR complex (i.e. Cas-like (e.g. Cas9-like or Cas12-like) binding to dead gRNA and target) the adapter proteins bind and, the functional domain on the adapter protein is positioned in a spatial orientation which is advantageous for the attributed function to be effective. For example, if the functional domain is a transcription activator (e.g. VP64 or p65), the transcription activator is placed in a spatial orientation which allows it to affect the transcription of the target. Likewise, a transcription repressor will be advantageously positioned to affect the transcription of the target and a nuclease (e.g. Fok1) will be advantageously positioned to cleave or partially cleave the target. 
     The skilled person will understand that modifications to the dead gRNA which allow for binding of the adapter+functional domain but not proper positioning of the adapter+functional domain (e.g. due to steric hindrance within the three-dimensional structure of the CRISPR complex) are modifications which are not intended. The one or more modified dead gRNA may be modified at the tetra loop, the stem loop 1, stem loop 2, or stem loop 3, as described herein, preferably at either the tetra loop or stem loop 2, and most preferably at both the tetra loop and stem loop 2. 
     As explained herein the functional domains may be, for example, one or more domains from the group consisting of methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, and molecular switches (e.g. light inducible). In some cases, it is advantageous that additionally at least one NLS is provided. In some instances, it is advantageous to position the NLS at the N terminus. When more than one functional domain is included, the functional domains may be the same or different. 
     The dead gRNA may be designed to include multiple binding recognition sites (e.g. aptamers) specific to the same or different adapter protein. The dead gRNA may be designed to bind to the promoter region −1000-+1 nucleic acids upstream of the transcription start site (i.e. TSS), preferably −200 nucleic acids. This positioning improves functional domains which affect gene activation (e.g. transcription activators) or gene inhibition (e.g. transcription repressors). The modified dead gRNA may be one or more modified dead gRNAs targeted to one or more target loci (e.g. at least 1 gRNA, at least 2 gRNA, at least 5 gRNA, at least 10 gRNA, at least 20 gRNA, at least 30 gRNA, at least 50 gRNA) comprised in a composition. 
     The adaptor protein may be any number of proteins that binds to an aptamer or recognition site introduced into the modified dead gRNA and which allows proper positioning of one or more functional domains, once the dead gRNA has been incorporated into the CRISPR complex, to affect the target with the attributed function. As explained in detail in this application such may be coat proteins, preferably bacteriophage coat proteins. The functional domains associated with such adaptor proteins (e.g. in the form of fusion protein) may include, for example, one or more domains from the group consisting of methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, and molecular switches (e.g. light inducible). Preferred domains are Fok1, VP64, P65, HSF1, MyoD1. In the event that the functional domain is a transcription activator or transcription repressor it is advantageous that additionally at least an NLS is provided and preferably at the N terminus. When more than one functional domain is included, the functional domains may be the same or different. The adaptor protein may utilize known linkers to attach such functional domains. 
     Thus, the modified dead gRNA, the (inactivated) Cas (with or without functional domains), and the binding protein with one or more functional domains, may each individually be comprised in a composition and administered to a host individually or collectively. Alternatively, these components may be provided in a single composition for administration to a host. Administration to a host may be performed via viral vectors known to the skilled person or described herein for delivery to a host (e.g. lentiviral vector, adenoviral vector, AAV vector). As explained herein, use of different selection markers (e.g. for lentiviral gRNA selection) and concentration of gRNA (e.g. dependent on whether multiple gRNAs are used) may be advantageous for eliciting an improved effect. 
     On the basis of this concept, several variations are appropriate to elicit a genomic locus event, including DNA cleavage, gene activation, or gene deactivation. Using the provided compositions, the person skilled in the art can advantageously and specifically target single or multiple loci with the same or different functional domains to elicit one or more genomic locus events. The compositions may be applied in a wide variety of methods for screening in libraries in cells and functional modeling in vivo (e.g. gene activation of lincRNA and identification of function; gain-of-function modeling; loss-of-function modeling; the use the compositions of the invention to establish cell lines and transgenic animals for optimization and screening purposes). 
     The current invention comprehends the use of the compositions of the current invention to establish and utilize conditional or inducible CRISPR transgenic cell/animals, which are not believed prior to the present invention or application. For example, the target cell comprises a Cas protein conditionally or inducibly (e.g. in the form of Cre dependent constructs) and/or the adapter protein conditionally or inducibly and, on expression of a vector introduced into the target cell, the vector expresses that which induces or gives rise to the condition of Cas expression and/or adaptor expression in the target cell. By applying the teaching and compositions of the current invention with the known method of creating a CRISPR complex, inducible genomic events affected by functional domains are also an aspect of the current invention. One example of this is the creation of a CRISPR knock-in/conditional transgenic animal (e.g. mouse comprising e.g. a Lox-Stop-polyA-Lox(LSL) cassette) and subsequent delivery of one or more compositions providing one or more modified dead gRNA (e.g. −200 nucleotides to TSS of a target gene of interest for gene activation purposes) as described herein (e.g. modified dead gRNA with one or more aptamers recognized by coat proteins, e.g. MS2), one or more adapter proteins as described herein (MS2 binding protein linked to one or more VP64) and means for inducing the conditional animal (e.g. Cre recombinase for rendering Cas expression inducible). Alternatively, the adaptor protein may be provided as a conditional or inducible element with a conditional or inducible Cas to provide an effective model for screening purposes, which advantageously only requires minimal design and administration of specific dead gRNAs for a broad number of applications. 
     In another aspect the dead guides are further modified to improve specificity. Protected dead guides may be synthesized, whereby secondary structure is introduced into the 3′ end of the dead guide to improve its specificity. A protected guide RNA (pgRNA) comprises a guide sequence capable of hybridizing to a target sequence in a genomic locus of interest in a cell and a protector strand, wherein the protector strand is optionally complementary to the guide sequence and wherein the guide sequence may in part be hybridizable to the protector strand. The pgRNA optionally includes an extension sequence. The thermodynamics of the pgRNA-target DNA hybridization is determined by the number of bases complementary between the guide RNA and target DNA. By employing ‘thermodynamic protection’, specificity of dead gRNA can be improved by adding a protector sequence. For example, one method adds a complementary protector strand of varying lengths to the 3′ end of the guide sequence within the dead gRNA. As a result, the protector strand is bound to at least a portion of the dead gRNA and provides for a protected gRNA (pgRNA). In turn, the dead gRNA references herein may be easily protected using the described embodiments, resulting in pgRNA. The protector strand can be either a separate RNA transcript or strand or a chimeric version joined to the 3′ end of the dead gRNA guide sequence 
     Tandem Guides and Multiplex (Tandem) Targeting Approaches 
     CRISPR enzymes as defined herein can employ more than one RNA guide without losing activity. This enables the use of the CRISPR enzymes, systems or complexes as defined herein for targeting multiple DNA targets, genes or gene loci, with a single enzyme, system or complex as defined herein. The guide RNAs may be tandemly arranged, optionally separated by a nucleotide sequence such as a direct repeat as defined herein. The position of the different guide RNAs is the tandem does not influence the activity. In preferred embodiments, said CRISPR enzyme, CRISPR-Cas enzyme or Cas enzyme is Cas-like protein, or any one of the modified or mutated variants thereof described herein elsewhere. 
     In one aspect, the invention provides a non-naturally occurring or engineered CRISPR enzyme, preferably a non-class I CRISPR enzyme, as is described herein, such as without limitation a Cas-like protein as described herein elsewhere, used for tandem or multiplex targeting. It is to be understood that any of the CRISPR (or CRISPR-Cas or Cas) enzymes, complexes, or systems according to the invention as described herein elsewhere may be used in such an approach. Any of the methods, products, compositions and uses as described herein elsewhere are equally applicable with the multiplex or tandem targeting approach further detailed below. By means of further guidance, the following particular aspects and embodiments are provided. 
     In one aspect, the invention provides for the use of a Cas enzyme, complex or system as defined herein for targeting multiple gene loci. In one embodiment, this can be established by using multiple (tandem or multiplex) guide RNA (gRNA) sequences. 
     In one aspect, the invention provides methods for using one or more elements of a Cas enzyme, complex or system as defined herein for tandem or multiplex targeting, wherein said CRISPR system comprises multiple guide RNA sequences. Preferably, said gRNA sequences are separated by a nucleotide sequence, such as a direct repeat as defined herein elsewhere. 
     The Cas enzyme, system or complex as defined herein provides an effective means for modifying multiple target polynucleotides. The Cas enzyme, system or complex as defined herein has a wide variety of utility including modifying (e.g., deleting, inserting, translocating, inactivating, activating) one or more target polynucleotides in a multiplicity of cell types. As such the Cas enzyme, system or complex as defined herein of the invention has a broad spectrum of applications in, e.g., gene therapy, drug screening, disease diagnosis, and prognosis, including targeting multiple gene loci within a single CRISPR system. 
     In one aspect, the invention provides a Cas enzyme, system or complex as defined herein, i.e. a non-class I Cas CRISPR-Cas complex having a Cas protein having at least one destabilization domain associated therewith, and multiple guide RNAs that target multiple nucleic acid molecules such as DNA molecules, whereby each of said multiple guide RNAs specifically targets its corresponding nucleic acid molecule, e.g., DNA molecule. Each nucleic acid molecule target, e.g., DNA molecule can encode a gene product or encompass a gene locus. Using multiple guide RNAs hence enables the targeting of multiple gene loci or multiple genes. In some embodiments the Cas enzyme may cleave the DNA molecule encoding the gene product. In some embodiments expression of the gene product is altered. The Cas protein and the guide RNAs do not naturally occur together. The invention comprehends the guide RNAs comprising tandemly arranged guide sequences. The invention further comprehends coding sequences for the Cas protein being codon optimized for expression in a eukaryotic cell. In a preferred embodiment the eukaryotic cell is a mammalian cell, a plant cell or a yeast cell and in a more preferred embodiment the mammalian cell is a human cell. Expression of the gene product may be decreased. The Cas enzyme may form part of a CRISPR system or complex, which further comprises tandemly arranged guide RNAs (gRNAs) comprising a series of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 25, 25, 30, or more than 30 guide sequences, each capable of specifically hybridizing to a target sequence in a genomic locus of interest in a cell. In some embodiments, the functional Cas, CRISPR system, or complex binds to the multiple target sequences. In some embodiments, the functional CRISPR system or complex may edit the multiple target sequences, e.g., the target sequences may comprise a genomic locus, and in some embodiments, there may be an alteration of gene expression. In some embodiments, the functional CRISPR system or complex may comprise further functional domains. In some embodiments, the invention provides a method for altering or modifying expression of multiple gene products. The method may comprise introducing into a cell containing said target nucleic acids, e.g., DNA molecules, or containing and expressing target nucleic acid, e.g., DNA molecules; for instance, the target nucleic acids may encode gene products or provide for expression of gene products (e.g., regulatory sequences). 
     In preferred embodiments, the CRISPR enzyme used for multiplex targeting is a Cas or Cas-like protein (Cas-like (e.g. Cas9-like or Cas12-like), or the CRISPR system or complex comprises a Cas protein. In some embodiments, a CRISPR enzyme used for multiplex targeting is AsCas9 protein or AsCas9-like protein. In some embodiments, the CRISPR enzyme is an LbCas9-like or LbCas9 protein. In some embodiments, the Cas enzyme used for multiplex targeting cleaves both strands of DNA to produce a double strand break (DSB). In some embodiments, the CRISPR enzyme used for multiplex targeting is a nickase. In some embodiments, the Cas enzyme used for multiplex targeting is a dual nickase. In some embodiments, the Cas enzyme used for multiplex targeting is a Cas enzyme such as a DD Cas9-like enzyme as defined herein elsewhere. 
     In some general embodiments, the Cas enzyme used for multiplex targeting is associated with one or more functional domains. In some more specific embodiments, the CRISPR enzyme used for multiplex targeting is a deadCas as defined herein elsewhere. Additional functional domains are described elsewhere herein. 
     In an aspect, the Cas enzyme, system or complex for use in multiple targeting as defined herein or the polynucleotides defined here and elsewhere herein can be delivered to a cell and/or a target polynucleotide using a suitable delivery vehicle. Exemplary suitable delivery vehicles are described in greater detail elsewhere herein. 
     The CRISPR-Cas systems and components thereof capable of multiple targeting can be used, for example, to treat a disease, confer or modify multiple traits/genes to a cell and/or, generate a model system, used for screening assays, agent development and the like. Such methods and others are described in greater detail elsewhere herein. In some embodiments where the CRISPR-Cas system contains multiple guide RNAs, they can be included in the system in a tandemly arranged format. The different guide RNAs may optionally be separated by nucleotide sequences such as direct repeats. 
     In some embodiments, the Cas protein (e.g. Cas and Cas-like protein) that can be used for multiple targeting may include further alterations or mutations of the Cas proteins as defined herein elsewhere, and can be a chimeric Cas protein. 
     Each gRNA may be designed to include multiple binding recognition sites (e.g., aptamers) specific to the same or different adapter protein. Each gRNA or sgRNA may be designed to bind to the promoter region −1000−+1 nucleic acids upstream of the transcription start site (i.e. TSS), preferably −200 nucleic acids. This positioning improves functional domains which affect gene activation (e.g., transcription activators) or gene inhibition (e.g., transcription repressors). The modified gRNA may be one or more modified gRNAs targeted to one or more target loci (e.g., at least 1 gRNA, at least 2 gRNA, at least 5 gRNA, at least 10 gRNA, at least 20 gRNA, at least 30 g RNA, at least 50 gRNA) comprised in a composition. Said multiple gRNA sequences can be tandemly arranged and are preferably separated by a direct repeat. 
     In an aspect, a CRISPR-Cas system capable of multiple targeting can include: I. two or more CRISPR-Cas system polynucleotide sequences comprising (a) a first guide sequence capable of hybridizing to a first target sequence in a polynucleotide locus, (b) a second guide sequence capable of hybridizing to a second target sequence in a polynucleotide locus, (c) a direct repeat sequence, and II. one or more Cas-like enzymes or one or more polynucleotide sequences encoding the Cas-like enzyme(s), wherein when transcribed, the first and the second guide sequences direct sequence-specific binding of a first and/or a second Cas CRISPR complex or domain to the first and second target sequences respectively, wherein the first CRISPR complex or domain comprises the Cas-like enzyme complexed with the first guide sequence that is hybridizable to the first target sequence, wherein the second CRISPR complex or domain comprises the Cas-like enzyme complexed with the second guide sequence that is hybridizable to the second target sequence, and wherein the first guide sequence directs cleavage of one strand of the DNA duplex near the first target sequence and the second guide sequence directs cleavage of the other strand near the second target sequence inducing a double strand break, thereby modifying the organism or the non-human or non-animal organism. Similarly, compositions comprising more than two guide RNAs can be envisaged e.g. each specific for one target, and arranged tandemly in the composition or CRISPR system or complex as described herein. 
     For multiplex targeting, in some embodiments, a template, such as a repair template, which may be dsODN or ssODN, can also be delivered or included in the CRISPR-Cas system. Repair templates and delivery of the repair templates are discussed in greater detail elsewhere herein. 
     The invention also comprehends products obtained from using CRISPR enzyme or Cas enzyme or Cas enzyme or CRISPR-CRISPR enzyme or CRISPR-Cas system or CRISPR-Cas system for use in tandem or multiple targeting as defined herein. Exemplary products are discussed in greater detail elsewhere herein. 
     Escorted Guides 
     In one aspect, the invention provides escorted CRISPR-Cas systems or complexes, especially such a system involving an escorted CRISPR-Cas system guide. By “escorted” is meant that CRISPR-Cas system or complex or guide is delivered to a selected time or place within a cell, so that activity of the CRISPR-Cas system or complex or guide is spatially or temporally controlled. For example, the activity and destination of the CRISPR-Cas system or complex or guide may be controlled by an escort RNA aptamer sequence that has binding affinity for an aptamer ligand, such as a cell surface protein or other localized cellular component. Alternatively, the escort aptamer may for example be responsive to an aptamer effector on or in the cell, such as a transient effector, such as an external energy source that is applied to the cell at a particular time. 
     The escorted CRISPR-Cas systems or complexes have a gRNA with a functional structure designed to improve gRNA structure, architecture, stability, genetic expression, or any combination thereof. Such a structure can include an aptamer. 
     Aptamers are biomolecules that can be designed or selected to bind tightly to other ligands, for example using a technique called systematic evolution of ligands by exponential enrichment (SELEX; Tuerk C, Gold L: “Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase.” Science 1990, 249:505-510). Nucleic acid aptamers can for example be selected from pools of random-sequence oligonucleotides, with high binding affinities and specificities for a wide range of biomedically relevant targets, suggesting a wide range of therapeutic utilities for aptamers (Keefe, Anthony D., Supriya Pai, and Andrew Ellington. “Aptamers as therapeutics.” Nature Reviews Drug Discovery 9.7 (2010): 537-550). These characteristics also suggest a wide range of uses for aptamers as drug delivery vehicles (Levy-Nissenbaum, Etgar, et al. “Nanotechnology and aptamers: applications in drug delivery.”  Trends in biotechnology  26.8 (2008): 442-449; and, Hicke B J, Stephens A W. “Escort aptamers: a delivery service for diagnosis and therapy.” J Clin Invest 2000, 106:923-928). Aptamers may also be constructed that function as molecular switches, responding to a que by changing properties, such as RNA aptamers that bind fluorophores to mimic the activity of green fluorescent protein (Paige, Jeremy S., Karen Y. Wu, and Samie R. Jaffrey. “RNA mimics of green fluorescent protein.” Science 333.6042 (2011): 642-646). It has also been suggested that aptamers may be used as components of targeted siRNA therapeutic delivery systems, for example targeting cell surface proteins (Zhou, Jiehua, and John J. Rossi. “Aptamer-targeted cell-specific RNA interference.” Silence 1.1 (2010): 4). 
     Accordingly, provided herein is a gRNA modified, e.g., by one or more aptamer(s) designed to improve gRNA delivery, including delivery across the cellular membrane, to intracellular compartments, or into the nucleus. Such a structure can include, either in addition to the one or more aptamer(s) or without such one or more aptamer(s), moiety(ies) so as to render the guide deliverable, inducible or responsive to a selected effector. The invention accordingly comprehends an gRNA that responds to normal or pathological physiological conditions, including without limitation pH, hypoxia, O 2  concentration, temperature, protein concentration, enzymatic concentration, lipid structure, light exposure, mechanical disruption (e.g. ultrasound waves), magnetic fields, electric fields, or electromagnetic radiation. 
     An aspect of the invention provides non-naturally occurring or engineered composition comprising an escorted guide RNA (egRNA) comprising: an RNA guide sequence capable of hybridizing to a target sequence in a genomic locus of interest in a cell; and, an escort RNA aptamer sequence, wherein the escort aptamer has binding affinity for an aptamer ligand on or in the cell, or the escort aptamer is responsive to a localized aptamer effector on or in the cell, wherein the presence of the aptamer ligand or effector on or in the cell is spatially or temporally restricted. 
     The escort aptamer may for example change conformation in response to an interaction with the aptamer ligand or effector in the cell. 
     The escort aptamer may have specific binding affinity for the aptamer ligand. 
     The aptamer ligand may be localized in a location or compartment of the cell, for example on or in a membrane of the cell. Binding of the escort aptamer to the aptamer ligand may accordingly direct the egRNA to a location of interest in the cell, such as the interior of the cell by way of binding to an aptamer ligand that is a cell surface ligand. In this way, a variety of spatially restricted locations within the cell may be targeted, such as the cell nucleus or mitochondria. 
     Once intended alterations have been introduced, such as by editing intended copies of a gene in the genome of a cell, continued CRISPR/Cas expression in that cell is no longer necessary. Indeed, sustained expression would be undesirable in certain casein case of off-target effects at unintended genomic sites, etc. Thus time-limited expression would be useful. Inducible expression offers one approach, but in addition Applicants have engineered a Self-Inactivating Cas CRISPR-Cas system that relies on the use of a non-coding guide target sequence within the CRISPR vector itself. Thus, after expression begins, the CRISPR system will lead to its own destruction, but before destruction is complete it will have time to edit the genomic copies of the target gene (which, with a normal point mutation in a diploid cell, requires at most two edits). Simply, the self-inactivating CRISPR-Cas system includes additional RNA (i.e., guide RNA) that targets the coding sequence for the CRISPR enzyme itself or that targets one or more non-coding guide target sequences complementary to unique sequences present in one or more of the following: (a) within the promoter driving expression of the non-coding RNA elements, (b) within the promoter driving expression of the Cas gene, (c) within 100 bp of the ATG translational start codon in the Cas coding sequence, (d) within the inverted terminal repeat (iTR) of a viral delivery vector, e.g., in an AAV genome. 
     The egRNA may include an RNA aptamer linking sequence, operably linking the escort RNA sequence to the RNA guide sequence. 
     In embodiments, the egRNA may include one or more photolabile bonds or non-naturally occurring residues. 
     In one aspect, the escort RNA aptamer sequence may be complementary to a target miRNA, which may or may not be present within a cell, so that only when the target miRNA is present is there binding of the escort RNA aptamer sequence to the target miRNA which results in cleavage of the egRNA by an RNA-induced silencing complex (RISC) within the cell. 
     The formation of a RISC is advantageous in some embodiments. In some embodiments, guide RNAs, including but not limited to protected and/or escorted guide RNAs, may be adapted to include RNA nucleotides that promote formation of a RISC, for example in combination with an siRNA or miRNA that may be provided or may, for instance, already be expressed in a cell. This may be useful, for instance, as a self-inactivating system to clear or degrade the guide. 
     Thus, the guide RNA may comprise a sequence complementary to a target miRNA or an siRNA, which may or may not be present within a cell. In this way, only when the miRNA or siRNA is present, for example through expression (by the cell or through human intervention), is there binding of the RNA sequence to the miRNA or siRNA which then results in cleavage of the guide RNA an RNA-induced silencing complex (RISC) within the cell. Therefore, in some embodiments, the guide RNA comprises an RNA sequence complementary to a target miRNA or siRNA, and binding of the guide RNA sequence to the target miRNA or siRNA results in cleavage of the guide RNA by an RNA-induced silencing complex (RISC) within the cell. 
     RISC formation through use of escorted guides is described in International Patent Publication No. WO 2016/094874, RISC formation through use of protected guides is described in International Patent Publication No. WO 2016/094867, which can be adapted for use with the CRISRP-Cas systems described herein. 
     In embodiments, the escort RNA aptamer sequence may for example be from 10 to 200 nucleotides in length, and the egRNA may include more than one escort RNA aptamer sequence. 
     It is to be understood that any of the RNA guide sequences as described herein elsewhere can be used in the egRNA described herein. In certain embodiments of the invention, the guide RNA or mature crRNA comprises, consists essentially of, or consists of a direct repeat sequence and a guide sequence or spacer sequence. In certain embodiments, the guide RNA or mature crRNA comprises, consists essentially of, or consists of a direct repeat sequence linked to a guide sequence or spacer sequence. In certain embodiments the guide RNA or mature crRNA comprises 19 nts of partial direct repeat followed by 23-25 nt of guide sequence or spacer sequence. In certain embodiments, the effector protein is a FnCas9-like or FnCas12-like and requires at least 16 nt of guide sequence to achieve detectable DNA cleavage and a minimum of 17 nt of guide sequence to achieve efficient DNA cleavage in vitro. In certain embodiments, the direct repeat sequence is located upstream (i.e., 5′) from the guide sequence or spacer sequence. In a preferred embodiment the seed sequence (i.e. the sequence essential critical for recognition and/or hybridization to the sequence at the target locus) of the FnCas9-like or FnCas12-like guide RNA is approximately within the first 5 nt on the 5′ end of the guide sequence or spacer sequence. 
     The sgRNA or egRNA may be included in a non-naturally occurring or engineered Cas CRISPR-Cas complex composition, together with a Cas which may include at least one mutation, for example a mutation so that the Cas has no more than 5% of the nuclease activity of a Cas not having the at least one mutation, for example having a diminished nuclease activity of at least 97%, or 100% as compared with the Cas not having the at least one mutation. The Cas may also include one or more nuclear localization sequences. Mutated Cas and engineered enzymes having modulated activity, such as diminished nuclease activity, are described herein elsewhere. 
     In embodiments, the compositions described herein comprise a CRISPR-Cas complex having at least three functional domains, at least one of which is associated with Cas and at least two of which are associated with egRNA. 
     With respect to mutations of the Cas enzyme, when the enzyme is not FnCas9, mutations may be as described herein elsewhere; conservative substitution for any of the replacement amino acids is also envisaged. In an aspect the invention provides as to any or each or all embodiments herein-discussed wherein the CRISPR enzyme comprises at least one or more, or at least two or more mutations, wherein the at least one or more mutation or the at least two or more mutations are selected from those described herein elsewhere. 
     Inducible Guides 
     The present invention provides compositions and methods by which gRNA-mediated gene editing activity can be adapted. The invention provides gRNA secondary structures that improve cutting efficiency by increasing gRNA and/or increasing the amount of RNA delivered into the cell. The gRNA may include light labile or inducible nucleotides. 
     To increase the effectiveness of gRNA, for example gRNA delivered with viral or non-viral technologies, Applicants added secondary structures into the gRNA that enhance its stability and improve gene editing. Separately, to overcome the lack of effective delivery, Applicants modified gRNAs with cell penetrating RNA aptamers; the aptamers bind to cell surface receptors and promote the entry of gRNAs into cells. Notably, the cell-penetrating aptamers can be designed to target specific cell receptors, in order to mediate cell-specific delivery. Applicants also have created guides that are inducible. 
     Light responsiveness of an inducible system may be achieved via the activation and binding of cryptochrome-2 and CIB1. Blue light stimulation induces an activating conformational change in cryptochrome-2, resulting in recruitment of its binding partner CIB1. This binding is fast and reversible, achieving saturation in &lt;15 sec following pulsed stimulation and returning to baseline &lt;15 min after the end of stimulation. These rapid binding kinetics result in a system temporally bound only by the speed of transcription/translation and transcript/protein degradation, rather than uptake and clearance of inducing agents. Crytochrome-2 activation is also highly sensitive, allowing for the use of low light intensity stimulation and mitigating the risks of phototoxicity. Further, in a context such as the intact mammalian brain, variable light intensity may be used to control the size of a stimulated region, allowing for greater precision than vector delivery alone may offer. 
     The invention contemplates energy sources such as electromagnetic radiation, sound energy or thermal energy to induce the guide. Advantageously, the electromagnetic radiation is a component of visible light. In a preferred embodiment, the light is a blue light with a wavelength of about 450 to about 495 nm. In an especially preferred embodiment, the wavelength is about 488 nm. In another preferred embodiment, the light stimulation is via pulses. The light power may range from about 0-9 mW/cm 2 . In a preferred embodiment, a stimulation paradigm of as low as 0.25 sec every 15 sec should result in maximal activation. The chemical or energy sensitive guide may undergo a conformational change upon induction by the binding of a chemical source or by the energy allowing it act as a guide and have the CRISPR-Cas system or complex function. The invention can involve applying the chemical source or energy so as to have the guide function and the Cas CRISPR-Cas system or complex function; and optionally further determining that the expression of the genomic locus is altered. 
     There are several different designs of this chemical inducible system: 1. ABI-PYL based system inducible by Abscisic Acid (ABA) (see, e.g., http://stke.sciencemag.org/cgi/content/abstract/sigtrans;4/164/r52), 2. FKBP-FRB based system inducible by rapamycin (or related chemicals based on rapamycin) (see, e.g., http://www.nature.com/nmeth/journal/v2/n6/full/nmeth763.html), 3. GID1-GAI based system inducible by Gibberellin (GA) (see, e.g., http://www.nature.com/nchembio/journal/v8/n5/full/nchembio.922.html). 
     Another system contemplated by the present invention is a chemical inducible system based on change in sub-cellular localization. Applicants also developed a system in which the polypeptide include a DNA binding domain comprising at least five or more Transcription activator-like effector (TALE) monomers and at least one or more half-monomers specifically ordered to target the genomic locus of interest linked to at least one or more effector domains are further linker to a chemical or energy sensitive protein. This protein will lead to a change in the sub-cellular localization of the entire polypeptide (i.e. transportation of the entire polypeptide from cytoplasm into the nucleus of the cells) upon the binding of a chemical or energy transfer to the chemical or energy sensitive protein. This transportation of the entire polypeptide from one sub-cellular compartments or organelles, in which its activity is sequestered due to lack of substrate for the effector domain, into another one in which the substrate is present would allow the entire polypeptide to come in contact with its desired substrate (i.e. genomic DNA in the mammalian nucleus) and result in activation or repression of target gene expression. 
     This type of system could also be used to induce the cleavage of a genomic locus of interest in a cell when the effector domain is a nuclease. 
     A chemical inducible system can be an estrogen receptor (ER) based system inducible by 4-hydroxytamoxifen (4OHT) (see, e.g., http://www.pnas.org/content/104/3/1027.abstract). A mutated ligand-binding domain of the estrogen receptor called ERT2 translocase into the nucleus of cells upon binding of 4-hydroxytamoxifen. In further embodiments of the invention any naturally occurring or engineered derivative of any nuclear receptor, thyroid hormone receptor, retinoic acid receptor, estrogen receptor, estrogen-related receptor, glucocorticoid receptor, progesterone receptor, androgen receptor may be used in inducible systems analogous to the ER based inducible system. 
     Another inducible system is based on the design using Transient receptor potential (TRP) ion channel-based system inducible by energy, heat or radio-wave (see, e.g., http://www.sciencemag.org/content/336/6081/604). These TRP family proteins respond to different stimuli, including light and heat. When this protein is activated by light or heat, the ion channel will open and allow the entering of ions such as calcium into the plasma membrane. This influx of ions will bind to intracellular ion interacting partners linked to a polypeptide including the guide and the other components of the CRISPR-Cas complex or system, and the binding will induce the change of sub-cellular localization of the polypeptide, leading to the entire polypeptide entering the nucleus of cells. Once inside the nucleus, the guide protein and the other components of the CRISPR-Cas complex will be active and modulating target gene expression in cells. 
     This type of system could also be used to induce the cleavage of a genomic locus of interest in a cell; and, in this regard, it is noted that the Cas enzyme is a nuclease or a nickase. The light could be generated with a laser or other forms of energy sources. The heat could be generated by raise of temperature results from an energy source, or from nano-particles that release heat after absorbing energy from an energy source delivered in the form of radio-wave. 
     While light activation may be an advantageous embodiment, sometimes it may be disadvantageous especially for in vivo applications in which the light may not penetrate the skin or other organs. In this instance, other methods of energy activation are contemplated, in particular, electric field energy and/or ultrasound which have a similar effect. 
     Electric field energy is preferably administered substantially as described in the art, using one or more electric pulses of from about 1 Volt/cm to about 10 kVolts/cm under in vivo conditions. Instead of or in addition to the pulses, the electric field may be delivered in a continuous manner. The electric pulse may be applied for between 1 μs and 500 milliseconds, preferably between 1 μs and 100 milliseconds. The electric field may be applied continuously or in a pulsed manner for 5 about minutes. 
     As used herein, ‘electric field energy’ is the electrical energy to which a cell is exposed. Preferably the electric field has a strength of from about 1 Volt/cm to about 10 kVolts/cm or more under in vivo conditions (see WO97/49450). 
     As used herein, the term “electric field” includes one or more pulses at variable capacitance and voltage and including exponential and/or square wave and/or modulated wave and/or modulated square wave forms. References to electric fields and electricity should be taken to include reference the presence of an electric potential difference in the environment of a cell. Such an environment may be set up by way of static electricity, alternating current (AC), direct current (DC), etc., as known in the art. The electric field may be uniform, non-uniform or otherwise, and may vary in strength and/or direction in a time dependent manner. 
     Single or multiple applications of electric field, as well as single or multiple applications of ultrasound are also possible, in any order and in any combination. The ultrasound and/or the electric field may be delivered as single or multiple continuous applications, or as pulses (pulsatile delivery). 
     Electroporation has been used in both in vitro and in vivo procedures to introduce foreign material into living cells. With in vitro applications, a sample of live cells is first mixed with the agent of interest and placed between electrodes such as parallel plates. Then, the electrodes apply an electrical field to the cell/implant mixture. Examples of systems that perform in vitro electroporation include the Electro Cell Manipulator ECM600 product, and the Electro Square Porator T820, both made by the BTX Division of Genetronics, Inc (see U.S. Pat. No. 5,869,326). 
     The known electroporation techniques (both in vitro and in vivo) function by applying a brief high voltage pulse to electrodes positioned around the treatment region. The electric field generated between the electrodes causes the cell membranes to temporarily become porous, whereupon molecules of the agent of interest enter the cells. In known electroporation applications, this electric field comprises a single square wave pulse on the order of 1000 V/cm, of about 100 .mu.s duration. Such a pulse may be generated, for example, in known applications of the Electro Square Porator T820. 
     Preferably, the electric field has a strength of from about 1 V/cm to about 10 kV/cm under in vitro conditions. Thus, the electric field may have a strength of 1 V/cm, 2 V/cm, 3 V/cm, 4 V/cm, 5 V/cm, 6 V/cm, 7 V/cm, 8 V/cm, 9 V/cm, 10 V/cm, 20 V/cm, 50 V/cm, 100 V/cm, 200 V/cm, 300 V/cm, 400 V/cm, 500 V/cm, 600 V/cm, 700 V/cm, 800 V/cm, 900 V/cm, 1 kV/cm, 2 kV/cm, 5 kV/cm, 10 kV/cm, 20 kV/cm, 50 kV/cm or more. More preferably from about 0.5 kV/cm to about 4.0 kV/cm under in vitro conditions. Preferably the electric field has a strength of from about 1 V/cm to about 10 kV/cm under in vivo conditions. However, the electric field strengths may be lowered where the number of pulses delivered to the target site are increased. Thus, pulsatile delivery of electric fields at lower field strengths is envisaged. 
     Preferably, the application of the electric field is in the form of multiple pulses such as double pulses of the same strength and capacitance or sequential pulses of varying strength and/or capacitance. As used herein, the term “pulse” includes one or more electric pulses at variable capacitance and voltage and including exponential and/or square wave and/or modulated wave/square wave forms. 
     Preferably, the electric pulse is delivered as a waveform selected from an exponential wave form, a square wave form, a modulated wave form and a modulated square wave form. 
     A preferred embodiment employs direct current at low voltage. Thus, Applicants disclose the use of an electric field which is applied to the cell, tissue or tissue mass at a field strength of between 1V/cm and 20V/cm, for a period of 100 milliseconds or more, preferably 15 minutes or more. 
     Ultrasound is advantageously administered at a power level of from about 0.05 W/cm 2  to about 100 W/cm 2 . Diagnostic or therapeutic ultrasound may be used, or combinations thereof. 
     As used herein, the term “ultrasound” refers to a form of energy which consists of mechanical vibrations the frequencies of which are so high they are above the range of human hearing. Lower frequency limit of the ultrasonic spectrum may generally be taken as about 20 kHz. Most diagnostic applications of ultrasound employ frequencies in the range 1 and 15 MHz′ (From Ultrasonics in Clinical Diagnosis, P. N. T. Wells, ed., 2nd. Edition, Publ. Churchill Livingstone [Edinburgh, London &amp; NY, 1977]). 
     Ultrasound has been used in both diagnostic and therapeutic applications. When used as a diagnostic tool (“diagnostic ultrasound”), ultrasound is typically used in an energy density range of up to about 100 mW/cm 2  (FDA recommendation), although energy densities of up to 750 mW/cm 2  have been used. In physiotherapy, ultrasound is typically used as an energy source in a range up to about 3 to 4 W/cm 2  (WHO recommendation). In other therapeutic applications, higher intensities of ultrasound may be employed, for example, HIFU at 100 W/cm up to 1 kW/cm 2  (or even higher) for short periods of time. The term “ultrasound” as used in this specification is intended to encompass diagnostic, therapeutic and focused ultrasound. 
     Focused ultrasound (FUS) allows thermal energy to be delivered without an invasive probe (see Morocz et al 1998 Journal of Magnetic Resonance Imaging Vol. 8, No. 1, pp. 136-142. Another form of focused ultrasound is high intensity focused ultrasound (HIFU) which is reviewed by Moussatov et al in Ultrasonics (1998) Vol. 36, No. 8, pp. 893-900 and TranHuuHue et al in Acustica (1997) Vol. 83, No. 6, pp. 1103-1106. 
     Preferably, a combination of diagnostic ultrasound and a therapeutic ultrasound is employed. This combination is not intended to be limiting, however, and the skilled reader will appreciate that any variety of combinations of ultrasound may be used. Additionally, the energy density, frequency of ultrasound, and period of exposure may be varied. 
     Preferably, the exposure to an ultrasound energy source is at a power density of from about 0.05 to about 100 Wcm −2 . Even more preferably, the exposure to an ultrasound energy source is at a power density of from about 1 to about 15 Wcm −2 . 
     Preferably, the exposure to an ultrasound energy source is at a frequency of from about 0.015 to about 10.0 MHz. More preferably the exposure to an ultrasound energy source is at a frequency of from about 0.02 to about 5.0 MHz or about 6.0 MHz. Most preferably, the ultrasound is applied at a frequency of 3 MHz. 
     Preferably, the exposure is for periods of from about 10 milliseconds to about 60 minutes. Preferably the exposure is for periods of from about 1 second to about 5 minutes. More preferably, the ultrasound is applied for about 2 minutes. Depending on the particular target cell to be disrupted, however, the exposure may be for a longer duration, for example, for 15 minutes. 
     Advantageously, the target tissue is exposed to an ultrasound energy source at an acoustic power density of from about 0.05 Wcm −2  to about 10 Wcm −2  with a frequency ranging from about 0.015 to about 10 MHz (see International Patent Publication No. WO 98/52609). However, alternatives are also possible, for example, exposure to an ultrasound energy source at an acoustic power density of above 100 Wcm −2 , but for reduced periods of time, for example, 1000 Wcm −2  for periods in the millisecond range or less. 
     Preferably the application of the ultrasound is in the form of multiple pulses; thus, both continuous wave and pulsed wave (pulsatile delivery of ultrasound) may be employed in any combination. For example, continuous wave ultrasound may be applied, followed by pulsed wave ultrasound, or vice versa. This may be repeated any number of times, in any order and combination. The pulsed wave ultrasound may be applied against a background of continuous wave ultrasound, and any number of pulses may be used in any number of groups. 
     Preferably, the ultrasound may comprise pulsed wave ultrasound. In a highly preferred embodiment, the ultrasound is applied at a power density of 0.7 Wcm −2  or 1.25 Wcm −2  as a continuous wave. Higher power densities may be employed if pulsed wave ultrasound is used. 
     Use of ultrasound is advantageous as, like light, it may be focused accurately on a target. Moreover, ultrasound is advantageous as it may be focused more deeply into tissues unlike light. It is therefore better suited to whole-tissue penetration (such as but not limited to a lobe of the liver) or whole organ (such as but not limited to the entire liver or an entire muscle, such as the heart) therapy. Another important advantage is that ultrasound is a non-invasive stimulus which is used in a wide variety of diagnostic and therapeutic applications. By way of example, ultrasound is well known in medical imaging techniques and, additionally, in orthopedic therapy. Furthermore, instruments suitable for the application of ultrasound to a subject vertebrate are widely available and their use is well known in the art. 
     The rapid transcriptional response and endogenous targeting of the instant invention make for an ideal system for the study of transcriptional dynamics. For example, the instant invention may be used to study the dynamics of variant production upon induced expression of a target gene. On the other end of the transcription cycle, mRNA degradation studies are often performed in response to a strong extracellular stimulus, causing expression level changes in a plethora of genes. The instant invention may be utilized to reversibly induce transcription of an endogenous target, after which point stimulation may be stopped and the degradation kinetics of the unique target may be tracked. 
     The temporal precision of the instant invention may provide the power to time genetic regulation in concert with experimental interventions. For example, targets with suspected involvement in long-term potentiation (LTP) may be modulated in organotypic or dissociated neuronal cultures, but only during stimulus to induce LTP, so as to avoid interfering with the normal development of the cells. Similarly, in cellular models exhibiting disease phenotypes, targets suspected to be involved in the effectiveness of a particular therapy may be modulated only during treatment. Conversely, genetic targets may be modulated only during a pathological stimulus. Any number of experiments in which timing of genetic cues to external experimental stimuli is of relevance may potentially benefit from the utility of the instant invention. 
     The in vivo context offers equally rich opportunities for the instant invention to control gene expression. Photoinducibility provides the potential for spatial precision. Taking advantage of the development of optrode technology, a stimulating fiber optic lead may be placed in a precise brain region. Stimulation region size may then be tuned by light intensity. This may be done in conjunction with the delivery of the CRISPR-Cas system or complex of the invention, or, in the case of transgenic Cas-animals, guide RNA of the invention may be delivered and the optrode technology can allow for the modulation of gene expression in precise brain regions. A transparent Cas-expressing organism, can have guide RNA of the invention administered to it and then there can be extremely precise laser induced local gene expression changes. 
     These embodiments can also offer valuable temporal precision in vivo. These embodiments may be used to alter gene expression during a particular stage of development. These embodiments may be used to time a genetic cue to a particular experimental window. For example, genes implicated in learning may be overexpressed or repressed only during the learning stimulus in a precise region of the intact rodent or primate brain. Further, these embodiments may be used to induce gene expression changes only during particular stages of disease development. For example, an oncogene may be overexpressed only once a tumor reaches a particular size or metastatic stage. Conversely, proteins suspected in the development of Alzheimer&#39;s or other disease may be knocked down only at defined time points in the animal&#39;s life and within a particular brain or other tissue region. Although these examples do not exhaustively list the potential applications of the invention, they highlight some of the areas in which the invention may be a powerful technology. 
     Protected Guides 
     Cas enzymes described herein can be used in combination with protected guide RNAs. In one aspect, an object of the current invention is to further enhance the specificity of Cas given individual guide RNAs through thermodynamic tuning of the binding specificity of the guide RNA to target DNA. This is a general approach of introducing mismatches, elongation or truncation of the guide sequence to increase/decrease the number of complimentary bases vs. mismatched bases shared between a genomic target and its potential off-target loci, in order to give thermodynamic advantage to targeted genomic loci over genomic off-targets. 
     In one aspect, the invention provides for the guide sequence being modified by secondary structure to increase the specificity of the CRISPR-Cas system and whereby the secondary structure can protect against exonuclease activity and allow for 3′ additions to the guide sequence. 
     In one aspect, the invention provides for hybridizing a “protector RNA” to a guide sequence, wherein the “protector RNA” is an RNA strand complementary to the 5′ end of the guide RNA (gRNA), to thereby generate a partially double-stranded gRNA. In an embodiment of the invention, protecting the mismatched bases with a perfectly complementary protector sequence decreases the likelihood of target DNA binding to the mismatched base pairs at the 3′ end. In embodiments of the invention, additional sequences comprising an extended length may also be present. 
     Guide RNA (gRNA) extensions matching the genomic target provide gRNA protection and enhance specificity. Extension of the gRNA with matching sequence distal to the end of the spacer seed for individual genomic targets is envisaged to provide enhanced specificity. Matching gRNA extensions that enhance specificity have been observed in cells without truncation. Prediction of gRNA structure accompanying these stable length extensions has shown that stable forms arise from protective states, where the extension forms a closed loop with the gRNA seed due to complimentary sequences in the spacer extension and the spacer seed. These results demonstrate that the protected guide concept also includes sequences matching the genomic target sequence distal of the 20mer spacer-binding region. Thermodynamic prediction can be used to predict completely matching or partially matching guide extensions that result in protected gRNA states. This extends the concept of protected gRNAs to interaction between X and Z, where X will generally be of length 17-20nt and Z is of length 1-30nt. Thermodynamic prediction can be used to determine the optimal extension state for Z, potentially introducing small numbers of mismatches in Z to promote the formation of protected conformations between X and Z. Throughout the present application, the terms “X” and seed length (SL) are used interchangeably with the term exposed length (EpL) which denotes the number of nucleotides available for target DNA to bind; the terms “Y” and protector length (PL) are used interchangeably to represent the length of the protector; and the terms “Z”, “E”, “E′” and “EL” are used interchangeably to correspond to the term extended length (ExL) which represents the number of nucleotides by which the target sequence is extended. 
     An extension sequence which corresponds to the extended length (ExL) may optionally be attached directly to the guide sequence at the 3′ end of the protected guide sequence. The extension sequence may be 2 to 12 nucleotides in length. Preferably ExL may be denoted as 0, 2, 4, 6, 8, 10 or 12 nucleotides in length. In a preferred embodiment the ExL is denoted as 0 or 4 nucleotides in length. In a more preferred embodiment the ExL is 4 nucleotides in length. The extension sequence may or may not be complementary to the target sequence. 
     An extension sequence may further optionally be attached directly to the guide sequence at the 5′ end of the protected guide sequence as well as to the 3′ end of a protecting sequence. As a result, the extension sequence serves as a linking sequence between the protected sequence and the protecting sequence. Without wishing to be bound by theory, such a link may position the protecting sequence near the protected sequence for improved binding of the protecting sequence to the protected sequence. It will be understood that the above-described relationship of seed, protector, and extension applies where the distal end (i.e., the targeting end) of the guide is the 5′ end, e.g. a guide that functions is a Cas system. In an embodiment where the distal end of the guide is the 3′ end, the relationship will be the reverse. In such an embodiment, the invention provides for hybridizing a “protector RNA” to a guide sequence, wherein the “protector RNA” is an RNA strand complementary to the 3′ end of the guide RNA (gRNA), to thereby generate a partially double-stranded gRNA. 
     Addition of gRNA mismatches to the distal end of the gRNA can demonstrate enhanced specificity. The introduction of unprotected distal mismatches in Y or extension of the gRNA with distal mismatches (Z) can demonstrate enhanced specificity. This concept as mentioned is tied to X, Y, and Z components used in protected gRNAs. The unprotected mismatch concept may be further generalized to the concepts of X, Y, and Z described for protected guide RNAs. 
     In one aspect, the invention provides for enhanced Cas specificity wherein the double stranded 3′ end of the protected guide RNA (pgRNA) allows for two possible outcomes: (1) the guide RNA-protector RNA to guide RNA-target DNA strand exchange will occur and the guide will fully bind the target, or (2) the guide RNA will fail to fully bind the target and because Cas target cleavage is a multiple step kinetic reaction that requires guide RNA:target DNA binding to activate Cas-catalyzed DSBs, wherein Cas cleavage does not occur if the guide RNA does not properly bind. According to particular embodiments, the protected guide RNA improves specificity of target binding as compared to a naturally occurring CRISPR-Cas system. According to particular embodiments, the protected modified guide RNA improves stability as compared to a naturally occurring CRISPR-Cas. According to particular embodiments, the protector sequence has a length between 3 and 120 nucleotides and comprises 3 or more contiguous nucleotides complementary to another sequence of guide or protector. According to particular embodiments, the protector sequence forms a hairpin. According to particular embodiments, the guide RNA further comprises a protected sequence and an exposed sequence. According to particular embodiments, the exposed sequence is 1 to 19 nucleotides. More particularly, the exposed sequence is at least 75%, at least 90% or about 100% complementary to the target sequence. According to particular embodiments, the guide sequence is at least 90% or about 100% complementary to the protector strand. According to particular embodiments, the guide sequence is at least 75%, at least 90% or about 100% complementary to the target sequence. According to particular embodiments, the guide RNA further comprises an extension sequence. More particularly, when the distal end of the guide is the 3′ end, the extension sequence is operably linked to the 3′ end of the protected guide sequence, and optionally directly linked to the 3′ end of the protected guide sequence. According to particular embodiments, the extension sequence is 1-12 nucleotides. According to particular embodiments the extension sequence is operably linked to the guide sequence at the 3′ end of the protected guide sequence and the 5′ end of the protector strand and optionally directly linked to the 3′ end of the protected guide sequence and the 53′ end of the protector strand, wherein the extension sequence is a linking sequence between the protected sequence and the protector strand. According to particular embodiments, the extension sequence is 100% not complementary to the protector strand, optionally at least 95%, at least 90%, at least 80%, at least 70%, at least 60%, or at least 50% not complementary to the protector strand. According to particular embodiments, the guide sequence further comprises mismatches appended to the end of the guide sequence, wherein the mismatches thermodynamically optimize specificity. 
     According to the invention, in certain embodiments, guide modifications that impede strand invasion will be desirable. For example, to minimize off-target activity, in certain embodiments, it will be desirable to design or modify a guide to impede strand invasion at off-target sites. In certain such embodiments, it may be acceptable or useful to design or modify a guide at the expense of on-target binding efficiency. In certain embodiments, guide-target mismatches at the target site may be tolerated that substantially reduce off-target activity. 
     In certain embodiments of the invention, it is desirable to adjust the binding characteristics of the protected guide to minimize off-target CRISPR activity. Accordingly, thermodynamic prediction algorithms are used to predict strengths of binding on target and off target. Alternatively, or in addition, selection methods are used to reduce or minimize off-target effects, by absolute measures or relative to on-target effects. 
     Design options include, without limitation, i) adjusting the length of protector strand that binds to the protected strand, ii) adjusting the length of the portion of the protected strand that is exposed, iii) extending the protected strand with a stem-loop located external (distal) to the protected strand (i.e. designed so that the stem loop is external to the protected strand at the distal end), iv) extending the protected strand by addition of a protector strand to form a stem-loop with all or part of the protected strand, v) adjusting binding of the protector strand to the protected strand by designing in one or more base mismatches and/or one or more non-canonical base pairings, vi) adjusting the location of the stem formed by hybridization of the protector strand to the protected strand, and vii) addition of a non-structured protector to the end of the protected strand. 
     In one aspect, the invention provides an engineered, non-naturally occurring CRISPR-Cas system comprising a Cas protein and a protected guide RNA that targets a DNA molecule encoding a gene product in a cell, whereby the protected guide RNA targets the DNA molecule encoding the gene product and the Cas protein cleaves the DNA molecule encoding the gene product, whereby expression of the gene product is altered; and, wherein the Cas protein and the protected guide RNA do not naturally occur together. The invention comprehends the protected guide RNA comprising a guide sequence fused to a direct repeat sequence. The invention further comprehends the CRISPR protein being codon optimized for expression in a eukaryotic cell. In a preferred embodiment the eukaryotic cell is a mammalian cell, a plant cell or a yeast cell and in a more preferred embodiment the mammalian cell is a human cell. In a further embodiment of the invention, the expression of the gene product is decreased. In some embodiments the CRISPR protein is Cas or Cas-like protein. In some embodiments the CRISPR protein is Cas9-like, Cas12-like, and/or Cas12a-like. In some embodiments, the Cas12a-like protein is  Acidaminococcus  sp. BV3L6 , Lachnospiraceae bacterium  or  Francisella Novicida  Cas12a, and may include mutated Cas12a-like derived from these organisms. The protein may be a further Cas9 or Cas12a homolog or ortholog. In some embodiments, the nucleotide sequence encoding the Csa9 or Cas12a protein is codon-optimized for expression in a eukaryotic cell. In some embodiments, the Cas9-like and/or Cas12a-like protein directs cleavage of one or two strands at the location of the target sequence. In some embodiments, the first regulatory element is a polymerase III promoter. In some embodiments, the second regulatory element is a polymerase II promoter. 
     In one aspect, the invention provides a recombinant polynucleotide comprising a protected guide sequence downstream of a direct repeat sequence, wherein the protected guide sequence when expressed directs sequence-specific binding of a CRISPR complex or AAV-CRISPR complex to a corresponding target sequence present in a eukaryotic cell. The polynucleotide can be carried within and expressed in vivo from the AAV-CRISPR enzyme. In some embodiments, the target sequence is a viral sequence present in a eukaryotic cell. In some embodiments, the target sequence is a proto-oncogene or an oncogene. 
     Governing Guides 
     In some embodiments, the CRISPR-Cas system can include one or more governing guide polynucleotides, e.g., governing gRNAs. Governing guides can be used in some embodiments to induce self-inactivation of the CRISPR-Cas system and/or provide other spatial temporal control of the CRISRP-Cas system and/or component(s) thereof. Some governing guides can also be referred to as “self-inactivating guides”. Self-inactivating and inducible CRISRP-Cas systems are described elsewhere herein. 
     The targeting sequence for the governing gRNA can be selected to increase regulation or control of one or more of the CRISPR-Cas system components (e.g. Cas effectors) and/or to reduce or minimize off-target effects of the system. For example, a governing gRNA can minimize undesirable cleavage, e.g., “recleavage” after CRISPR-Cas system mediated alteration of a target nucleic acid or off-target cutting of a Cas effector of the system, by inactivating (e.g., cleaving) a nucleic acid that encodes a Cas-like (e.g. Cas9-like and/or Cas12-like) or other Cas effector molecule present in the system. In an embodiment, a governing gRNA can place temporal or other limit(s) on the level of expression or activity of the Cas-like (e.g. Cas9-like and/or Cas12-like) or other Cas effector molecule/gRNA molecule complex. In an embodiment, the governing gRNA can reduce off-target or other unwanted activity. 
     Suitable target sequences for the governing gRNA can be, for instance, near to or within the translational start codon for the Cas effector (e.g. Cas, Cas-like, Cas9-like, and/or Cas12-like) coding sequence(s), in a non-coding sequence in the promoter driving expression of the non-coding RNA elements, within the promoter driving expression of the Cas effector gene(s), within 100 bp of the ATG translational start codon in the Cas effector coding sequence(s), and/or within the inverted terminal repeat (iTR) of a viral delivery vector, e.g., in the AAV genome. A double stranded break near this region can induce a frame shift in the Cas effector coding sequence(s), causing a loss of protein expression. An alternative target sequence for the “self-inactivating” guide RNA would aim to edit/inactivate regulatory regions/sequences needed for the expression of the CRISPR-Cas system or components thereof or for the stability of the vector. For instance, if the promoter for the Cas effector coding sequence is disrupted then transcription can be inhibited or prevented. Similarly, if a vector includes sequences for replication, maintenance or stability then it is possible to target these. For instance, in an AAV vector a useful target sequence is within the iTR. Other useful sequences to target can be promoter sequences, polyadenylation sites, etc. 
     Addition of non-targeting nucleotides to the 5′ end (e.g. 1-10 nucleotides, preferably 1-5 nucleotides) of the “self-inactivating” guide RNA or governing guide RNA can be used to delay its processing and/or modify its efficiency as a means of ensuring editing at the targeted genomic locus prior to CRISPR-Cas-like (e.g. Cas9-like and/or Cas12-like) shutdown. 
     Recombination Templates 
     In some embodiments, the composition for engineering cells comprise a template, e.g., a recombination template. A template may be a component of another vector as described herein, contained in a separate vector, or provided as a separate polynucleotide. In some embodiments, a recombination template is designed to serve as a template in homologous recombination, such as within or near a target sequence nicked or cleaved by a nucleic acid-targeting effector protein as a part of a nucleic acid-targeting complex. 
     In an embodiment, the template nucleic acid alters the sequence of the target position. In an embodiment, the template nucleic acid results in the incorporation of a modified, or non-naturally occurring base into the target nucleic acid. 
     The template sequence may undergo a breakage mediated or catalyzed recombination with the target sequence. In an embodiment, the template nucleic acid may include sequence that corresponds to a site on the target sequence that is cleaved by a Cas protein mediated cleavage event. In an embodiment, the template nucleic acid may include a sequence that corresponds to both, a first site on the target sequence that is cleaved in a first Cas protein mediated event, and a second site on the target sequence that is cleaved in a second Cas protein mediated event. 
     In certain embodiments, the template nucleic acid can include a sequence which results in an alteration in the coding sequence of a translated sequence, e.g., one which results in the substitution of one amino acid for another in a protein product, e.g., transforming a mutant allele into a wild type allele, transforming a wild type allele into a mutant allele, and/or introducing a stop codon, insertion of an amino acid residue, deletion of an amino acid residue, or a nonsense mutation. In certain embodiments, the template nucleic acid can include a sequence which results in an alteration in a non-coding sequence, e.g., an alteration in an exon or in a 5′ or 3′ non-translated or non-transcribed region. Such alterations include an alteration in a control element, e.g., a promoter, enhancer, and an alteration in a cis-acting or trans-acting control element. 
     A template nucleic acid having homology with a target position in a target gene may be used to alter the structure of a target sequence. The template sequence may be used to alter an unwanted structure, e.g., an unwanted or mutant nucleotide. The template nucleic acid may include a sequence which, when integrated, results in decreasing the activity of a positive control element; increasing the activity of a positive control element; decreasing the activity of a negative control element; increasing the activity of a negative control element; decreasing the expression of a gene; increasing the expression of a gene; increasing resistance to a disorder or disease; increasing resistance to viral entry; correcting a mutation or altering an unwanted amino acid residue conferring, increasing, abolishing or decreasing a biological property of a gene product, e.g., increasing the enzymatic activity of an enzyme, or increasing the ability of a gene product to interact with another molecule. 
     The template nucleic acid may include a sequence which results in a change in sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more nucleotides of the target sequence. 
     A template polynucleotide may be of any suitable length, such as about or more than about 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000, or more nucleotides in length. In an embodiment, the template nucleic acid may be 20+/−10, 30+/−10, 40+/−10, 50+/−10, 60+/−10, 70+/−10, 80+/−10, 90+/−10, 100+/−10, 1 10+/−10, 120+/−10, 130+/−10, 140+/−10, 150+/−10, 160+/−10, 170+/−10, 1 80+/−10, 190+/−10, 200+/−10, 210+/−10, of 220+/−10 nucleotides in length. In an embodiment, the template nucleic acid may be 30+/−20, 40+/−20, 50+/−20, 60+/−20, 70+/−20, 80+/−20, 90+/−20, 100+/−20, 1 10+/−20, 120+/−20, 130+/−20, 140+/−20, I 50+/−20, 160+/−20, 170+/−20, 180+/−20, 190+/−20, 200+/−20, 210+/−20, of 220+/−20 nucleotides in length. In an embodiment, the template nucleic acid is 10 to 1,000, 20 to 900, 30 to 800, 40 to 700, 50 to 600, 50 to 500, 50 to 400, 50 to 300, 50 to 200, or 50 to 100 nucleotides in length. 
     In some embodiments, the template polynucleotide is complementary to a portion of a polynucleotide comprising the target sequence. When optimally aligned, a template polynucleotide might overlap with one or more nucleotides of a target sequences (e.g. about or more than about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more nucleotides). In some embodiments, when a template sequence and a polynucleotide comprising a target sequence are optimally aligned, the nearest nucleotide of the template polynucleotide is within about 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, 5000, 10000, or more nucleotides from the target sequence. 
     In some embodiments, a template nucleic acid comprises the following components: [5′ homology arm]-[replacement sequence]-[3′ homology arm]. The homology arms provide for recombination into the chromosome, thus replacing the undesired element, e.g., a mutation or signature, with the replacement sequence. In an embodiment, the homology arms flank the most distal cleavage sites. In an embodiment, the 3′ end of the 5′ homology arm is the position next to the 5′ end of the replacement sequence. In an embodiment, the 5′ homology arm can extend at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides 5′ from the 5′ end of the replacement sequence. In an embodiment, the 5′ end of the 3′ homology arm is the position next to the 3′ end of the replacement sequence. In an embodiment, the 3′ homology arm can extend at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides 3′ from the 3′ end of the replacement sequence. 
     In certain embodiments, one or both homology arms may be shortened to avoid including certain sequence repeat elements. For example, a 5′ homology arm may be shortened to avoid a sequence repeat element. In other embodiments, a 3′ homology arm may be shortened to avoid a sequence repeat element. In some embodiments, both the 5′ and the 3′ homology arms may be shortened to avoid including certain sequence repeat elements. 
     The exogenous polynucleotide template comprises a sequence to be integrated (e.g., a mutated gene). The sequence for integration may be a sequence endogenous or exogenous to the cell. Examples of a sequence to be integrated include polynucleotides encoding a protein or a non-coding RNA (e.g., a microRNA). Thus, the sequence for integration may be operably linked to an appropriate control sequence or sequences. Alternatively, the sequence to be integrated may provide a regulatory function. 
     An upstream or downstream sequence may comprise from about 20 bp to about 2500 bp, for example, about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, or 2500 bp. In some methods, the exemplary upstream or downstream sequence have about 200 bp to about 2000 bp, about 600 bp to about 1000 bp, or more particularly about 700 bp to about 1000. 
     In certain embodiments, one or both homology arms may be shortened to avoid including certain sequence repeat elements. For example, a 5′ homology arm may be shortened to avoid a sequence repeat element. In other embodiments, a 3′ homology arm may be shortened to avoid a sequence repeat element. In some embodiments, both the 5′ and the 3′ homology arms may be shortened to avoid including certain sequence repeat elements. 
     In some methods, the exogenous polynucleotide template may further comprise a marker. Such a marker may make it easy to screen for targeted integrations. Examples of suitable markers include restriction sites, fluorescent proteins, or selectable markers. The exogenous polynucleotide template of the disclosure can be constructed using recombinant techniques (see, for example, Sambrook et al., 2001 and Ausubel et al., 1996). 
     In certain embodiments, a template nucleic acid for correcting a mutation may designed for use as a single-stranded oligonucleotide. When using a single-stranded oligonucleotide, 5′ and 3′ homology arms may range up to about 200 base pairs (bp) in length, e.g., at least 25, 50, 75, 100, 125, 150, 175, or 200 bp in length. 
     Suzuki et al. describe in vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration (2016, Nature 540:144-149). 
     Target Polynucleotides and Target Sequences 
     The target sequence can be a sequence of target polynucleotide sequence. The target sequence may be DNA. The target sequence may be any RNA sequence. In some embodiments, the target sequence may be a sequence within a RNA molecule selected from the group consisting of messenger RNA (mRNA), pre-mRNA, ribosomal RNA (rRNA), transfer RNA (tRNA), micro-RNA (miRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), double stranded RNA (dsRNA), non-coding RNA (ncRNA), long non-coding RNA (lncRNA), and small cytoplasmic RNA (scRNA). In some preferred embodiments, the target sequence may be a sequence within a RNA molecule selected from the group consisting of mRNA, pre-mRNA, and rRNA. In some preferred embodiments, the target sequence may be a sequence within a RNA molecule selected from the group consisting of ncRNA, and lncRNA. In some more preferred embodiments, the target sequence may be a sequence within an mRNA molecule or a pre-mRNA molecule. 
     In the context of formation of a CRISPR complex, “target sequence” or “target polynucleotide sequence” refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. A target sequence may include RNA polynucleotides. The term “target RNA” refers to a RNA polynucleotide being or containing the target sequence. In other words, the target RNA may be a RNA polynucleotide or a part of a RNA polynucleotide to which a part of the gRNA, i.e. the guide sequence, is designed to have complementarity and to which the effector function mediated by the complex containing a CRISPR effector protein (including, but not limited to, those Cas-like polypeptides described herein) and a gRNA or other nucleic acid component is to be directed. In some embodiments, a target polynucleotide having a target polynucleotide sequence is located in the nucleus or cytoplasm of a cell. 
     In the context of formation of a CRISPR complex, such as the multi-component nucleic acid targeting system described herein, “target sequence” refers to a polynucleotide sequence to which a guide sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. The section of the guide sequence through which complementarity to the target sequence is important for cleavage activity is referred to herein as the seed sequence. A target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides. In some embodiments, a target sequence is located in the nucleus or cytoplasm of a cell, and may include nucleic acids in or from mitochondrial, organelles, vesicles, liposomes or particles present within the cell. In some embodiments, especially for non-nuclear uses, NLSs are not preferred. In some embodiments, a CRISPR system comprises one or more nuclear exports signals (NESs). In some embodiments, a CRISPR system comprises one or more NLSs and one or more NESs. In some embodiments, direct repeats may be identified in silico by searching for repetitive motifs that fulfill any or all of the following criteria: 1. found in a 2 Kb window of genomic sequence flanking the type II CRISPR locus; 2. span from 20 to 50 bp; and 3. interspaced by 20 to 50 bp. In some embodiments, 2 of these criteria may be used, for instance 1 and 2, 2 and 3, or 1 and 3. In some embodiments, all 3 criteria may be used. 
     In certain embodiments, a protospacer adjacent motif (PAM) or PAM-like motif directs recognition and/or binding of one or more of the polypeptides capable of allosterically interacting as disclosed herein to the target sequence. In some embodiments, the PAM may be a 5′ PAM (i.e., located upstream of the 5′ end of the protospacer). In other embodiments, the PAM may be a 3′ PAM (i.e., located downstream of the 5′ end of the protospacer). The term “PAM” may be used interchangeably with the term “PFS” or “protospacer flanking site” or “protospacer flanking sequence”. In certain embodiments, one or more of the polypeptides capable of allosterically interacting as described herein may recognize a 3′ PAM. In certain embodiments, one or more of the polypeptides capable of allosterically interacting may recognize a 3′ PAM which is 5′H, wherein H is A, C or U. 
     PAM and PFS Elements 
     PAM elements are sequences that can be recognized and bound by Cas proteins. Cas proteins/effector complexes can then unwind the dsDNA at a position adjacent to the PAM element. It will be appreciated that Cas proteins and systems that include them that target RNA do not require PAM sequences (Marraffini et al. 2010. Nature. 463:568-571). Instead, many rely on PFSs, which are discussed elsewhere herein. In certain embodiments, the target sequence should be associated with a PAM (protospacer adjacent motif) or PFS (protospacer flanking sequence or site), that is, a short sequence recognized by the CRISPR complex. Depending on the nature of the CRISPR-Cas protein, the target sequence should be selected, such that its complementary sequence in the DNA duplex (also referred to herein as the non-target sequence) is upstream or downstream of the PAM. In the embodiments, the complementary sequence of the target sequence is downstream or 3′ of the PAM or upstream or 5′ of the PAM. The precise sequence and length requirements for the PAM differ depending on the Cas protein used, but PAMs are typically 2-5 base pair sequences adjacent the protospacer (that is, the target sequence). Examples of the natural PAM sequences for different Cas proteins are provided herein below and the skilled person will be able to identify further PAM sequences for use with a given Cas protein. 
     The ability to recognize different PAM sequences depends on the Cas polypeptide(s) included in the system. See e.g., Gleditzsch et al. 2019. RNA Biology. 16(4):504-517. Table 2 (from Gleditzsch et al. 2019) below shows several Cas polypeptides and the PAM sequence they recognize. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Example PAM Sequences 
               
            
           
           
               
               
               
            
               
                   
                 Cas Protein 
                 PAM Sequence 
               
               
                   
               
               
                   
                 SpCas9 
                 NGG/NRG 
               
               
                   
               
               
                   
                 SaCas9 
                 NGRRT or NGRRN 
               
               
                   
               
               
                   
                 NmeCas9 
                 NNNNGATT 
               
               
                   
               
               
                   
                 CjCas9 
                 NNNNRYAC 
               
               
                   
               
               
                   
                 StCas9 
                 NNAGAAW 
               
               
                   
               
               
                   
                 Cas12a (Cpf1) 
                 TTTV 
               
               
                   
                 (including bCpf1 
                   
               
               
                   
                 and AsCpf1) 
                   
               
               
                   
               
               
                   
                 Cas12b (C2c1) 
                 TTT, TTA, and TTC 
               
               
                   
               
               
                   
                 Cas12c (C2c3) 
                 TA 
               
               
                   
               
               
                   
                 Cas12d (CasY) 
                 TA 
               
               
                   
               
               
                   
                 Cas12e (CasX) 
                 5′-TTCN-3′ 
               
               
                   
               
            
           
         
       
     
     In a preferred embodiment, the CRISPR effector protein may recognize a 3′ PAM. In certain embodiments, the CRISPR effector protein may recognize a 3′ PAM which is 5′H, wherein H is A, C or U. 
     Further, engineering of the PAM Interacting (PI) domain on the Cas protein may allow programing of PAM specificity, improve target site recognition fidelity, and increase the versatility of the CRISPR-Cas protein, for example as described for Cas9 in Kleinstiver B P et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature. 2015 Jul. 23; 523(7561):481-5. doi: 10.1038/nature14592. As further detailed herein, the skilled person will understand that Cas13 proteins may be modified analogously. Gao et al, “Engineered Cpfl Enzymes with Altered PAM Specificities,” bioRxiv 091611; doi: http://dx.doi.org/10.1101/091611 (Dec. 4, 2016). Doench et al. created a pool of sgRNAs, tiling across all possible target sites of a panel of six endogenous mouse and three endogenous human genes and quantitatively assessed their ability to produce null alleles of their target gene by antibody staining and flow cytometry. The authors showed that optimization of the PAM improved activity and also provided an on-line tool for designing sgRNAs. 
     PAM sequences can be identified in a polynucleotide using an appropriate design tool, which are commercially available as well as online. Such freely available tools include, but are not limited to, CRISPRFinder and CRISPRTarget. Mojica et al. 2009. Microbiol. 155 (Pt. 3):733-740; Atschul et al. 1990. J. Mol. Biol. 215:403-410; Biswass et al. 2013 RNA Biol. 10:817-827; and Grissa et al. 2007. Nucleic Acid Res. 35:W52-57. Experimental approaches to PAM identification can include, but are not limited to, plasmid depletion assays (Jiang et al. 2013. Nat. Biotechnol. 31:233-239; Esvelt et al. 2013. Nat. Methods. 10:1116-1121; Kleinstiver et al. 2015 . Nature.  523:481-485), screened by a high-throughput in vivo model called PAM-SCNAR (Pattanayak et al. 2013. Nat. Biotechnol. 31:839-843 and Leenay et al. 2016. Mol. Cell. 16:253), and negative screening (Zetsche et al. 2015. Cell. 163:759-771). 
     As previously mentioned, CRISPR-Cas systems that target RNA do not typically rely on PAM sequences. Instead such systems typically recognize protospacer flanking sites (PFSs) instead of PAMs Thus, Type VI CRISPR-Cas systems typically recognize protospacer flanking sites (PFSs) instead of PAMs. PFSs represents an analogue to PAMs for RNA targets. Type VI CRISPR-Cas systems employ a Cas13. Some Cas13 proteins analyzed to date, such as Cas13a (C2c2) identified from  Leptotrichia shahii  (LShCAs13a) have a specific discrimination against G at the 3′ end of the target RNA. The presence of a C at the corresponding crRNA repeat site can indicate that nucleotide pairing at this position is rejected. However, some Cas13 proteins (e.g., LwaCAs13a and PspCas13b) do not seem to have a PFS preference. See e.g., Gleditzsch et al. 2019. RNA Biology. 16(4):504-517. 
     Some Type VI proteins, such as subtype B, have 5′-recognition of D (G, T, A) and a 3′-motif requirement of NAN or NNA. One example is the Cas13b protein identified in  Bergeyella zoohelcum  (BzCas13b). See e.g., Gleditzsch et al. 2019 . RNA Biology.  16(4):504-517. 
     Overall Type VI CRISPR-Cas systems appear to have less restrictive rules for substrate (e.g., target sequence) recognition than those that target DNA (e.g., Type V and type II). 
     Determination of PAM 
     Determination of PAM can be ensured as follows. This experiment closely parallels similar work in  E. coli  for the heterologous expression of StCas9 (Sapranauskas, R. et al. Nucleic Acids Res 39, 9275-9282 (2011)). Applicants introduce a plasmid containing both a PAM and a resistance gene into the heterologous  E. coli , and then plate on the corresponding antibiotic. If there is DNA cleavage of the plasmid, Applicants observe no viable colonies. 
     In further detail, the assay is as follows for a DNA target, but can be appropriately adapted for an RNA target by one of ordinary skill in the art. Two  E. coli  strains are used in this assay. One carries a plasmid that encodes the endogenous effector protein locus from the bacterial strain. The other strain carries an empty plasmid (e.g. pACYC184, control strain). All possible 7 or 8 bp PAM sequences are presented on an antibiotic resistance plasmid (pUC19 with ampicillin resistance gene). The PAM is located next to the sequence of proto-spacer 1 (the DNA target to the first spacer in the endogenous effector protein locus). Two PAM libraries were cloned. One has a 8 random bp 5′ of the proto-spacer (e.g. total of 65536 different PAM sequences=complexity). The other library has 7 random bp 3′ of the proto-spacer (e.g. total complexity is 16384 different PAMs). Both libraries were cloned to have in average 500 plasmids per possible PAM. Test strain and control strain were transformed with 5′PAM and 3′PAM library in separate transformations and transformed cells were plated separately on ampicillin plates. Recognition and subsequent cutting/interference with the plasmid renders a cell vulnerable to ampicillin and prevents growth. Approximately 12 h after transformation, all colonies formed by the test and control strains where harvested and plasmid DNA was isolated. Plasmid DNA was used as template for PCR amplification and subsequent deep sequencing. Representation of all PAMs in the untransformed libraries showed the expected representation of PAMs in transformed cells. Representation of all PAMs found in control strains showed the actual representation. Representation of all PAMs in test strain showed which PAMs are not recognized by the enzyme and comparison to the control strain allows extracting the sequence of the depleted PAM. 
     PAM Interacting Domain 
     In some aspects, one or more of the Cas-like proteins in a CRISPR-Cas system described herein comprise at least one PAM interacting domain, including but not limited to PAM interacting domains described herein, PAM interacting domains known in the art, and domains recognized to be PAM interacting domains by comparison to consensus sequences and motifs. The PAM interacting domain can interact with, associated with, and/or bind, a PAM motif of a nucleic acid component and/or target polynucleotide. 
     Specialized Cas-Based Systems 
     Inducible and Split CRISPR-Cas Systems 
     In some embodiments, the CRISPR-Cas system is a split CRISPR-Cas system. See e.g., Zetche et al., 2015. Nat. Biotechnol. 33(2): 139-142 and International Patent Publication WO 2019/018423, the compositions and techniques of which can be used in and/or adapted for use with the present invention. Split CRISPR-Cas proteins are set forth herein and in documents incorporated herein by reference in further detail herein. In certain embodiments, each part of a split CRISPR protein are attached to a member of a specific binding pair, and when bound with each other, the members of the specific binding pair maintain the parts of the CRISPR protein in proximity. In certain embodiments, each part of a split CRISPR protein is associated with an inducible binding pair. An inducible binding pair is one which is capable of being switched “on” or “off” by a protein or small molecule that binds to both members of the inducible binding pair. In some embodiments, CRISPR proteins may preferably split between domains, leaving domains intact. In particular embodiments, said Cas split domains (e.g., RuvC and HNH domains in the case of Cas9) can be simultaneously or sequentially introduced into the cell such that said split Cas domain(s) process the target nucleic acid sequence in the algae cell. The reduced size of the split Cas compared to the wild type Cas allows other methods of delivery of the systems to the cells, such as the use of cell penetrating peptides as described herein. 
     In some embodiments, the CRISPR-Cas system can include one or more inducible CRISPR-Cas system effectors that can be composed of a first Cas effector fusion construct attached to a first half of an inducible dimer and a second Cas effector fusion construct attached to a second half of the inducible dimer, where the first Cas effector (e.g. Cas9-like and/or Cas12-like) fusion construct is operably linked to one or more nuclear localization signals, where the second Cas effector fusion construct is operably linked to one or more nuclear export signals, where contact with an inducer energy source brings the first and second halves of the inducible dimer together, where bringing the first and second halves of the inducible dimer together allows the first and second CRISPR effector fusion constructs to constitute a functional CRISPR effector (optionally wherein the CRISPR-Cas system comprises a guide RNA (gRNA) comprising a guide sequence capable of hybridizing to a target sequence in a genomic locus of interest in a cell, and where the functional CRISPR-Cas system binds to the target sequence and, optionally, edits the genomic locus to alter gene expression). 
     In some embodiments, the inducible CRISPR-Cas system, the inducible dimer is or comprises, consists essentially of, or consists of an inducible heterodimer. In an aspect, an inducible CRISPR-Cas system, the first half or a first portion or a first fragment of the inducible heterodimer is, comprises, consists of, or consists essentially of an FKBP, optionally FKBP12. In some embodiments, in the inducible CRISPR-Cas system, the second half or a second portion or a second fragment of the inducible heterodimer is, comprises, consists of, or consists essentially of FRB. In some embodiments, in the inducible CRISPR-Cas system, the arrangement of the first CRISPR fusion construct is, comprises, consists of, or consists essentially of N′ terminal CRISPR part-FRB-NES. In some embodiments, in the inducible CRISPR-Cas system, the arrangement of the first CRISPR fusion construct is or comprises or consists of or consists essentially of NES-N′ terminal CRISPR part-FRB-NES. In some embodiments, in the inducible CRISPR-Cas system, the arrangement of the second CRISP fusion construct is or comprises or consists essentially of or consists of C′ terminal CRISP part-FKBP-NLS. In some embodiments, in the inducible CRISPR-Cas-Cas system, the arrangement of the second CRISP fusion construct is or comprises or consists of or consists essentially of NLS-C′ terminal CRISP part-FKBP-NLS. In an aspect, in inducible CRISPR-Cas system there can be a linker that separates the CRISP part from the half or portion or fragment of the inducible dimer. In an aspect, in the inducible CRISPR-Cas system, the inducer energy source is or comprises or consists essentially of or consists of rapamycin. In an aspect, in inducible CRISPR-Cas system, the inducible dimer is an inducible homodimer. 
     In an aspect, the inducible CRISPR-Cas system, is composed of a first CRISPR fusion construct attached to a first half of an inducible heterodimer and a second CRISPR fusion construct attached to a second half of the inducible heterodimer, where the first CRISPR fusion construct is operably linked to one or more nuclear localization signals, where the second CRISPR fusion construct is operably linked to a nuclear export signal, wherein contact with an inducer energy source brings the first and second halves of the inducible heterodimer together, where bringing the first and second halves of the inducible heterodimer together allows the first and second CRISPR fusion constructs to constitute a functional CRISPR-Cas system or Cas effector, and optionally where the CRISPR-Cas system comprises a guide RNA (gRNA) comprising a guide sequence capable of hybridizing to a target sequence in a genomic locus of interest in a cell, and wherein the functional CRISPR-Cas system edits the genomic locus to alter gene expression. 
     Accordingly, an inducible or split CRISPR-Cas system or effector thereof can be/include homodimers as well as heterodimers, dead-CRISPR or CRISPR protein having essentially no nuclease activity, e.g., through mutation, systems or complexes wherein there is one or more NLS and/or one or more NES; functional domain(s) linked to split Cas effector (e.g. a Cas-like effector such as a Cas9-like and/or Cas12-like). 
     An inducer energy source may be considered to be simply an inducer or a dimerizing agent. The term ‘inducer energy source’ is used herein throughout for consistency. The inducer energy source (or inducer) acts to reconstitute the enzyme. In some embodiments, the inducer energy source brings the two parts of the enzyme together through the action of the two halves of the inducible dimer. The two halves of the inducible dimer therefore are brought tougher in the presence of the inducer energy source. The two halves of the dimer will not form into the dimer (dimerize) without the inducer energy source. In some embodiments, a CRISPR enzyme may form a component of an inducible system. The inducible nature of the system would allow for spatiotemporal control of gene editing or gene expression using a form of energy. The form of energy may include but is not limited to electromagnetic radiation, sound energy, chemical energy, biological energy, and thermal energy. Examples of inducible system include tetracycline inducible promoters (Tet-On or Tet-Off), small molecule two-hybrid transcription activations systems (FKBP, ABA, etc.), or light inducible systems (Phytochrome, LOV domains, or cryptochrome). In one embodiment, the CRISPR enzyme may be a part of a Light Inducible Transcriptional Effector (LITE) to direct changes in transcriptional activity in a sequence-specific manner. The components of a light may include a CRISPR enzyme, a light-responsive cytochrome heterodimer (e.g. from  Arabidopsis thaliana ), and a transcriptional activation/repression domain. Other examples of inducible DNA binding proteins and methods for their use are provided in U.S. 61/736,465, U.S. Provisional Application No. 61/721,283, and International Patent Publication No. WO 2014/018423 A2 which is hereby incorporated by reference in its entirety. 
     Thus, the two halves of the inducible dimer cooperate with the inducer energy source to dimerize the dimer. This in turn reconstitutes the CRISPR-Cas system or effector thereof by bringing the first and second parts of the CRISPR-Cas system and/or Cas effector together. 
     The CRISPR-Cas protein fusion constructs each comprise one part of the split CRISPR effector protein. These are fused, preferably via a linker such as a GlySer linker described herein (see e.g., SEQ ID NOS: 6-20), to one of the two halves of the dimer. Other suitable linkers are described in International Patent Publication No. WO 2015/089427. The two halves of the dimer may be substantially the same two monomers that together that form the homodimer, or they may be different monomers that together form the heterodimer. As such, the two monomers can be thought of as one half of the full dimer. 
     The CRISPR-Cas effector protein is split in the sense that the two parts of the CRISPR-Cas effector protein substantially comprise a functioning CRISPR protein. That CRISPR protein may function as a genome editing enzyme (when forming a complex with the target DNA and the guide), such as a nickase or a nuclease (cleaving both strands of the DNA), or it may be a dead-CRISPR protein which is essentially a DNA-binding protein with very little or no catalytic activity, due to typically mutation(s) in its catalytic domains. 
     The two parts of the split CRISPR effector protein can be thought of as the N′ terminal part and the C′ terminal part of the split CRISPR effector protein. The fusion is typically at the split point of the CRISPR protein. In other words, the C′ terminal of the N′ terminal part of the split CRISPR protein is fused to one of the dimer halves, whilst the N′ terminal of the C′ terminal part is fused to the other dimer half. 
     The CRISPR protein does not have to be split in the sense that the break is newly created. The split point can be designed in silico and cloned into the constructs. Together, the two parts of the split CRISPR protein, the N′ terminal and C′ terminal parts, form a full CRISPR protein, comprising preferably at least 70% or more of the wildtype amino acids (or nucleotides encoding them), preferably at least 80% or more, preferably at least 90% or more, preferably at least 95% or more, and most preferably at least 99% or more of the wildtype amino acids (or nucleotides encoding them). Some trimming may be possible, and mutants are envisaged. Non-functional domains may be removed entirely. What is important is that the two parts may be brought together and that the desired CRISPR protein function is restored or reconstituted. The dimer may be a homodimer or a heterodimer. 
     One or more, preferably two, NLSs may be used in operable linkage to the first CRISPR protein construct. One or more, preferably two, NESs may be used in operable linkage to the first Cas construct. The NLSs and/or the NESs preferably flank the split Cas effector (e.g. Cas-like, Cas9-like and/or Cas12-like)-dimer (i.e., half dimer) fusion, i.e., one NLS may be positioned at the N′ terminal of the first CRISPR protein construct and one NLS may be at the C′ terminal of the first CRISPR protein construct. Similarly, one NES may be positioned at the N′ terminal of the second CRISPR construct and one NES may be at the C′ terminal of the second CRISPR-Cas effector construct. Where reference is made to N′ or C′ terminals, it will be appreciated that these correspond to 5′ ad 3′ ends in the corresponding nucleotide sequence. 
     A preferred arrangement is that the first CRISPR-Cas effector protein construct is arranged 5′-NLS-(N′ terminal CRISPR-Cas effector protein part)-linker-(first half of the dimer)-NLS-3′. A preferred arrangement is that the second CRISPR-Cas effector protein construct is arranged 5′-NES—(second half of the dimer)-linker-(C′ terminal CRISPR-Cas effector protein part)-NES-3′. A suitable promoter is preferably upstream of each of these constructs. The two constructs may be delivered separately or together. 
     In some embodiments, one or all of the NES(s) in operable linkage to the second Cas effector (e.g. Cas-like, Cas9-like and/or Cas12-like) construct may be swapped out for an NLS. In other embodiments, the localization signal can be in operable linkage to the second Cas effector (e.g., Cas-like, Cas9-like and/or Cas12-like) construct is one or more NES(s). 
     It will also be appreciated that the NES may be operably linked to the N′ terminal fragment of the split CRISPR-Cas effector protein and that the NLS may be operably linked to the C′ terminal fragment of the split CRISPR-Cas effector protein. However, the arrangement where the NLS is operably linked to the N′ terminal fragment of the split Cas effector (e.g. Cas-like, Cas9-like, and/or Cas12-like) and that the NES is operably linked to the C′ terminal fragment of the split CRISPR-Cas effector protein may be preferred. 
     The NES functions to localize the second CRISPR-Cas effector protein fusion construct outside of the nucleus, at least until the inducer energy source is provided (e.g., at least until an energy source is provided to the inducer to perform its function). The presence of the inducer stimulates dimerization of the two CRISPR-Cas effector protein fusions within the cytoplasm and makes it thermodynamically worthwhile for the dimerized, first and second, CRISPR-Cas effector protein fusions to localize to the nucleus. Without being bound by theory, the NES can sequester the second CRISPR protein fusion to the cytoplasm (i.e., outside of the nucleus). The NLS on the first CRISPR protein fusion can localize it to the nucleus. In both cases, the NES or NLS can shift an equilibrium (the equilibrium of nuclear transport) to a desired direction. The dimerization typically occurs outside of the nucleus (a very small fraction might happen in the nucleus) and the NLSs on the dimerized complex can shift the equilibrium of nuclear transport to nuclear localization, so the dimerized and hence reconstituted CRISPR-Cas protein enters the nucleus. 
     The split-effector approach and/or inducible approach can be used with other techniques to add layers of further control of the CRISPR-Cas systems described herein. Different localization sequences can be used (i.e., the NES and NLS as preferred) to reduce background activity from auto-assembled complexes. Tissue specific promoters, for example one for each of the first and second CRISPR protein fusion constructs, may also be used for tissue-specific targeting, thus providing spatial control. Two different tissue specific promoters may be used to exert a finer degree of control if required. The same approach may be used in respect of stage-specific promoters or there may a mixture of stage and tissue specific promoters, where one of the first and second Cas effectors (e.g., Cas-like, Cas9-like, and/or Cas12-like) fusion constructs is under the control of (i.e. operably linked to or comprises) a tissue-specific promoter, whilst the other of the first and second Cas-like (e.g. Cas9-like and/or Cas12-like) fusion constructs is under the control of (i.e. operably linked to or comprises) a stage-specific promoter. 
     The number of NLS or NES associated with each of the Cas effectors can by any suitable number. In some embodiments, the first and/or the second Cas effector can be operably linked to 1, 2, 3, or more NLS or NES. 
     Where the FRB and FKBP system are used, the FKBP is preferably flanked by nuclear localization sequences (NLSs). Where the FRB and FKBP system are used, the preferred arrangement is N′ terminal CRISPR protein—FRB-NES: C′ terminal Cas-like (e.g. Cas9-like and/or Cas12-like)-FKBP-NLS. Thus, the first CRISPR protein fusion construct can, in some embodiments, comprise the C′ terminal CRISPR protein part and the second CRISPR protein fusion construct would comprise the N′ terminal CRISPR protein part. 
     In some embodiments, the inducible CRISPR-Cas system can be turned on quickly, i.e. can have a rapid response. Without being bound by theory, that CRISPR effector protein activity can be induced through dimerization of existing (already present) fusion constructs (through contact with the inducer energy source) more rapidly than through the expression (especially translation) of new fusion constructs. As such, the first and second CRISPR protein fusion constructs may be expressed in the target cell ahead of time, i.e. before CRISPR protein activity is required. CRISPR protein activity can then be temporally controlled and then quickly constituted through addition of the inducer energy source, which ideally acts more quickly (to dimerize the heterodimer and thereby provide CRISPR protein activity) than through expression (including induction of transcription) of CRISPR protein delivered by a vector, for example. 
     In some embodiments, the inducible CRISPR-Cas effectors can include one or more rapamycin or chemically sensitive dimerization domains, which can allow for temporal control of the CRISPR-Cas system by controlling exposure of the CRISPR-Cas system to the rapamycin or chemical inducer. In some embodiments, inducement can be accomplished by delivery of Rapamycin to a subject or cell containing an inducible CRISPR-Cas system containing one or more rapamycin domains. Rapamycin treatments can last 12 days. In some embodiments, the dose of rapamycin can be about 200 nM. This temporal and/or molar dosage is an example of an appropriate dose for Human embryonic kidney 293FT (HEK293FT) cell lines and this may also be used in other cell lines. This figure can be extrapolated out for therapeutic use in vivo into, for example, mg/kg. However, it is also envisaged that the standard dosage for administering rapamycin to a subject is used here as well. By the “standard dosage”, it is meant the dosage under rapamycin&#39;s normal therapeutic use or primary indication (i.e. the dose used when rapamycin is administered for use to prevent organ rejection). 
     In some embodiments, the arrangement of CRISPR protein-FRB/FKBP pieces are separate and inactive until rapamycin-induced dimerization of FRB and FKBP results in reassembly of a functional full-length CRISPR protein nuclease. Thus, it is preferred that first CRISPR protein fusion construct attached to a first half of an inducible heterodimer is delivered separately and/or is localized separately from the second Cas effector (e.g., Cas-like, Cas9-like, and/or Cas12-like) fusion construct attached to a first half of an inducible heterodimer. 
     To sequester the CRISPR protein (N)-FRB fragment in the cytoplasm, where it is less likely to dimerize with the nuclear-localized Cas-like (e.g. Cas9-like and/or Cas12-like) (C)-FKBP fragment, it is preferable to use on CRISPR protein (N)-FRB a single nuclear export sequence (NES) from the human protein tyrosine kinase 2 (CRISPR protein (N)—FRB-NES). In the presence of rapamycin, CRISPR protein (N)—FRB-NES dimerizes with CRISPR protein (C)-FKBP-2×NLS to reconstitute a complete CRISPR protein, which shifts the balance of nuclear trafficking toward nuclear import and allows DNA targeting. 
     An exemplary first and second light-inducible dimer halves is the CIB1 and CRY2 system. The CIB1 domain is a heterodimeric binding partner of the light-sensitive Cryptochrome 2 (CRY2). In another example, the blue light-responsive Magnet dimerization system (pMag and nMag) may be fused to the two parts of a split Cas-like (e.g. Cas9-like and/or Cas12-like) protein. In response to light stimulation, pMag and nMag dimerize and Cas-like (e.g. Cas9-like and/or Cas12-like) reassembles. For example, such system is described in connection with Cas9 in Nihongaki et al. (Nat. Biotechnol. 33, 755-790, 2015). 
     In some embodiments, the inducer energy source may be an antibiotic, a small molecule, a hormone, a hormone derivative, a steroid or a steroid derivative. In a more preferred embodiment, the inducer energy source maybe abscisic acid (ABA), doxycycline (DOX), cumate, rapamycin, 4-hydroxytamoxifen (4OHT), estrogen or ecdysone. The at least one switch may be selected from the group consisting of antibiotic based inducible systems, electromagnetic energy based inducible systems, small molecule based inducible systems, nuclear receptor based inducible systems and hormone based inducible systems. In a more preferred embodiment the at least one switch may be selected from the group consisting of tetracycline (Tet)/DOX inducible systems, light inducible systems, ABA inducible systems, cumate repressor/operator systems, 4OHT/estrogen inducible systems, ecdysone-based inducible systems and FKBP12/FRAP (FKBP12-rapamycin complex) inducible systems. Such inducers are also discussed herein and in PCT/US2013/051418, incorporated herein by reference. 
     Further it is described that one or more functional domains can be associated with one or both parts of the effector protein, International Patent Publication No. WO 2015/089427 identifies split points within SpCas9, incorporated herein by reference. This approach can be adapted for the CRISPR-Cas systems described herein. 
     The first and second fusion constructs of the CRISPR effector protein described herein of a split CRISPR-Cas system can be delivered in the same or separate vectors and/or complexes. 
     In some embodiments, the inducible system can include an “on switch” and/or an “off switch”. In particular embodiments, it may be possible to make use of specific inhibitors and/or agonist of Cas effector (e.g. Cas-like, Cas9-like, Cas12-like, or other Cas effector). Off-switches and on-switches may be any molecules (i.e. peptides, proteins, small molecules, nucleic acids, organic compounds, inorganic compounds, and the like) capable of interfering with any aspect of the Cas effector protein. For instance, Pawluck et al. 2016 (Cell 167, 1-10) describe mobile elements from bacteria that encode protein inhibitors of Cas9, which can be adapted and/or applied to the CRISPR-Cas systems described herein. Three families of anti-CRISPRs were found to inhibit  N. meningitidis  Cas9 in vivo and in vitro. The anti-CRISPRs bind directly to NmeCas9. These proteins are described to be potent “off-switches” for NmeCas9 genome editing in human cells. Methods for identifying small molecules which affect efficiency of Cas9 are described for example by Yu et al. (Cell Stem Cell 16, 142-147, 2015), which can be adapted and/or applied to the CRISPR-Cas systems described herein. In certain embodiments small molecules may be used for control the Cas effector(s) present in the system. Maji et al. describe a small molecule-regulated protein degron domain to control CRISRP-Cas system editing. Maji et al. “Multidimensional chemical control of CRISPR-Cas9” Nature Chemical Biology (2017) 13:9-12, which can be adapted and/or applied to the CRISPR-Cas systems described herein. In certain example embodiments, the inhibitor may be a bacteriophage derived protein. See Rauch et al. “Inhibition of CRISPR-Cas9 with Bacteriophage Proteins” Cell (2017) 168(2):150-158, which can be adapted and/or applied to the CRISPR-Cas-like (e.g. Cas9-like and/or Cas12-like) systems described herein. In certain example embodiments, the anti-CRISPR may inhibit CRISPR-Cas systems descried herein by binding to guide molecules. See Shin et al. “Disabling Cas9 by an anti-CRISPR DNA mimic” bioRxiv, Apr. 22, 2017, doi:http://dx.doi.org/10.1101/129627, which can be adapted and/or applied to the CRISPR-Cas systems described herein. 
     In particular embodiments, intracellular DNA is removed by genetically encoded DNai, which responds to a transcriptional input and degrades user-defined DNA as described in Caliando &amp; Voigt, Nature Communications 6: 6989 (2015), which can be adapted and/or applied to the CRISPR-Cas systems described herein. 
     Self-Inactivating CRISPR-Cas Systems 
     Once all copies of a gene in the genome of a cell have been edited, continued CRISPR-Cas expression in that cell is no longer necessary. Indeed, sustained expression is undesirable to avoid off-target effects and other toxicity issues. International Patent Publication No. WO 2015/089351 describes self-Inactivating CRISPR-Cas systems which rely on the use of a non-coding guide target sequence within the CRISPR vector itself. Thus, after expression begins, the CRISPR system can lead to its own destruction, but before destruction is complete it will have time to edit the genomic copies of the target gene (which, with a normal point mutation in a diploid cell, requires at most two edits). In some embodiments, the CRISPR-Cas system described herein can be a self-inactivating CRISPR-Cas system, which includes one additional RNA (i.e., guide RNA) that targets the coding sequence for one or more of the CRISPR-Cas enzyme itself or that targets one or more non-coding guide target sequences complementary to unique sequences present in within the promoter driving expression of the non-coding RNA elements, within the promoter driving expression of the Cas effector (e.g., Cas-like, Cas9-like, and/or Cas12-like) gene(s), within 100 bp of the ATG translational start codon in the Cas effector (e.g., Cas-like, Cas9-like, and/or Cas12-like) coding sequence, or within the inverted terminal repeat (iTR) of a viral delivery vector, e.g., in the AAV genome. 
     Similarly, self-inactivating CRISPR-Cas systems which make use of “governing guides” are exemplified in relation to Cas9 in International Patent Publication No. WO 2015/070083, which is incorporated herein by reference, and may be extrapolated to Cas-like (e.g. Cas9-like and/or Cas12-like) or other Cas effectors described herein. More particularly methods and compositions that use, or include, a nucleic acid, e.g., a DNA, that encodes a Cas-like (e.g. Cas9-like and/or Cas12-like) or other Cas effector molecule or a gRNA molecule, can, in addition, use or include a “governing gRNA molecule.” The governing gRNA molecule can complex with the Cas-like (e.g. Cas9-like and/or Cas12-like) or other Cas effector molecule to inactivate or silence a component of a Cas-like (e.g. Cas9-like and/or Cas12-like) system. The additional gRNA molecule, referred to herein as a governing gRNA molecule, comprises a targeting domain which targets a component of the Cas-like (e.g. Cas9-like and/or Cas12-like) system. In an embodiment, the governing gRNA molecule targets and silences (1) a nucleic acid that encodes a Cas-like (e.g. Cas9-like and/or Cas12-like) and/or other Cas effector molecule(s) (i.e., a Cas-like (e.g. Cas9-like and/or Cas12-like)-targeting gRNA molecule), (2) a nucleic acid that encodes a gRNA molecule (i.e., a gRNA-targeting gRNA molecule), or (3) a nucleic acid sequence engineered into one or more of the CRISPR-Cas system components that is designed with minimal homology to other nucleic acid sequences in the cell to minimize off-target cleavage (i.e., an engineered control sequence-targeting gRNA molecule). Governing guides and their targets are discussed in greater detail herein, such as in the context of the nucleic acid components of the CRISPR-Cas systems described herein. 
     The additional guide RNA (e.g. governing gRNA) can be delivered via a vector, e.g., a separate vector or the same vector that is encoding the CRISPR-Cas complex. When provided by a separate vector, the CRISPR RNA (e.g. governing gRNA) that targets Cas effector (e.g., a Cas-like, Cas9-like and/or Cas12-like) expression can be administered sequentially or simultaneously. When administered sequentially, the CRISPR RNA that targets Cas effector expression is to be delivered after the CRISPR RNA that is intended for e.g. gene editing or gene engineering. This period may be a period of minutes (e.g. 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes). This period may be a period of hours (e.g. 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours). This period may be a period of days (e.g. 2 days, 3 days, 4 days, 7 days). This period may be a period of weeks (e.g. 2 weeks, 3 weeks, 4 weeks). This period may be a period of months (e.g. 2 months, 4 months, 8 months, 12 months). This period may be a period of years (2 years, 3 years, 4 years). In this fashion, the Cas enzyme associates with a first gRNA capable of hybridizing to a first target, such as a genomic locus or loci of interest and undertakes the function(s) desired of the CRISPR-Cas system (e.g., gene engineering); and subsequently the Cas effector enzyme(s) may then associate with the second gRNA capable of hybridizing to the sequence comprising at least part of the Cas-effector or CRISPR cassette, when present. Where the gRNA targets the sequence(s) encoding expression of the Cas effector(s) of the CRISPR-Cas system the enzyme becomes impeded and the system becomes self-inactivating. In the same manner, CRISPR RNA that targets Cas effector expression can be delivered via, for example liposome, lipofection, nanoparticles, microvesicles as described elsewhere herein, may be administered sequentially or simultaneously. Similarly, self-inactivation can be used for inactivation of one or more guide RNA used to target one or more targets. 
     In some embodiments, a single gRNA is provided that is capable of hybridization to a sequence downstream of a CRISPR enzyme start codon, whereby after a period of time there is a loss of the CRISPR enzyme expression and self-inactivation of the CRISPR-Cas system. In some embodiments, one or more gRNA(s) are provided that are capable of hybridization to one or more coding or non-coding regions of the polynucleotide encoding the CRISPR-Cas system, whereby after a period of time there is inactivation of one or more, or in some cases all, of the CRISPR-Cas systems. In some aspects of the system, and not to be limited by theory, the cell may comprise a plurality of CRISPR-Cas complexes, wherein a first subset of CRISPR complexes comprise a first chiRNA (chimericRNA) capable of targeting a genomic locus or loci to be edited, and a second subset of CRISPR complexes comprise at least one second chiRNA capable of targeting the polynucleotide encoding the CRISPR-Cas system, wherein the first subset of CRISPR-Cas complexes mediate editing of the targeted genomic locus or loci and the second subset of CRISPR complexes eventually inactivate the CRISPR-Cas system, thereby inactivating further CRISPR-Cas expression in the cell. 
     The components of the self-inactivating CRISPR-Cas system can be included in a vector or vector systems. The various coding sequences (CRISPR enzyme, guide RNAs, tracr and tracr mate) can be included on a single vector or on multiple vectors. For instance, it is possible to encode the enzyme on one vector and the various RNA sequences on another vector, or to encode the enzyme and one chiRNA on one vector, and the remaining chiRNA on another vector, or any other permutation. In general, a system using a total of one or two different vectors is preferred. Where multiple vectors are used, it is possible to deliver them in unequal numbers, and ideally with an excess of a vector which encodes the first guide RNA relative to the second guide RNA, thereby assisting in delaying final inactivation of the CRISPR system until genome editing has had a chance to occur. 
     In some embodiments, one or more vectors can include a polynucleotide encoding (i) a CRISPR enzyme; (ii) a first guide RNA capable of hybridizing to a target sequence in the cell; (iii) a second guide RNA capable of hybridizing to one or more target sequence(s) in the vector which encodes the CRISPR enzyme; (iv) at least one tracr mate sequence; (v) at least one tracr sequence; (iv) or a combination thereof. The first and second complexes can use the same tracr and tracr mate, thus differing only by the guide sequence, wherein, when expressed within the cell: the first guide RNA directs sequence-specific binding of a first CRISPR complex to the target sequence in the cell; the second guide RNA directs sequence-specific binding of a second CRISPR complex to the target sequence in the vector which encodes the CRISPR enzyme; the CRISPR complexes comprise (a) a tracr mate sequence hybridized to a tracr sequence and (b) a CRISPR enzyme bound to a guide RNA, such that a guide RNA can hybridize to its target sequence; and the second CRISPR complex inactivates the CRISPR-Cas system to prevent continued expression of the CRISPR enzyme by the cell. The CRISPR enzyme can be Cas-like (e.g. Cas9-like and/or Cas12-like). In some embodiments the CRISPR enzyme can be SpCas9, SpCas9-like, SaCas9, SaCas9-like, StCas9, or StCas9-like. 
     Further characteristics of the vector(s), the encoded enzyme, the guide sequences, etc. are disclosed elsewhere herein. For instance, one or both of the guide sequence(s) can be part of a chiRNA sequence which provides the guide, tracr mate and tracr sequences within a single RNA, such that the system can encode (i) a CRISPR enzyme; (ii) a first chiRNA comprising a sequence capable of hybridizing to a first target sequence in the cell, a first tracr mate sequence, and a first tracr sequence; (iii) a second guide RNA capable of hybridizing to the vector which encodes the CRISPR enzyme, a second tracr mate sequence, and a second tracr sequence. Similarly, the enzyme can include one or more NLS, etc. 
     Furthermore, if the guide RNAs are expressed in array format, the “self-inactivating” guide RNAs that target both promoters simultaneously will result in the excision of the intervening nucleotides from within the CRISPR-Cas expression construct, effectively leading to its complete inactivation. Similarly, excision of the intervening nucleotides will result where the guide RNAs target both ITRs, or targets two or more other CRISPR-Cas components simultaneously. Self-inactivation as explained herein is applicable, in general, with CRISPR-Cas systems, such as any of those described herein, in order to provide regulation of the CRISPR-Cas system. For example, self-inactivation as explained herein may be applied to the CRISPR repair of mutations, for example expansion disorders, as explained herein. As a result of this self-inactivation, CRISPR repair is only transiently active. 
     In some embodiments of a self-inactivating CRISPR-Cas system, plasmids that co-express one or more sgRNA targeting genomic sequences of interest (e.g. 1-2, 1-5, 1-10, 1-15, 1-20, 1-30) can be established with “self-inactivating” sgRNAs that target an SpCas9-like sequence at or near the engineered ATG start site (e.g. within 5 nucleotides, within 15 nucleotides, within 30 nucleotides, within 50 nucleotides, within 100 nucleotides). A regulatory sequence in the U6 promoter region can also be targeted with an sgRNA. The U6-driven sgRNAs may be designed in an array format such that multiple sgRNA sequences can be simultaneously released. When first delivered into target tissue/cells (left cell) sgRNAs begin to accumulate while Cas-effector levels rise in the nucleus. Cas effector complexes with all of the sgRNAs to mediate genome editing and self-inactivation of the CRISPR-Cas system plasmids. 
     In some embodiments, a self-inactivating CRISPR-Cas system described herein can express or include in single or in tandem array format from 1 up to 4 or more different guide sequences; e.g. up to about 20 or about 30 guides sequences. Each individual self-inactivating guide sequence may target a different target. Such may be processed from, e.g. one chimeric pol3 transcript. Pol3 promoters such as U6 or H1 promoters may be used. Pol2 promoters such as those mentioned throughout herein. Inverted terminal repeat (iTR) sequences may flank the Pol3 promoter-sgRNA(s)-Pol2 promoter-Cas effector protein(s). 
     In particular embodiments one or more guide(s) can edit or otherwise modify one or more target(s) while one or more self-inactivating guides inactivate the CRISPR-Cas system. Thus, for example, a CRISPR-Cas system described herein capable of repairing expansion disorders can be directly combined with the self-inactivating CRISPR-Cas system described herein. Such a system may, for example, have two guides directed to the target region for repair as well as at least a third guide directed to self-inactivation of a CRISPR effector of the CRISRP-Cas system. Further examples are set forth in International Patent Publication No. WO 2015/089351, which can be adapted for and/or applied to the CRISPR-Cas systems described herein. 
     Another approach to achieve self-inactivation is the incorporation of a passcode kill switch into the CRISRP-Cas system or its host cell (such as in an adoptive therapy context). A passcode kill switch is a mechanism which efficiently kills the host cell when the conditions of the cell are altered. An exemplary passcode kill switch is the introduction of a hybrid LacI-GalR family transcription factors, which require the presence of IPTG to be switched on (Chan et al. 2015 Nature Chemical Biology doi:10.1038/nchembio.1979 which can be used to drive a gene encoding an enzyme critical for cell-survival. By combining different transcription factors sensitive to different chemicals, a “code” can be generated, this system can be used to spatially and temporally control the extent of CRISPR-induced genetic modifications, which can be of interest in different fields including therapeutic applications and may also be of interest to avoid the “escape” of GMOs from their intended environment. 
     Base Editors 
     General Overview 
     The present disclosure also provides for a base editing system that can include a Cas-like protein or system thereof described elsewhere herein. In general, such a system may comprise a deaminase (e.g., an adenosine deaminase or cytidine deaminase) fused with a Cas protein, such as a Cas-like protein described herein. The Cas protein may be a Cas-like, dead Cas protein, and/or a Cas nickase protein. In certain embodiments, the system comprises a mutated form of an adenosine deaminase fused with a dead CRISPR-Cas or CRISPR-Cas nickase. The mutated form of the adenosine deaminase may have both adenosine deaminase and cytidine deaminase activities. 
     Thus, in some embodiments the Cas-based system described herein can be a base editing system. As used herein, “base editing” refers generally to the process of polynucleotide modification via a CRISPR-Cas-based or Cas-based system that does not include excising nucleotides to make the modification. Base editing can convert base pairs at precise locations without generating excess undesired editing byproducts that can be made using traditional CRISPR-Cas systems. 
     In certain example embodiments, a Cas-like protein include a deaminase domain (e.g. an adenosine deaminase, cytosine deaminase and/or cytidine deaminase), as described elsewhere herein for base-editing purposes. The deaminase domain can be configured as an activate able functional domain or matched pair thereof as previously described elsewhere herein. In some aspects the deaminase into a matched pair of activatable functional domains as a “split protein” with each portion of the deaminase being incorporated into the engineered CRISPR-Cas system described herein into activatable functional domains that are attached to, integrated in, and/or fused with one or more Cas-like proteins described herein. 
     Cytosine Deaminase 
     In some aspects the deaminase is a cytosine deaminase. Programmable deamination of cytosine has been reported and may be used for correction of A→G and T→C point mutations. For example, Komor et al., Nature (2016) 533:420-424 reports targeted deamination of cytosine by APOBEC1 cytidine deaminase in a non-targeted DNA stranded displaced by the binding of a Cas-guide RNA complex to a targeted DNA strand, which results in conversion of cytosine to uracil. See also Kim et al., Nature Biotechnology (2017) 35:371-376; Shimatani et al., Nature Biotechnology (2017) doi:10.1038/nbt.3833; Zong et al., Nature Biotechnology (2017) doi:10.1038/nbt.3811; Yang Nature Communication (2016) doi:10.1038/ncomms13330. 
     Adenosine Deaminase 
     The term “adenosine deaminase” or “adenosine deaminase protein” as used herein refers to a protein, a polypeptide, or one or more functional domain(s) of a protein or a polypeptide that is capable of catalyzing a hydrolytic deamination reaction that converts an adenine (or an adenine moiety of a molecule) to a hypoxanthine (or a hypoxanthine moiety of a molecule), as shown below. In some embodiments, the adenine-containing molecule is an adenosine (A), and the hypoxanthine-containing molecule is an inosine (I). The adenine-containing molecule can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). 
     
       
         
         
             
             
         
       
     
     In one aspect, the present disclosure provides an engineered adenosine deaminase. The engineered adenosine deaminase may comprise one or more mutations herein. In some embodiments, the engineered adenosine deaminase has cytidine deaminase activity. In certain examples, the engineered adenosine deaminase has both cytidine deaminase activity and adenosine deaminase. 
     According to the present disclosure, adenosine deaminases that can be used in connection with the present disclosure include, but are not limited to, members of the enzyme family known as adenosine deaminases that act on RNA (ADARs), members of the enzyme family known as adenosine deaminases that act on tRNA (ADATs), and other adenosine deaminase domain-containing (ADAD) family members. According to the present disclosure, the adenosine deaminase is capable of targeting adenine in a RNA/DNA and RNA duplexes. Indeed, Zheng et al. (Nucleic Acids Res. 2017, 45(6): 3369-3377) demonstrate that ADARs can carry out adenosine to inosine editing reactions on RNA/DNA and RNA/RNA duplexes. In particular embodiments, the adenosine deaminase has been modified to increase its ability to edit DNA in an RNA/DNA heteroduplex of in an RNA duplex as detailed elsewhere herein. 
     In some embodiments, the adenosine deaminase is derived from one or more metazoa species, including but not limited to, mammals, birds, frogs, squids, fish, flies and worms. In some embodiments, the adenosine deaminase is a human, squid or  Drosophila  adenosine deaminase. 
     In some embodiments, the adenosine deaminase is a human ADAR, including hADAR1, hADAR2, hADAR3. In some embodiments, the adenosine deaminase is a  Caenorhabditis elegans  ADAR protein, including ADR-1 and ADR-2. In some embodiments, the adenosine deaminase is a  Drosophila  ADAR protein, including dAdar. In some embodiments, the adenosine deaminase is a squid  Loligo pealeii  ADAR protein, including sqADAR2a and sqADAR2b. In some embodiments, the adenosine deaminase is a human ADAT protein. In some embodiments, the adenosine deaminase is a  Drosophila  ADAT protein. In some embodiments, the adenosine deaminase is a human ADAD protein, including TENR (hADAD1) and TENRL (hADAD2). 
     In some embodiments, the adenosine deaminase is a TadA protein such as  E. coli  TadA. See Kim et al., Biochemistry 45:6407-6416 (2006); Wolf et al., EMBO J. 21:3841-3851 (2002). In some embodiments, the adenosine deaminase is mouse ADA. See Grunebaum et al., Curr. Opin. Allergy Clin. Immunol. 13:630-638 (2013). In some embodiments, the adenosine deaminase is human ADAT2. See Fukui et al., J. Nucleic Acids 2010:260512 (2010)). In some embodiments, the deaminase (e.g., adenosine or cytidine deaminase) is one or more of those described in Cox et al., Science. 2017, Nov. 24; 358(6366): 1019-1027; Komore et al., Nature. 2016 May 19; 533(7603):420-4; and Gaudelli et al., Nature. 2017 Nov. 23; 551(7681):464-471. 
     In some embodiments, the adenosine deaminase protein recognizes and converts one or more target adenosine residue(s) in a double-stranded nucleic acid substrate into inosine residues (s). In some embodiments, the double-stranded nucleic acid substrate is an RNA-DNA hybrid duplex. In some embodiments, the adenosine deaminase protein recognizes a binding window on the double-stranded substrate. In some embodiments, the binding window contains at least one target adenosine residue(s). In some embodiments, the binding window is in the range of about 3 bp to about 100 bp. In some embodiments, the binding window is in the range of about 5 bp to about 50 bp. In some embodiments, the binding window is in the range of about 10 bp to about 30 bp. In some embodiments, the binding window is about 1 bp, 2 bp, 3 bp, 5 bp, 7 bp, 10 bp, 15 bp, 20 bp, 25 bp, 30 bp, 40 bp, 45 bp, 50 bp, 55 bp, 60 bp, 65 bp, 70 bp, 75 bp, 80 bp, 85 bp, 90 bp, 95 bp, or 100 bp. 
     In some embodiments, the adenosine deaminase protein comprises one or more deaminase domains. Not intended to be bound by a particular theory, it is contemplated that the deaminase domain functions to recognize and convert one or more target adenosine (A) residue(s) contained in a double-stranded nucleic acid substrate into inosine (I) residue(s). In some embodiments, the deaminase domain comprises an active center. In some embodiments, the active center comprises a zinc ion. In some embodiments, during the A-to-I editing process, base pairing at the target adenosine residue is disrupted, and the target adenosine residue is “flipped” out of the double helix to become accessible by the adenosine deaminase. In some embodiments, amino acid residues in or near the active center interact with one or more nucleotide(s) 5′ to a target adenosine residue. In some embodiments, amino acid residues in or near the active center interact with one or more nucleotide(s) 3′ to a target adenosine residue. In some embodiments, amino acid residues in or near the active center further interact with the nucleotide complementary to the target adenosine residue on the opposite strand. In some embodiments, the amino acid residues form hydrogen bonds with the 2′ hydroxyl group of the nucleotides. 
     In some embodiments, the adenosine deaminase comprises human ADAR2 full protein (hADAR2) or the deaminase domain thereof (hADAR2-D). In some embodiments, the adenosine deaminase is an ADAR family member that is homologous to hADAR2 or hADAR2-D. 
     Particularly, in some embodiments, the homologous ADAR protein is human ADAR1 (hADAR1) or the deaminase domain thereof (hADAR1-D). In some embodiments, glycine 1007 of hADAR1-D corresponds to glycine 487 hADAR2-D, and glutamic Acid 1008 of hADAR1-D corresponds to glutamic acid 488 of hADAR2-D. 
     In some embodiments, the adenosine deaminase comprises the wild-type amino acid sequence of hADAR2-D. In some embodiments, the adenosine deaminase comprises one or more mutations in the hADAR2-D sequence, such that the editing efficiency, and/or substrate editing preference of hADAR2-D is changed according to specific needs. 
     The engineered adenosine deaminase may be fused or otherwise attached to, coupled to, or integrated with a Cas protein, e.g., Cas-like (e.g. Cas9-like, Cas12-like), Cas9, Cas 12 (e.g., Cas12a, Cas12b, Cas12c, Cas12d, etc.), Cas13 (e.g., Cas13a, Cas13b (such as Cas13b-t1, Cas13b-t2, Cas13b-t3), Cas13c, Cas13d, etc.), Cas14, CasX, CasY, or an engineered form of the Cas protein (e.g., an invective, dead form, a nickase form). In some examples, provided herein include an engineered adenosine deaminase fused with a Cas-like protein such as Cas9-like and/or Cas12-like. 
     Certain mutations of hADAR1 and hADAR2 proteins have been described in Kuttan et al., Proc Natl Acad Sci USA. (2012) 109(48):E3295-304; Want et al. ACS Chem Biol. (2015) 10(11):2512-9; and Zheng et al. Nucleic Acids Res. (2017) 45(6):3369-337, each of which is incorporated herein by reference in its entirety. 
     In some embodiments, the adenosine deaminase comprises a mutation at glycine336 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the glycine residue at position 336 is replaced by an aspartic acid residue (G336D). 
     In some embodiments, the adenosine deaminase comprises a mutation at Glycine487 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the glycine residue at position 487 is replaced by a non-polar amino acid residue with relatively small side chains. For example, in some embodiments, the glycine residue at position 487 is replaced by an alanine residue (G487A). In some embodiments, the glycine residue at position 487 is replaced by a valine residue (G487V). In some embodiments, the glycine residue at position 487 is replaced by an amino acid residue with relatively large side chains. In some embodiments, the glycine residue at position 487 is replaced by a arginine residue (G487R). In some embodiments, the glycine residue at position 487 is replaced by a lysine residue (G487K). In some embodiments, the glycine residue at position 487 is replaced by a tryptophan residue (G487W). In some embodiments, the glycine residue at position 487 is replaced by a tyrosine residue (G487Y). 
     In some embodiments, the adenosine deaminase comprises a mutation at glutamic acid488 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the glutamic acid residue at position 488 is replaced by a glutamine residue (E488Q). In some embodiments, the glutamic acid residue at position 488 is replaced by a histidine residue (E488H). In some embodiments, the glutamic acid residue at position 488 is replace by an arginine residue (E488R). In some embodiments, the glutamic acid residue at position 488 is replace by a lysine residue (E488K). In some embodiments, the glutamic acid residue at position 488 is replace by an asparagine residue (E488N). In some embodiments, the glutamic acid residue at position 488 is replace by an alanine residue (E488A). In some embodiments, the glutamic acid residue at position 488 is replace by a Methionine residue (E488M). In some embodiments, the glutamic acid residue at position 488 is replace by a serine residue (E488S). In some embodiments, the glutamic acid residue at position 488 is replace by a phenylalanine residue (E488F). In some embodiments, the glutamic acid residue at position 488 is replace by a lysine residue (E488L). In some embodiments, the glutamic acid residue at position 488 is replace by a tryptophan residue (E488W). 
     In some embodiments, the adenosine deaminase comprises a mutation at threonine490 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the threonine residue at position 490 is replaced by a cysteine residue (T490C). In some embodiments, the threonine residue at position 490 is replaced by a serine residue (T490S). In some embodiments, the threonine residue at position 490 is replaced by an alanine residue (T490A). In some embodiments, the threonine residue at position 490 is replaced by a phenylalanine residue (T490F). In some embodiments, the threonine residue at position 490 is replaced by a tyrosine residue (T490Y). In some embodiments, the threonine residue at position 490 is replaced by a serine residue (T490R). In some embodiments, the threonine residue at position 490 is replaced by an alanine residue (T490K). In some embodiments, the threonine residue at position 490 is replaced by a phenylalanine residue (T490P). In some embodiments, the threonine residue at position 490 is replaced by a tyrosine residue (T490E). 
     In some embodiments, the adenosine deaminase comprises a mutation at valine493 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the valine residue at position 493 is replaced by an alanine residue (V493A). In some embodiments, the valine residue at position 493 is replaced by a serine residue (V493S). In some embodiments, the valine residue at position 493 is replaced by a threonine residue (V493T). In some embodiments, the valine residue at position 493 is replaced by an arginine residue (V493R). In some embodiments, the valine residue at position 493 is replaced by an aspartic acid residue (V493D). In some embodiments, the valine residue at position 493 is replaced by a proline residue (V493P). In some embodiments, the valine residue at position 493 is replaced by a glycine residue (V493G). 
     In some embodiments, the adenosine deaminase comprises a mutation at alanine589 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the alanine residue at position 589 is replaced by a valine residue (A589V). 
     In some embodiments, the adenosine deaminase comprises a mutation at asparagine597 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the asparagine residue at position 597 is replaced by a lysine residue (N597K). In some embodiments, the adenosine deaminase comprises a mutation at position 597 of the amino acid sequence, which has an asparagine residue in the wild type sequence. In some embodiments, the asparagine residue at position 597 is replaced by an arginine residue (N597R). In some embodiments, the adenosine deaminase comprises a mutation at position 597 of the amino acid sequence, which has an asparagine residue in the wild type sequence. In some embodiments, the asparagine residue at position 597 is replaced by an alanine residue (N597A). In some embodiments, the adenosine deaminase comprises a mutation at position 597 of the amino acid sequence, which has an asparagine residue in the wild type sequence. In some embodiments, the asparagine residue at position 597 is replaced by a glutamic acid residue (N597E). In some embodiments, the adenosine deaminase comprises a mutation at position 597 of the amino acid sequence, which has an asparagine residue in the wild type sequence. In some embodiments, the asparagine residue at position 597 is replaced by a histidine residue (N597H). In some embodiments, the adenosine deaminase comprises a mutation at position 597 of the amino acid sequence, which has an asparagine residue in the wild type sequence. In some embodiments, the asparagine residue at position 597 is replaced by a glycine residue (N597G). In some embodiments, the adenosine deaminase comprises a mutation at position 597 of the amino acid sequence, which has an asparagine residue in the wild type sequence. In some embodiments, the asparagine residue at position 597 is replaced by a tyrosine residue (N597Y). In some embodiments, the asparagine residue at position 597 is replaced by a phenylalanine residue (N597F). In some embodiments, the adenosine deaminase comprises mutation N597I. In some embodiments, the adenosine deaminase comprises mutation N597L. In some embodiments, the adenosine deaminase comprises mutation N597V. In some embodiments, the adenosine deaminase comprises mutation N597M. In some embodiments, the adenosine deaminase comprises mutation N597C. In some embodiments, the adenosine deaminase comprises mutation N597P. In some embodiments, the adenosine deaminase comprises mutation N597T. In some embodiments, the adenosine deaminase comprises mutation N597S. In some embodiments, the adenosine deaminase comprises mutation N597W. In some embodiments, the adenosine deaminase comprises mutation N597Q. In some embodiments, the adenosine deaminase comprises mutation N597D. In certain example embodiments, the mutations at N597 described above are further made in the context of an E488Q background 
     In some embodiments, the adenosine deaminase comprises a mutation at serine599 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the serine residue at position 599 is replaced by a threonine residue (S599T). 
     In some embodiments, the adenosine deaminase comprises a mutation at asparagine613 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the asparagine residue at position 613 is replaced by a lysine residue (N613K). In some embodiments, the adenosine deaminase comprises a mutation at position 613 of the amino acid sequence, which has an asparagine residue in the wild type sequence. In some embodiments, the asparagine residue at position 613 is replaced by an arginine residue (N613R). In some embodiments, the adenosine deaminase comprises a mutation at position 613 of the amino acid sequence, which has an asparagine residue in the wild type sequence. In some embodiments, the asparagine residue at position 613 is replaced by an alanine residue (N613A) In some embodiments, the adenosine deaminase comprises a mutation at position 613 of the amino acid sequence, which has an asparagine residue in the wild type sequence. In some embodiments, the asparagine residue at position 613 is replaced by a glutamic acid residue (N613E). In some embodiments, the adenosine deaminase comprises mutation N613I. In some embodiments, the adenosine deaminase comprises mutation N613L. In some embodiments, the adenosine deaminase comprises mutation N613V. In some embodiments, the adenosine deaminase comprises mutation N613F. In some embodiments, the adenosine deaminase comprises mutation N613M. In some embodiments, the adenosine deaminase comprises mutation N613C. In some embodiments, the adenosine deaminase comprises mutation N613G. In some embodiments, the adenosine deaminase comprises mutation N613P. In some embodiments, the adenosine deaminase comprises mutation N613T. In some embodiments, the adenosine deaminase comprises mutation N613S. In some embodiments, the adenosine deaminase comprises mutation N613Y. In some embodiments, the adenosine deaminase comprises mutation N613W. In some embodiments, the adenosine deaminase comprises mutation N613Q. In some embodiments, the adenosine deaminase comprises mutation N613H. In some embodiments, the adenosine deaminase comprises mutation N613D. In some embodiments, the mutations at N613 described above are further made in combination with a E488Q mutation. 
     In some embodiments, to improve editing efficiency, the adenosine deaminase may comprise one or more of the mutations: G336D, G487A, G487V, E488Q, E488H, E488R, E488N, E488A, E488S, E488M, T490C, T490S, V493T, V493S, V493A, V493R, V493D, V493P, V493G, N597K, N597R, N597A, N597E, N597H, N597G, N597Y, A589V, S599T, N613K, N613R, N613A, N613E, based on amino acid sequence positions of hADAR2-D, and mutations in a homologous ADAR protein corresponding to the above. 
     In some embodiments, to reduce editing efficiency, the adenosine deaminase may comprise one or more of the mutations: E488F, E488L, E488W, T490A, T490F, T490Y, T490R, T490K, T490P, T490E, N597F, based on amino acid sequence positions of hADAR2-D, and mutations in a homologous ADAR protein corresponding to the above. In particular embodiments, it can be of interest to use an adenosine deaminase enzyme with reduced efficacy to reduce off-target effects. 
     In some embodiments, to reduce off-target effects, the adenosine deaminase comprises one or more of mutations at R348, V351, T375, K376, E396, C451, R455, N473, R474, K475, R477, R481, S486, E488, T490, S495, R510, based on amino acid sequence positions of hADAR2-D, and mutations in a homologous ADAR protein corresponding to the above. In some embodiments, the adenosine deaminase comprises mutation at E488 and one or more additional positions selected from R348, V351, T375, K376, E396, C451, R455, N473, R474, K475, R477, R481, S486, T490, S495, R510. In some embodiments, the adenosine deaminase comprises mutation at T375, and optionally at one or more additional positions. In some embodiments, the adenosine deaminase comprises mutation at N473, and optionally at one or more additional positions. In some embodiments, the adenosine deaminase comprises mutation at V351, and optionally at one or more additional positions. In some embodiments, the adenosine deaminase comprises mutation at E488 and T375, and optionally at one or more additional positions. In some embodiments, the adenosine deaminase comprises mutation at E488 and N473, and optionally at one or more additional positions. In some embodiments, the adenosine deaminase comprises mutation E488 and V351, and optionally at one or more additional positions. In some embodiments, the adenosine deaminase comprises mutation at E488 and one or more of T375, N473, and V351. 
     In some embodiments, to reduce off-target effects, the adenosine deaminase comprises one or more of mutations selected from R348E, V351L, T375G, T375S, R455G, R455S, R455E, N473D, R474E, K475Q, R477E, R481E, S486T, E488Q, T490A, T490S, S495T, and R510E, based on amino acid sequence positions of hADAR2-D, and mutations in a homologous ADAR protein corresponding to the above. In some embodiments, the adenosine deaminase comprises mutation E488Q and one or more additional mutations selected from R348E, V351L, T375G, T375S, R455G, R455S, R455E, N473D, R474E, K475Q, R477E, R481E, S486T, T490A, T490S, S495T, and R510E. In some embodiments, the adenosine deaminase comprises mutation T375G or T375S, and optionally one or more additional mutations. In some embodiments, the adenosine deaminase comprises mutation N473D, and optionally one or more additional mutations. In some embodiments, the adenosine deaminase comprises mutation V351L, and optionally one or more additional mutations. In some embodiments, the adenosine deaminase comprises mutation E488Q, and T375G or T375G, and optionally one or more additional mutations. In some embodiments, the adenosine deaminase comprises mutation E488Q and N473D, and optionally one or more additional mutations. In some embodiments, the adenosine deaminase comprises mutation E488Q and V351L, and optionally one or more additional mutations. In some embodiments, the adenosine deaminase comprises mutation E488Q and one or more of T375G/S, N473D and V351L. 
     In certain examples, the adenosine deaminase protein or catalytic domain thereof has been modified to comprise a mutation at E488, preferably E488Q, of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein and/or wherein the adenosine deaminase protein or catalytic domain thereof has been modified to comprise a mutation at T375, preferably T375G of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In certain examples, the adenosine deaminase protein or catalytic domain thereof has been modified to comprise a mutation at E1008, preferably E1008Q, of the hADAR1d amino acid sequence, or a corresponding position in a homologous ADAR protein. 
     Crystal structures of the human ADAR2 deaminase domain bound to duplex RNA reveal a protein loop that binds the RNA on the 5′ side of the modification site. This 5′ binding loop is one contributor to substrate specificity differences between ADAR family members. See Wang et al., Nucleic Acids Res., 44(20):9872-9880 (2016), the content of which is incorporated herein by reference in its entirety. In addition, an ADAR2-specific RNA-binding loop was identified near the enzyme active site. See Mathews et al., Nat. Struct. Mol. Biol., 23(5):426-33 (2016), the content of which is incorporated herein by reference in its entirety. In some embodiments, the adenosine deaminase comprises one or more mutations in the RNA binding loop to improve editing specificity and/or efficiency. 
     In some embodiments, the adenosine deaminase comprises a mutation at alanine454 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the alanine residue at position 454 is replaced by a serine residue (A454S). In some embodiments, the alanine residue at position 454 is replaced by a cysteine residue (A454C). In some embodiments, the alanine residue at position 454 is replaced by an aspartic acid residue (A454D). 
     In some embodiments, the adenosine deaminase comprises a mutation at arginine455 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the arginine residue at position 455 is replaced by an alanine residue (R455A). In some embodiments, the arginine residue at position 455 is replaced by a valine residue (R455V). In some embodiments, the arginine residue at position 455 is replaced by a histidine residue (R455H). In some embodiments, the arginine residue at position 455 is replaced by a glycine residue (R455G). In some embodiments, the arginine residue at position 455 is replaced by a serine residue (R455S). In some embodiments, the arginine residue at position 455 is replaced by a glutamic acid residue (R455E). In some embodiments, the adenosine deaminase comprises mutation R455C. In some embodiments, the adenosine deaminase comprises mutation R455I. In some embodiments, the adenosine deaminase comprises mutation R455K. In some embodiments, the adenosine deaminase comprises mutation R455L. In some embodiments, the adenosine deaminase comprises mutation R455M. In some embodiments, the adenosine deaminase comprises mutation R455N. In some embodiments, the adenosine deaminase comprises mutation R455Q. In some embodiments, the adenosine deaminase comprises mutation R455F. In some embodiments, the adenosine deaminase comprises mutation R455W. In some embodiments, the adenosine deaminase comprises mutation R455P. In some embodiments, the adenosine deaminase comprises mutation R455Y. In some embodiments, the adenosine deaminase comprises mutation R455E. In some embodiments, the adenosine deaminase comprises mutation R455D. In some embodiments, the mutations at R455 described above are further made in combination with a E488Q mutation. 
     In some embodiments, the adenosine deaminase comprises a mutation at isoleucine456 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the isoleucine residue at position 456 is replaced by a valine residue (I456V). In some embodiments, the isoleucine residue at position 456 is replaced by a leucine residue (I456L). In some embodiments, the isoleucine residue at position 456 is replaced by an aspartic acid residue (I456D). 
     In some embodiments, the adenosine deaminase comprises a mutation at phenylalanine457 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the phenylalanine residue at position 457 is replaced by a tyrosine residue (F457Y). In some embodiments, the phenylalanine residue at position 457 is replaced by an arginine residue (F457R). In some embodiments, the phenylalanine residue at position 457 is replaced by a glutamic acid residue (F457E). 
     In some embodiments, the adenosine deaminase comprises a mutation at serine458 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the serine residue at position 458 is replaced by a valine residue (S458V). In some embodiments, the serine residue at position 458 is replaced by a phenylalanine residue (S458F). In some embodiments, the serine residue at position 458 is replaced by a proline residue (S458P). In some embodiments, the adenosine deaminase comprises mutation S458I. In some embodiments, the adenosine deaminase comprises mutation S458L. In some embodiments, the adenosine deaminase comprises mutation S458M. In some embodiments, the adenosine deaminase comprises mutation S458C. In some embodiments, the adenosine deaminase comprises mutation S458A. In some embodiments, the adenosine deaminase comprises mutation S458G. In some embodiments, the adenosine deaminase comprises mutation S458T. In some embodiments, the adenosine deaminase comprises mutation S458Y. In some embodiments, the adenosine deaminase comprises mutation S458W. In some embodiments, the adenosine deaminase comprises mutation S458Q. In some embodiments, the adenosine deaminase comprises mutation S458N. In some embodiments, the adenosine deaminase comprises mutation S458H. In some embodiments, the adenosine deaminase comprises mutation S458E. In some embodiments, the adenosine deaminase comprises mutation S458D. In some embodiments, the adenosine deaminase comprises mutation S458K. In some embodiments, the adenosine deaminase comprises mutation S458R. In some embodiments, the mutations at S458 described above are further made in combination with a E488Q mutation. 
     In some embodiments, the adenosine deaminase comprises a mutation at proline459 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the proline residue at position 459 is replaced by a cysteine residue (P459C). In some embodiments, the proline residue at position 459 is replaced by a histidine residue (P459H). In some embodiments, the proline residue at position 459 is replaced by a tryptophan residue (P459W). 
     In some embodiments, the adenosine deaminase comprises a mutation at histidine460 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the histidine residue at position 460 is replaced by an arginine residue (H460R). In some embodiments, the histidine residue at position 460 is replaced by an isoleucine residue (H460I). In some embodiments, the histidine residue at position 460 is replaced by a proline residue (H460P). In some embodiments, the adenosine deaminase comprises mutation H460L. In some embodiments, the adenosine deaminase comprises mutation H460V. In some embodiments, the adenosine deaminase comprises mutation H460F. In some embodiments, the adenosine deaminase comprises mutation H460M. In some embodiments, the adenosine deaminase comprises mutation H460C. In some embodiments, the adenosine deaminase comprises mutation H460A. In some embodiments, the adenosine deaminase comprises mutation H460G. In some embodiments, the adenosine deaminase comprises mutation H460T. In some embodiments, the adenosine deaminase comprises mutation H460S. In some embodiments, the adenosine deaminase comprises mutation H460Y. In some embodiments, the adenosine deaminase comprises mutation H460W. In some embodiments, the adenosine deaminase comprises mutation H460Q. In some embodiments, the adenosine deaminase comprises mutation H460N. In some embodiments, the adenosine deaminase comprises mutation H460E. In some embodiments, the adenosine deaminase comprises mutation H460D. In some embodiments, the adenosine deaminase comprises mutation H460K. In some embodiments, the mutations at H460 described above are further made in combination with a E488Q mutation. 
     In some embodiments, the adenosine deaminase comprises a mutation at proline462 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the proline residue at position 462 is replaced by a serine residue (P462S). In some embodiments, the proline residue at position 462 is replaced by a tryptophan residue (P462W). In some embodiments, the proline residue at position 462 is replaced by a glutamic acid residue (P462E). 
     In some embodiments, the adenosine deaminase comprises a mutation at aspartic acid469 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the aspartic acid residue at position 469 is replaced by a glutamine residue (D469Q). In some embodiments, the aspartic acid residue at position 469 is replaced by a serine residue (D469S). In some embodiments, the aspartic acid residue at position 469 is replaced by a tyrosine residue (D469Y). 
     In some embodiments, the adenosine deaminase comprises a mutation at arginine470 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the arginine residue at position 470 is replaced by an alanine residue (R470A). In some embodiments, the arginine residue at position 470 is replaced by an isoleucine residue (R470I). In some embodiments, the arginine residue at position 470 is replaced by an aspartic acid residue (R470D). 
     In some embodiments, the adenosine deaminase comprises a mutation at histidine471 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the histidine residue at position 471 is replaced by a lysine residue (H471K). In some embodiments, the histidine residue at position 471 is replaced by a threonine residue (H471T). In some embodiments, the histidine residue at position 471 is replaced by a valine residue (H471V). 
     In some embodiments, the adenosine deaminase comprises a mutation at proline472 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the proline residue at position 472 is replaced by a lysine residue (P472K). In some embodiments, the proline residue at position 472 is replaced by a threonine residue (P472T). In some embodiments, the proline residue at position 472 is replaced by an aspartic acid residue (P472D). 
     In some embodiments, the adenosine deaminase comprises a mutation at asparagine473 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the asparagine residue at position 473 is replaced by an arginine residue (N473R). In some embodiments, the asparagine residue at position 473 is replaced by a tryptophan residue (N473W). In some embodiments, the asparagine residue at position 473 is replaced by a proline residue (N473P). In some embodiments, the asparagine residue at position 473 is replaced by an aspartic acid residue (N473D). 
     In some embodiments, the adenosine deaminase comprises a mutation at arginine 474 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the arginine residue at position 474 is replaced by a lysine residue (R474K). In some embodiments, the arginine residue at position 474 is replaced by a glycine residue (R474G). In some embodiments, the arginine residue at position 474 is replaced by an aspartic acid residue (R474D). In some embodiments, the arginine residue at position 474 is replaced by a glutamic acid residue (R474E). 
     In some embodiments, the adenosine deaminase comprises a mutation at lysine475 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the lysine residue at position 475 is replaced by a glutamine residue (K475Q). In some embodiments, the lysine residue at position 475 is replaced by an asparagine residue (K475N). In some embodiments, the lysine residue at position 475 is replaced by an aspartic acid residue (K475D). 
     In some embodiments, the adenosine deaminase comprises a mutation at alanine476 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the alanine residue at position 476 is replaced by a serine residue (A476S). In some embodiments, the alanine residue at position 476 is replaced by an arginine residue (A476R). In some embodiments, the alanine residue at position 476 is replaced by a glutamic acid residue (A476E). 
     In some embodiments, the adenosine deaminase comprises a mutation at arginine477 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the arginine residue at position 477 is replaced by a lysine residue (R477K). In some embodiments, the arginine residue at position 477 is replaced by a threonine residue (R477T). In some embodiments, the arginine residue at position 477 is replaced by a phenylalanine residue (R477F). In some embodiments, the arginine residue at position 474 is replaced by a glutamic acid residue (R477E). 
     In some embodiments, the adenosine deaminase comprises a mutation at glycine478 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the glycine residue at position 478 is replaced by an alanine residue (G478A). In some embodiments, the glycine residue at position 478 is replaced by an arginine residue (G478R). In some embodiments, the glycine residue at position 478 is replaced by a tyrosine residue (G478Y). In some embodiments, the adenosine deaminase comprises mutation G4781. In some embodiments, the adenosine deaminase comprises mutation G478L. In some embodiments, the adenosine deaminase comprises mutation G478V. In some embodiments, the adenosine deaminase comprises mutation G478F. In some embodiments, the adenosine deaminase comprises mutation G478M. In some embodiments, the adenosine deaminase comprises mutation G478C. In some embodiments, the adenosine deaminase comprises mutation G478P. In some embodiments, the adenosine deaminase comprises mutation G478T. In some embodiments, the adenosine deaminase comprises mutation G478S. In some embodiments, the adenosine deaminase comprises mutation G478W. In some embodiments, the adenosine deaminase comprises mutation G478Q. In some embodiments, the adenosine deaminase comprises mutation G478N. In some embodiments, the adenosine deaminase comprises mutation G478H. In some embodiments, the adenosine deaminase comprises mutation G478E. In some embodiments, the adenosine deaminase comprises mutation G478D. In some embodiments, the adenosine deaminase comprises mutation G478K. In some embodiments, the mutations at G478 described above are further made in combination with a E488Q mutation. 
     In some embodiments, the adenosine deaminase comprises a mutation at glutamine479 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the glutamine residue at position 479 is replaced by an asparagine residue (Q479N). In some embodiments, the glutamine residue at position 479 is replaced by a serine residue (Q479S). In some embodiments, the glutamine residue at position 479 is replaced by a proline residue (Q479P). 
     In some embodiments, the adenosine deaminase comprises a mutation at arginine348 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the arginine residue at position 348 is replaced by an alanine residue (R348A). In some embodiments, the arginine residue at position 348 is replaced by a glutamic acid residue (R348E). 
     In some embodiments, the adenosine deaminase comprises a mutation at valine351 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the valine residue at position 351 is replaced by a leucine residue (V351L). In some embodiments, the adenosine deaminase comprises mutation V351Y. In some embodiments, the adenosine deaminase comprises mutation V351M. In some embodiments, the adenosine deaminase comprises mutation V351T. In some embodiments, the adenosine deaminase comprises mutation V351G. In some embodiments, the adenosine deaminase comprises mutation V351A. In some embodiments, the adenosine deaminase comprises mutation V351F. In some embodiments, the adenosine deaminase comprises mutation V351E. In some embodiments, the adenosine deaminase comprises mutation V351I. In some embodiments, the adenosine deaminase comprises mutation V351C. In some embodiments, the adenosine deaminase comprises mutation V351H. In some embodiments, the adenosine deaminase comprises mutation V351P. In some embodiments, the adenosine deaminase comprises mutation V351S. In some embodiments, the adenosine deaminase comprises mutation V351K. In some embodiments, the adenosine deaminase comprises mutation V351N. In some embodiments, the adenosine deaminase comprises mutation V351W. In some embodiments, the adenosine deaminase comprises mutation V351Q. In some embodiments, the adenosine deaminase comprises mutation V351D. In some embodiments, the adenosine deaminase comprises mutation V351R. In some embodiments, the mutations at V351 described above are further made in combination with a E488Q mutation. 
     In some embodiments, the adenosine deaminase comprises a mutation at threonine375 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the threonine residue at position 375 is replaced by a glycine residue (T375G). In some embodiments, the threonine residue at position 375 is replaced by a serine residue (T375S). In some embodiments, the adenosine deaminase comprises mutation T375H. In some embodiments, the adenosine deaminase comprises mutation T375Q. In some embodiments, the adenosine deaminase comprises mutation T375C. In some embodiments, the adenosine deaminase comprises mutation T375N. In some embodiments, the adenosine deaminase comprises mutation T375M. In some embodiments, the adenosine deaminase comprises mutation T375A. In some embodiments, the adenosine deaminase comprises mutation T375W. In some embodiments, the adenosine deaminase comprises mutation T375V. In some embodiments, the adenosine deaminase comprises mutation T375R. In some embodiments, the adenosine deaminase comprises mutation T375E. In some embodiments, the adenosine deaminase comprises mutation T375K. In some embodiments, the adenosine deaminase comprises mutation T375F. In some embodiments, the adenosine deaminase comprises mutation T375I. In some embodiments, the adenosine deaminase comprises mutation T375D. In some embodiments, the adenosine deaminase comprises mutation T375P. In some embodiments, the adenosine deaminase comprises mutation T375L. In some embodiments, the adenosine deaminase comprises mutation T375Y. In some embodiments, the mutations at T375Y described above are further made in combination with an E488Q mutation. 
     In some embodiments, the adenosine deaminase comprises a mutation at Arg481 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the arginine residue at position 481 is replaced by a glutamic acid residue (R481E). 
     In some embodiments, the adenosine deaminase comprises a mutation at Ser486 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the serine residue at position 486 is replaced by a threonine residue (S486T). 
     In some embodiments, the adenosine deaminase comprises a mutation at Thr490 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the threonine residue at position 490 is replaced by an alanine residue (T490A). In some embodiments, the threonine residue at position 490 is replaced by a serine residue (T490S). 
     In some embodiments, the adenosine deaminase comprises a mutation at Ser495 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the serine residue at position 495 is replaced by a threonine residue (S495T). 
     In some embodiments, the adenosine deaminase comprises a mutation at Arg510 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the arginine residue at position 510 is replaced by a glutamine residue (R510Q). In some embodiments, the arginine residue at position 510 is replaced by an alanine residue (R510A). In some embodiments, the arginine residue at position 510 is replaced by a glutamic acid residue (R510E). 
     In some embodiments, the adenosine deaminase comprises a mutation at Gly593 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the glycine residue at position 593 is replaced by an alanine residue (G593A). In some embodiments, the glycine residue at position 593 is replaced by a glutamic acid residue (G593E). 
     In some embodiments, the adenosine deaminase comprises a mutation at Lys594 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the lysine residue at position 594 is replaced by an alanine residue (K594A). 
     In some embodiments, the adenosine deaminase comprises a mutation at any one or more of positions A454, R455, 1456, F457, S458, P459, H460, P462, D469, R470, H471, P472, N473, R474, K475, A476, R477, G478, Q479, R348, R510, G593, K594 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. 
     In some embodiments, the adenosine deaminase comprises any one or more of mutations A454S, A454C, A454D, R455A, R455V, R455H, I456V, I456L, I456D, F457Y, F457R, F457E, S458V, S458F, S458P, P459C, P459H, P459W, H460R, H460I, H460P, P462S, P462W, P462E, D469Q, D469S, D469Y, R470A, R470I, R470D, H471K, H471T, H471V, P472K, P472T, P472D, N473R, N473W, N473P, R474K, R474G, R474D, K475Q, K475N, K475D, A476S, A476R, A476E, R477K, R477T, R477F, G478A, G478R, G478Y, Q479N, Q479S, Q479P, R348A, R510Q, R510A, G593A, G593E, K594A of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. 
     In some embodiments, the adenosine deaminase comprises a mutation at any one or more of positions T375, V351, G478, S458, H460 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein, optionally in combination a mutation at E488. In some embodiments, the adenosine deaminase comprises one or more of mutations selected from T375G, T375C, T375H, T375Q, V351M, V351T, V351Y, G478R, S458F, H460I, optionally in combination with E488Q. 
     In some embodiments, the adenosine deaminase comprises one or more of mutations selected from T375H, T375Q, V351M, V351Y, H460P, optionally in combination with E488Q. 
     In some embodiments, the adenosine deaminase comprises mutations T375S and S458F, optionally in combination with E488Q. 
     In some embodiments, the adenosine deaminase comprises a mutation at two or more of positions T375, N473, R474, G478, S458, P459, V351, R455, R455, T490, R348, Q479 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein, optionally in combination a mutation at E488. In some embodiments, the adenosine deaminase comprises two or more of mutations selected from T375G, T375S, N473D, R474E, G478R, S458F, P459W, V351L, R455G, R455S, T490A, R348E, Q479P, optionally in combination with E488Q. 
     In some embodiments, the adenosine deaminase comprises mutations T375G and V351L. In some embodiments, the adenosine deaminase comprises mutations T375G and R455G. In some embodiments, the adenosine deaminase comprises mutations T375G and R455S. In some embodiments, the adenosine deaminase comprises mutations T375G and T490A. In some embodiments, the adenosine deaminase comprises mutations T375G and R348E. In some embodiments, the adenosine deaminase comprises mutations T375S and V351L. In some embodiments, the adenosine deaminase comprises mutations T375S and R455G. In some embodiments, the adenosine deaminase comprises mutations T375S and R455S. In some embodiments, the adenosine deaminase comprises mutations T375S and T490A. In some embodiments, the adenosine deaminase comprises mutations T375S and R348E. In some embodiments, the adenosine deaminase comprises mutations N473D and V351L. In some embodiments, the adenosine deaminase comprises mutations N473D and R455G. In some embodiments, the adenosine deaminase comprises mutations N473D and R455S. In some embodiments, the adenosine deaminase comprises mutations N473D and T490A. In some embodiments, the adenosine deaminase comprises mutations N473D and R348E. In some embodiments, the adenosine deaminase comprises mutations R474E and V351L. In some embodiments, the adenosine deaminase comprises mutations R474E and R455G. In some embodiments, the adenosine deaminase comprises mutations R474E and R455S. In some embodiments, the adenosine deaminase comprises mutations R474E and T490A. In some embodiments, the adenosine deaminase comprises mutations R474E and R348E. In some embodiments, the adenosine deaminase comprises mutations S458F and T375G. In some embodiments, the adenosine deaminase comprises mutations S458F and T375S. In some embodiments, the adenosine deaminase comprises mutations S458F and N473D. In some embodiments, the adenosine deaminase comprises mutations S458F and R474E. In some embodiments, the adenosine deaminase comprises mutations S458F and G478R. In some embodiments, the adenosine deaminase comprises mutations G478R and T375G. In some embodiments, the adenosine deaminase comprises mutations G478R and T375S. In some embodiments, the adenosine deaminase comprises mutations G478R and N473D. In some embodiments, the adenosine deaminase comprises mutations G478R and R474E. In some embodiments, the adenosine deaminase comprises mutations P459W and T375G. In some embodiments, the adenosine deaminase comprises mutations P459W and T375S. In some embodiments, the adenosine deaminase comprises mutations P459W and N473D. In some embodiments, the adenosine deaminase comprises mutations P459W and R474E. In some embodiments, the adenosine deaminase comprises mutations P459W and G478R. In some embodiments, the adenosine deaminase comprises mutations P459W and S458F. In some embodiments, the adenosine deaminase comprises mutations Q479P and T375G. In some embodiments, the adenosine deaminase comprises mutations Q479P and T375S. In some embodiments, the adenosine deaminase comprises mutations Q479P and N473D. In some embodiments, the adenosine deaminase comprises mutations Q479P and R474E. In some embodiments, the adenosine deaminase comprises mutations Q479P and G478R. In some embodiments, the adenosine deaminase comprises mutations Q479P and S458F. In some embodiments, the adenosine deaminase comprises mutations Q479P and P459W. All mutations described in this paragraph may also further be made in combination with a E488Q mutations. 
     In some embodiments, the adenosine deaminase comprises a mutation at any one or more of positions K475, Q479, P459, G478, S458 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein, optionally in combination a mutation at E488. In some embodiments, the adenosine deaminase comprises one or more of mutations selected from K475N, Q479N, P459W, G478R, S458P, S458F, optionally in combination with E488Q. 
     In some embodiments, the adenosine deaminase comprises a mutation at any one or more of positions T375, V351, R455, H460, A476 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein, optionally in combination a mutation at E488. In some embodiments, the adenosine deaminase comprises one or more of mutations selected from T375G, T375C, T375H, T375Q, V351M, V351T, V351Y, R455H, H460P, H460I, A476E, optionally in combination with E488Q. 
     ADAR has been known to demonstrate a preference for neighboring nucleotides on either side of the edited A (www.nature.com/nsmb/journal/v23/n5/full/nsmb.3203.html, Matthews et al. (2017), Nature Structural Mol Biol, 23(5): 426-433, incorporated herein by reference in its entirety). Accordingly, in certain embodiments, the gRNA, target, and/or ADAR is selected optimized for motif preference. 
     Intentional mismatches have been demonstrated in vitro to allow for editing of non-preferred motifs (https://academic.oup.com/nar/article-lookup/doi/10.1093/nar/gku272; Schneider et al (2014), Nucleic Acid Res, 42(10):e87); Fukuda et al. (2017), Scientific Reports, 7, doi:10.1038/srep41478, incorporated herein by reference in its entirety). Accordingly, in certain embodiments, to enhance RNA editing efficiency on non-preferred 5′ or 3′ neighboring bases, intentional mismatches in neighboring bases are introduced. 
     In some embodiments, the adenosine deaminase may be a tRNA-specific adenosine deaminase or a variant thereof. In some embodiments, the adenosine deaminase may comprise one or more of the mutations: W23L, W23R, R26G, H36L, N37S, P48S, P48T, P48A, I49V, R51L, N72D, L84F, S97C, A106V, D108N, H123Y, G125A, A142N, S146C, D147Y, R152H, R152P, E155V, I156F, K157N, K161T, based on amino acid sequence positions of  E. coli  TadA, and mutations in a homologous deaminase protein corresponding to the above. In some embodiments, the adenosine deaminase may comprise one or more of the mutations: D108N based on amino acid sequence positions of  E. coli  TadA, and mutations in a homologous deaminase protein corresponding to the above. In some embodiments, the adenosine deaminase may comprise one or more of the mutations: A106V, D108N, based on amino acid sequence positions of  E. coli  TadA, and mutations in a homologous deaminase protein corresponding to the above. In some embodiments, the adenosine deaminase may comprise one or more of the mutations: A106V, D108N, D147Y, E155V, based on amino acid sequence positions of  E. coli  TadA, and mutations in a homologous deaminase protein corresponding to the above. In some embodiments, the adenosine deaminase may comprise one or more of the mutations: A106V, D108N, based on amino acid sequence positions of  E. coli  TadA, and mutations in a homologous deaminase protein corresponding to the above. In some embodiments, the adenosine deaminase may comprise one or more of the mutations: A106V, D108N, D147Y, E155V, L84F, H123Y, I156F, based on amino acid sequence positions of  E. coli  TadA, and mutations in a homologous deaminase protein corresponding to the above. In some embodiments, the adenosine deaminase may comprise one or more of the mutations: A106V, D108N, D147Y, E155V, L84F, H123Y, I156F, A142N, based on amino acid sequence positions of  E. coli  TadA, and mutations in a homologous deaminase protein corresponding to the above. In some embodiments, the adenosine deaminase may comprise one or more of the mutations: A106V, D108N, D147Y, E155V, L84F, H123Y, I156F, H36L, R51L, S146C, K157N, based on amino acid sequence positions of  E. coli  TadA, and mutations in a homologous deaminase protein corresponding to the above. In some embodiments, the adenosine deaminase may comprise one or more of the mutations: A106V, D108N, D147Y, E155V, L84F, H123Y, I156F, H36L, R51L, S146C, K157N, P48S, based on amino acid sequence positions of  E. coli  TadA, and mutations in a homologous deaminase protein corresponding to the above. In some embodiments, the adenosine deaminase may comprise one or more of the mutations: A106V, D108N, D147Y, E155V, L84F, H123Y, I156F, H36L, R51L, S146C, K157N, P48S, A142N, based on amino acid sequence positions of  E. coli  TadA, and mutations in a homologous deaminase protein corresponding to the above. In some embodiments, the adenosine deaminase may comprise one or more of the mutations: A106V, D108N, D147Y, E155V, L84F, H123Y, I156F, H36L, R51L, S146C, K157N, P48S, W23R, P48A, based on amino acid sequence positions of  E. coli  TadA, and mutations in a homologous deaminase protein corresponding to the above. In some embodiments, the adenosine deaminase may comprise one or more of the mutations: A106V, D108N, D147Y, E155V, L84F, H123Y, I156F, H36L, R51L, S146C, K157N, P48S, W23R, P48A, A142N, based on amino acid sequence positions of  E. coli  TadA, and mutations in a homologous deaminase protein corresponding to the above. In some embodiments, the adenosine deaminase may comprise one or more of the mutations: A106V, D108N, D147Y, E155V, L84F, H123Y, I156F, H36L, R51L, S146C, K157N, P48S, W23R, P48A, R152P, based on amino acid sequence positions of  E. coli  TadA, and mutations in a homologous deaminase protein corresponding to the above. In some embodiments, the adenosine deaminase may comprise one or more of the mutations: A106V, D108N, D147Y, E155V, L84F, H123Y, I156F, H36L, R51L, S146C, K157N, P48S, W23R, P48A, R152P, A142N, based on amino acid sequence positions of  E. coli  TadA, and mutations in a homologous deaminase protein corresponding to the above. 
     A&#39;s opposite C&#39;s in the targeting window of the ADAR deaminase domain can be preferentially edited over other bases. Additionally, A&#39;s base-paired with U&#39;s within a few bases of the targeted base can have low levels of editing by CRISPR-Cas-ADAR fusions, suggesting that there is flexibility for the enzyme to edit multiple A&#39;s. These two observations suggest that multiple A&#39;s in the activity window of CRISPR-Cas-ADAR fusions could be specified for editing by mismatching all A&#39;s to be edited with C&#39;s. Accordingly, in certain embodiments, multiple A:C mismatches in the activity window are designed to create multiple A:I edits. In certain embodiments, to suppress potential off-target editing in the activity window, non-target A&#39;s are paired with A&#39;s or G&#39;s. 
     The terms “editing specificity” and “editing preference” are used interchangeably herein to refer to the extent of A-to-I editing at a particular adenosine site in a double-stranded substrate. In some embodiment, the substrate editing preference is determined by the 5′ nearest neighbor and/or the 3′ nearest neighbor of the target adenosine residue. In some embodiments, the adenosine deaminase has preference for the 5′ nearest neighbor of the substrate ranked as U&gt;A&gt;C&gt;G (“&gt;” indicates greater preference). In some embodiments, the adenosine deaminase has preference for the 3′ nearest neighbor of the substrate ranked as G&gt;C˜A&gt;U (“&gt;” indicates greater preference; “˜” indicates similar preference). In some embodiments, the adenosine deaminase has preference for the 3′ nearest neighbor of the substrate ranked as G&gt;C&gt;U˜A (“&gt;” indicates greater preference; “˜” indicates similar preference). In some embodiments, the adenosine deaminase has preference for the 3′ nearest neighbor of the substrate ranked as G&gt;C&gt;A&gt;U (“&gt;” indicates greater preference). In some embodiments, the adenosine deaminase has preference for the 3′ nearest neighbor of the substrate ranked as C˜G˜A&gt;U (“&gt;” indicates greater preference; “˜” indicates similar preference). In some embodiments, the adenosine deaminase has preference for a triplet sequence containing the target adenosine residue ranked as TAG&gt;AAG&gt;CAC&gt;AAT&gt;GAA&gt;GAC (“&gt;” indicates greater preference), the center A being the target adenosine residue. 
     In some embodiments, the substrate editing preference of an adenosine deaminase is affected by the presence or absence of a nucleic acid binding domain in the adenosine deaminase protein. In some embodiments, to modify substrate editing preference, the deaminase domain is connected with a double-strand RNA binding domain (dsRBD) or a double-strand RNA binding motif (dsRBM). In some embodiments, the dsRBD or dsRBM may be derived from an ADAR protein, such as hADAR1 or hADAR2. In some embodiments, a full-length ADAR protein that comprises at least one dsRBD and a deaminase domain is used. In some embodiments, the one or more dsRBM or dsRBD is at the N-terminus of the deaminase domain. In other embodiments, the one or more dsRBM or dsRBD is at the C-terminus of the deaminase domain. 
     In some embodiments, the substrate editing preference of an adenosine deaminase is affected by amino acid residues near or in the active center of the enzyme. In some embodiments, to modify substrate editing preference, the adenosine deaminase may comprise one or more of the mutations: G336D, G487R, G487K, G487W, G487Y, E488Q, E488N, T490A, V493A, V493T, V493S, N597K, N597R, A589V, S599T, N613K, N613R, based on amino acid sequence positions of hADAR2-D, and mutations in a homologous ADAR protein corresponding to the above. 
     Particularly, in some embodiments, to reduce editing specificity, the adenosine deaminase can comprise one or more of mutations E488Q, V493A, N597K, N613K, based on amino acid sequence positions of hADAR2-D, and mutations in a homologous ADAR protein corresponding to the above. In some embodiments, to increase editing specificity, the adenosine deaminase can comprise mutation T490A. 
     In some embodiments, to increase editing preference for target adenosine (A) with an immediate 5′ G, such as substrates comprising the triplet sequence GAC, the center A being the target adenosine residue, the adenosine deaminase can comprise one or more of mutations G336D, E488Q, E488N, V493T, V493S, V493A, A589V, N597K, N597R, S599T, N613K, N613R, based on amino acid sequence positions of hADAR2-D, and mutations in a homologous ADAR protein corresponding to the above. 
     Particularly, in some embodiments, the adenosine deaminase comprises mutation E488Q or a corresponding mutation in a homologous ADAR protein for editing substrates comprising the following triplet sequences: GAC, GAA, GAU, GAG, CAU, AAU, UAC, the center A being the target adenosine residue. 
     In some embodiments, the adenosine deaminase comprises the wild-type amino acid sequence of hADAR1-D. In some embodiments, the adenosine deaminase comprises one or more mutations in the hADAR1-D sequence, such that the editing efficiency, and/or substrate editing preference of hADAR1-D is changed according to specific needs. 
     In some embodiments, the adenosine deaminase comprises a mutation at Glycine1007 of the hADAR1-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the glycine residue at position 1007 is replaced by a non-polar amino acid residue with relatively small side chains. For example, in some embodiments, the glycine residue at position 1007 is replaced by an alanine residue (G1007A). In some embodiments, the glycine residue at position 1007 is replaced by a valine residue (G1007V). In some embodiments, the glycine residue at position 1007 is replaced by an amino acid residue with relatively large side chains. In some embodiments, the glycine residue at position 1007 is replaced by an arginine residue (G1007R). In some embodiments, the glycine residue at position 1007 is replaced by a lysine residue (G1007K). In some embodiments, the glycine residue at position 1007 is replaced by a tryptophan residue (G1007W). In some embodiments, the glycine residue at position 1007 is replaced by a tyrosine residue (G1007Y). Additionally, in other embodiments, the glycine residue at position 1007 is replaced by a leucine residue (G1007L). In other embodiments, the glycine residue at position 1007 is replaced by a threonine residue (G1007T). In other embodiments, the glycine residue at position 1007 is replaced by a serine residue (G1007S). 
     In some embodiments, the adenosine deaminase comprises a mutation at glutamic acid1008 of the hADAR1-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the glutamic acid residue at position 1008 is replaced by a polar amino acid residue having a relatively large side chain. In some embodiments, the glutamic acid residue at position 1008 is replaced by a glutamine residue (E1008Q). In some embodiments, the glutamic acid residue at position 1008 is replaced by a histidine residue (E1008H). In some embodiments, the glutamic acid residue at position 1008 is replaced by an arginine residue (E1008R). In some embodiments, the glutamic acid residue at position 1008 is replaced by a lysine residue (E1008K). In some embodiments, the glutamic acid residue at position 1008 is replaced by a nonpolar or small polar amino acid residue. In some embodiments, the glutamic acid residue at position 1008 is replaced by a phenylalanine residue (E1008F). In some embodiments, the glutamic acid residue at position 1008 is replaced by a tryptophan residue (E1008W). In some embodiments, the glutamic acid residue at position 1008 is replaced by a glycine residue (E1008G). In some embodiments, the glutamic acid residue at position 1008 is replaced by an isoleucine residue (E10081). In some embodiments, the glutamic acid residue at position 1008 is replaced by a valine residue (E1008V). In some embodiments, the glutamic acid residue at position 1008 is replaced by a proline residue (E1008P). In some embodiments, the glutamic acid residue at position 1008 is replaced by a serine residue (E1008S). In other embodiments, the glutamic acid residue at position 1008 is replaced by an asparagine residue (E1008N). In other embodiments, the glutamic acid residue at position 1008 is replaced by an alanine residue (E1008A). In other embodiments, the glutamic acid residue at position 1008 is replaced by a Methionine residue (E1008M). In some embodiments, the glutamic acid residue at position 1008 is replaced by a leucine residue (E1008L). 
     In some embodiments, to improve editing efficiency, the adenosine deaminase may comprise one or more of the mutations: E1007S, E1007A, E1007V, E1008Q, E1008R, E1008H, E1008M, E1008N, E1008K, based on amino acid sequence positions of hADAR1-D, and mutations in a homologous ADAR protein corresponding to the above. 
     In some embodiments, to reduce editing efficiency, the adenosine deaminase may comprise one or more of the mutations: E1007R, E1007K, E1007Y, E1007L, E1007T, E1008G, E10081, E1008P, E1008V, E1008F, E1008W, E1008S, E1008N, E1008K, based on amino acid sequence positions of hADAR1-D, and mutations in a homologous ADAR protein corresponding to the above. 
     In some embodiments, the substrate editing preference, efficiency and/or selectivity of an adenosine deaminase is affected by amino acid residues near or in the active center of the enzyme. In some embodiments, the adenosine deaminase comprises a mutation at the glutamic acid 1008 position in hADAR1-D sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the mutation is E1008R, or a corresponding mutation in a homologous ADAR protein. In some embodiments, the E1008R mutant has an increased editing efficiency for target adenosine residue that has a mismatched G residue on the opposite strand. 
     In some embodiments, the adenosine deaminase protein further comprises or is connected to one or more double-stranded RNA (dsRNA) binding motifs (dsRBMs) or domains (dsRBDs) for recognizing and binding to double-stranded nucleic acid substrates. In some embodiments, the interaction between the adenosine deaminase and the double-stranded substrate is mediated by one or more additional proteins, including a CRISPR/CAS protein described elsewhere herein, including but not limited to one or more Cas-like (e.g. Cas9-like and/or Cas12-like) proteins. In some embodiments, the interaction between the adenosine deaminase and the double-stranded substrate is further mediated by one or more nucleic acid component(s), including a guide RNA. 
     In certain example embodiments, directed evolution may be used to design modified ADAR proteins capable of catalyzing additional reactions besides deamination of an adenine to a hypoxanthine. 
     Modified Adenosine Deaminase Having C to U Deamination Activity 
     In certain example embodiments, directed evolution may be used to design modified ADAR proteins capable of catalyzing additional reactions besides deamination of an adenine to a hypoxanthine. For example, the modified ADAR protein may be capable of catalyzing deamination of a cytidine to a uracil. While not bound by a particular theory, mutations that improve C to U activity may alter the shape of the binding pocket to be more amenable to the smaller cytidine base. 
     In certain embodiments the adenosine deaminase is engineered to convert the activity to cytidine deaminase. Such engineered adenosine deaminase may also retain its adenosine deaminase activity, i.e., such mutated adenosine deaminase may have both adenosine deaminase and cytidine deaminase activities. Accordingly, in some embodiments, the adenosine deaminase comprises one or more mutations in positions selected from E396, C451, V351, R455, T375, K376, S486, Q488, R510, K594, R348, G593, 5397, H443, L444, Y445, F442, E438, T448, A353, V355, T339, P539, T339, P539, V525 I520, P462 and N579. In particular embodiments, the adenosine deaminase comprises one or more mutations in a position selected from V351, L444, V355, V525 and I520. In some embodiments, the adenosine deaminase may comprise one or more of mutations at E488, V351, S486, T375, S370, P462, N597, based on amino acid sequence positions of hADAR2-D, and mutations in a homologous ADAR protein corresponding to the above. 
     In some embodiments, the adenosine deaminase may comprise one or more of the mutations: E488Q based on amino acid sequence positions of hADAR2-D, and mutations in a homologous ADAR protein corresponding to the above. In some embodiments, the adenosine deaminase may comprise one or more of the mutations: E488Q, V351G, based on amino acid sequence positions of hADAR2-D, and mutations in a homologous ADAR protein corresponding to the above. In some embodiments, the adenosine deaminase may comprise one or more of the mutations: E488Q, V351G, S486A, based on amino acid sequence positions of hADAR2-D, and mutations in a homologous ADAR protein corresponding to the above. In some embodiments, the adenosine deaminase may comprise one or more of the mutations: E488Q, V351G, S486A, T375S, based on amino acid sequence positions of hADAR2-D, and mutations in a homologous ADAR protein corresponding to the above. In some embodiments, the adenosine deaminase may comprise one or more of the mutations: E488Q, V351G, S486A, T375S, S370C, based on amino acid sequence positions of hADAR2-D, and mutations in a homologous ADAR protein corresponding to the above. In some embodiments, the adenosine deaminase may comprise one or more of the mutations: E488Q, V351G, S486A, T375S, S370C, P462A, based on amino acid sequence positions of hADAR2-D, and mutations in a homologous ADAR protein corresponding to the above. In some embodiments, the adenosine deaminase may comprise one or more of the mutations: E488Q, V351G, S486A, T375S, S370C, P462A, N597I, based on amino acid sequence positions of hADAR2-D, and mutations in a homologous ADAR protein corresponding to the above. In some embodiments, the adenosine deaminase may comprise one or more of the mutations: E488Q, V351G, S486A, T375S, S370C, P462A, N597I, L332I, based on amino acid sequence positions of hADAR2-D, and mutations in a homologous ADAR protein corresponding to the above. In some embodiments, the adenosine deaminase may comprise one or more of the mutations: E488Q, V351G, S486A, T375S, S370C, P462A, N597I, L332I, I398V, based on amino acid sequence positions of hADAR2-D, and mutations in a homologous ADAR protein corresponding to the above. In some embodiments, the adenosine deaminase may comprise one or more of the mutations: E488Q, V351G, S486A, T375S, S370C, P462A, N597I, L332I, I398V, K350I, based on amino acid sequence positions of hADAR2-D, and mutations in a homologous ADAR protein corresponding to the above. In some embodiments, the adenosine deaminase may comprise one or more of the mutations: E488Q, V351G, S486A, T375S, S370C, P462A, N597I, L332I, I398V, K350I, M383L, based on amino acid sequence positions of hADAR2-D, and mutations in a homologous ADAR protein corresponding to the above. In some embodiments, the adenosine deaminase may comprise one or more of the mutations: E488Q, V351G, S486A, T375S, S370C, P462A, N597I, L332I, I398V, K350I, M383L, D619G, based on amino acid sequence positions of hADAR2-D, and mutations in a homologous ADAR protein corresponding to the above. In some embodiments, the adenosine deaminase may comprise one or more of the mutations: E488Q, V351G, S486A, T375S, S370C, P462A, N597I, L332I, I398V, K350I, M383L, D619G, S582T, based on amino acid sequence positions of hADAR2-D, and mutations in a homologous ADAR protein corresponding to the above. In some embodiments, the adenosine deaminase may comprise one or more of the mutations: E488Q, V351G, S486A, T375S, S370C, P462A, N597I, L332I, I398V, K350I, M383L, D619G, S582T, V440I based on amino acid sequence positions of hADAR2-D, and mutations in a homologous ADAR protein corresponding to the above. In some embodiments, the adenosine deaminase may comprise one or more of the mutations: E488Q, V351G, S486A, T375S, S370C, P462A, N597I, L332I, I398V, K350I, M383L, D619G, S582T, V440I, S495N based on amino acid sequence positions of hADAR2-D, and mutations in a homologous ADAR protein corresponding to the above. In some embodiments, the adenosine deaminase may comprise one or more of the mutations: E488Q, V351G, S486A, T375S, S370C, P462A, N597I, L332I, I398V, K350I, M383L, D619G, S582T, V440I, S495N, K418E based on amino acid sequence positions of hADAR2-D, and mutations in a homologous ADAR protein corresponding to the above. In some embodiments, the adenosine deaminase may comprise one or more of the mutations: E488Q, V351G, S486A, T375S, S370C, P462A, N597I, L332I, I398V, K350I, M383L, D619G, S582T, V440I, S495N, K418E, S661T based on amino acid sequence positions of hADAR2-D, and mutations in a homologous ADAR protein corresponding to the above. In some examples, provided herein includes a mutated adenosine deaminase e.g., an adenosine deaminase comprising one or more mutations of E488Q, V351G, S486A, T375S, S370C, P462A, N597I, L332I, I398V, K350I, M383L, D619G, S582T, V440I, S495N, K418E, S661T, fused with a CRISPR-Cas protein (e.g. a Cas-like protein (e.g. Cas9-like and/or Cas12-like), dead CRISPR-Cas protein and/or CRISPR-Cas nickase) described elsewhere herein. In a particular example, provided herein includes a mutated adenosine deaminase e.g., an adenosine deaminase comprising E488Q, V351G, S486A, T375S, S370C, P462A, N597I, L332I, I398V, K350I, M383L, D619G, S582T, V440I, S495N, K418E, and S661T, fused with a CRISPR-Cas protein (e.g. a Cas-like protein (e.g. Cas9-like and/or Cas12-like), dead CRISPR-Cas protein and/or CRISPR-Cas nickase) described elsewhere herein. 
     In some embodiments, the modified adenosine deaminase having C-to-U deamination activity comprises a mutation at any one or more of positions V351, T375, R455, and E488 of the hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein. In some embodiments, the adenosine deaminase comprises mutation E488Q. In some embodiments, the adenosine deaminase comprises one or more of mutations selected from V351I, V351L, V351F, V351M, V351C, V351A, V351G, V351P, V351T, V351S, V351Y, V351W, V351Q, V351N, V351H, V351E, V351D, V351K, V351R, T375I, T375L, T375V, T375F, T375M, T375C, T375A, T375G, T375P, T375S, T375Y, T375W, T375Q, T375N, T375H, T375E, T375D, T375K, T375R, R455I, R455L, R455V, R455F, R455M, R455C, R455A, R455G, R455P, R455T, R455S, R455Y, R455W, R455Q, R455N, R455H, R455E, R455D, R455K. In some embodiments, the adenosine deaminase comprises mutation E488Q, and further comprises one or more of mutations selected from V351I, V351L, V351F, V351M, V351C, V351A, V351G, V351P, V351T, V351S, V351Y, V351W, V351Q, V351N, V351H, V351E, V351D, V351K, V351R, T375I, T375L, T375V, T375F, T375M, T375C, T375A, T375G, T375P, T375S, T375Y, T375W, T375Q, T375N, T375H, T375E, T375D, T375K, T375R, R455I, R455L, R455V, R455F, R455M, R455C, R455A, R455G, R455P, R455T, R455S, R455Y, R455W, R455Q, R455N, R455H, R455E, R455D, R455K. 
     In connection with the aforementioned deaminases, including modified ADAR proteins having C-to-U deamination activity, the invention described herein also relates to a method for deaminating a C in a target RNA sequence of interest, comprising delivering to a target RNA or DNA an AD-functionalized composition disclosed herein. 
     In certain example embodiments, the method for deaminating a C in a target RNA sequence comprising delivering to said target RNA: (a) a Cas protein described herein; (b) a guide molecule which comprises a guide sequence linked to a direct repeat sequence; and (c) a deaminase, (including but not limited to an ADAR protein (including but not limited to a modified ADAR protein having C-to-U deamination activity or catalytic domain thereof); wherein said modified ADAR protein or catalytic domain thereof is covalently or non-covalently linked to said Cas protein or said guide molecule or is adapted to link thereto after delivery; wherein guide molecule forms a complex with said Cas protein and directs said complex to bind said target RNA sequence of interest; wherein said guide sequence is capable of hybridizing with a target sequence comprising said C to form an RNA duplex; wherein, optionally, said guide sequence comprises a non-pairing A or U at a position corresponding to said C resulting in a mismatch in the RNA duplex formed; and wherein said modified ADAR protein or catalytic domain thereof deaminates said C in said RNA duplex. 
     In connection with the aforementioned modified ADAR protein having C-to-U deamination activity, the invention described herein further relates to an engineered, non-naturally occurring system suitable for deaminating a C in a target locus of interest, comprising: (a) a guide molecule which comprises a guide sequence linked to a direct repeat sequence, or a nucleotide sequence encoding said guide molecule; (b) a catalytically inactive CRISPR-Cas protein, or a nucleotide sequence encoding said catalytically inactive CRISPR-Cas protein; (c) a modified ADAR protein having C-to-U deamination activity or catalytic domain thereof, or a nucleotide sequence encoding said modified ADAR protein or catalytic domain thereof; wherein said modified ADAR protein or catalytic domain thereof is covalently or non-covalently linked to said CRISPR-Cas protein or said guide molecule or is adapted to link thereto after delivery; wherein said guide sequence is capable of hybridizing with a target RNA sequence comprising a C to form an RNA duplex; wherein, optionally, said guide sequence comprises a non-pairing A or U at a position corresponding to said C resulting in a mismatch in the RNA duplex formed; wherein, optionally, the system is a vector system comprising one or more vectors comprising: (a) a first regulatory element operably linked to a nucleotide sequence encoding said guide molecule which comprises said guide sequence, (b) a second regulatory element operably linked to a nucleotide sequence encoding said catalytically inactive CRISPR-Cas protein; and (c) a nucleotide sequence encoding a modified ADAR protein having C-to-U deamination activity or catalytic domain thereof which is under control of said first or second regulatory element or operably linked to a third regulatory element; wherein, if said nucleotide sequence encoding a modified ADAR protein or catalytic domain thereof is operably linked to a third regulatory element, said modified ADAR protein or catalytic domain thereof is adapted to link to said guide molecule or said CRISPR-Cas protein after expression; wherein components (a), (b) and (c) are located on the same or different vectors of the system, optionally wherein said first, second, and/or third regulatory element is an inducible promoter. 
     In an embodiment of the invention, the substrate of the adenosine deaminase is an RNA/DNA heteroduplex formed upon binding of the guide molecule to its DNA target which then forms the CRISPR-Cas complex with the CRISPR-Cas enzyme. The RNA/DNA or DNA/RNA heteroduplex is also referred to herein as the “RNA/DNA hybrid”, “DNA/RNA hybrid” or “double-stranded substrate”. 
     According to the present invention, the substrate of the adenosine deaminase is an RNA/DNAn RNA duplex formed upon binding of the guide molecule to its DNA target which then forms the CRISPR-Cas complex with the CRISPR-Cas enzyme. The substrate of the adenosine deaminase can also be an RNA/RNA duplex formed upon binding of the guide molecule to its RNA target which then forms the CRISPR-Cas complex with the CRISPR-Cas enzyme. The RNA/DNA or DNA/RNAn RNA duplex is also referred to herein as the “RNA/DNA hybrid”, “DNA/RNA hybrid” or “double-stranded substrate”. The particular features of the guide molecule and CRISPR-Cas enzyme are detailed below. 
     The term “editing selectivity” as used herein refers to the fraction of all sites on a double-stranded substrate that is edited by an adenosine deaminase. Without being bound by theory, it is contemplated that editing selectivity of an adenosine deaminase is affected by the double-stranded substrate&#39;s length and secondary structures, such as the presence of mismatched bases, bulges and/or internal loops. 
     In some embodiments, when the substrate is a perfectly base-paired duplex longer than 50 bp, the adenosine deaminase may be able to deaminate multiple adenosine residues within the duplex (e.g., 50% of all adenosine residues). In some embodiments, when the substrate is shorter than 50 bp, the editing selectivity of an adenosine deaminase is affected by the presence of a mismatch at the target adenosine site. Particularly, in some embodiments, adenosine (A) residue having a mismatched cytidine (C) residue on the opposite strand is deaminated with high efficiency. In some embodiments, adenosine (A) residue having a mismatched guanosine (G) residue on the opposite strand is skipped without editing. 
     In particular embodiments, the adenosine deaminase protein or catalytic domain thereof is delivered to the cell or expressed within the cell as a separate protein, but is modified so as to be able to link to either the Cas protein described herein (e.g. Cas-like (e.g. Cas9-lik and/or Cas12-like) protein or the guide molecule. In particular embodiments, this is ensured by the use of orthogonal RNA-binding protein or adaptor protein/aptamer combinations that exist within the diversity of bacteriophage coat proteins. Examples of such coat proteins include but are not limited to: MS2, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, 7s and PRR1. Aptamers can be naturally occurring or synthetic oligonucleotides that have been engineered through repeated rounds of in vitro selection or SELEX (systematic evolution of ligands by exponential enrichment) to bind to a specific target. 
     In particular embodiments, the guide molecule is provided with one or more distinct RNA loop(s) or distinct sequence(s) that can recruit an adaptor protein. A guide molecule may be extended, without colliding with the Cas protein described herein (e.g. Cas-like (e.g. Cas9-like and/or Cas12-like) protein by the insertion of distinct RNA loop(s) or distinct sequence(s) that may recruit adaptor proteins that can bind to the distinct RNA loop(s) or distinct sequence(s). Examples of modified guides and their use in recruiting effector domains to the C2c1 complex are provided in Konermann (Nature 2015, 517(7536): 583-588), which can be used to similarly design and construct guides for use with a Cas protein described herein (e.g. Cas-like (e.g. Cas9-like and/or Cas12-like) protein in view of the description provided herein. In particular embodiments, the aptamer is a minimal hairpin aptamer which selectively binds dimerized MS2 bacteriophage coat proteins in mammalian cells and is introduced into the guide molecule, such as in the stemloop and/or in a tetraloop. In these embodiments, the adenosine deaminase protein is fused to MS2. The adenosine deaminase protein is then co-delivered together with the C2c1 protein and corresponding guide RNA. 
     In some embodiments, the C2c1-ADAR, Cas-ADAR, Cas-like protein-ADAR base editing system described herein comprises (a) one Cas protein described herein (e.g. Cas-like (e.g. Cas9-like and/or Cas12-like, and/or C2c1) which is catalytically inactive or a nickase; (b) a guide molecule which comprises a guide sequence; and (c) an adenosine deaminase protein or catalytic domain thereof; wherein the adenosine deaminase protein or catalytic domain thereof is covalently or non-covalently linked to the Cas protein described herein (e.g. Cas-like (e.g. Cas9-like and/or Cas12-like, and/or C2c1) or the guide molecule or is adapted to link thereto after delivery; wherein the guide sequence is substantially complementary to the target sequence but comprises a non-pairing C corresponding to the A being targeted for deamination, resulting in a A-C mismatch in a DNA-RNA or RNA-RNA duplex formed by the guide sequence and the target sequence. For application in eukaryotic cells, the Cas protein described herein (e.g. Cas-like (e.g. Cas9-like and/or Cas12-like, and/or C2c1) and/or the adenosine deaminase are preferably NLS-tagged. 
     In some embodiments, the components (a), (b) and (c) are delivered to the cell as a ribonucleoprotein complex. The ribonucleoprotein complex can be delivered via one or more lipid nanoparticles. 
     In some embodiments, the components (a), (b) and (c) are delivered to the cell as one or more RNA molecules, such as one or more guide RNAs and one or more mRNA molecules encoding the Cas, ADAR, or Cas-ADAR protein, the adenosine deaminase protein, and optionally the adaptor protein. The RNA molecules can be delivered via one or more lipid nanoparticles. 
     In some embodiments, the components (a), (b) and (c) are delivered to the cell as one or more DNA molecules. In some embodiments, the one or more DNA molecules are comprised within one or more vectors such as viral vectors (e.g., AAV). In some embodiments, the one or more DNA molecules comprise one or more regulatory elements operably configured to express the Cas, ADAR, or Cas-ADAR protein, the guide molecule, and the adenosine deaminase protein or catalytic domain thereof, optionally wherein the one or more regulatory elements comprise inducible promoters. 
     In some embodiments of the guide molecule is capable of hybridizing with a target sequence comprising the Adenine to be deaminated within a first DNA strand or an RNA strand at the target locus to form a DNA-RNA or RNA-RNA duplex which comprises a non-pairing Cytosine opposite to said Adenine. In some aspects, upon duplex formation, the guide molecule forms a complex with one or more Cas proteins described herein and directs the complex to bind said first DNA strand or said RNA strand at the target locus of interest. Details on the aspect of the guide of the C2c1-ADAR base editing system are provided herein below. 
     In some embodiments, a C2c1 guide RNA having a canonical length (e.g., about 20 nt for AacC2c1) is used to form a DNA-RNA or RNA-RNA duplex with the target DNA or RNA. In some embodiments, a C2c1 guide molecule longer than the canonical length (e.g., &gt;20 nt for AacC2c1) is used to form a DNA-RNA or RNA-RNA duplex with the target DNA or RNA including outside of the C2c1-guide RNA-target DNA complex. In certain example embodiments, the guide sequence has a length of about 29-53 nt capable of forming a DNA-RNA or RNA-RNA duplex with said target sequence. In certain other example embodiments, the guide sequence has a length of about 40-50 nt capable of forming a DNA-RNA or RNA-RNA duplex with said target sequence. In certain example embodiments, the distance between said non-pairing C and the 5′ end of said guide sequence is 20-30 nucleotides. In certain example embodiments, the distance between said non-pairing C and the 3′ end of said guide sequence is 20-30 nucleotides. 
     In at least a first design, the Cas protein (includes any cas protein, including but not limited to C2c1 and Cas-like proteins)-ADAR system comprises (a) an adenosine deaminase fused or linked to a Cas protein, wherein the Cas protein is catalytically inactive or a nickase, and (b) a guide molecule comprising a guide sequence designed to introduce a A-C mismatch in a DNA-RNA or RNA-RNA duplex formed between the guide sequence and the target sequence. In some embodiments, the Cas protein and/or the adenosine deaminase are NLS-tagged, on either the N- or C-terminus or both. 
     In at least a second design, the Cas-ADAR system comprises (a) a Cas protein that is catalytically inactive or a nickase, (b) a guide molecule comprising a guide sequence designed to introduce a A-C mismatch in a DNA-RNA or RNA-RNA duplex formed between the guide sequence and the target sequence, and an aptamer sequence (e.g., MS2 RNA motif or PP7 RNA motif) capable of binding to an adaptor protein (e.g., MS2 coating protein or PP7 coat protein), and (c) an adenosine deaminase fused or linked to an adaptor protein, wherein the binding of the aptamer and the adaptor protein recruits the adenosine deaminase to the DNA-RNA or RNA-RNA duplex formed between the guide sequence and the target sequence for targeted deamination at the A of the A-C mismatch. In some embodiments, the adaptor protein and/or the adenosine deaminase are NLS-tagged, on either the N- or C-terminus or both. The Cas protein can also be NLS-tagged. 
     The use of different aptamers and corresponding adaptor proteins also allows orthogonal gene editing to be implemented. In one example in which adenosine deaminase are used in combination with cytidine deaminase for orthogonal gene editing/deamination, sgRNA targeting different loci are modified with distinct RNA loops in order to recruit MS2-adenosine deaminase and PP7-cytidine deaminase (or PP7-adenosine deaminase and MS2-cytidine deaminase), respectively, resulting in orthogonal deamination of A or C at the target loci of interested, respectively. PP7 is the RNA-binding coat protein of the bacteriophage  Pseudomonas . Like MS2, it binds a specific RNA sequence and secondary structure. The PP7 RNA-recognition motif is distinct from that of MS2. Consequently, PP7 and MS2 can be multiplexed to mediate distinct effects at different genomic loci simultaneously. For example, an sgRNA targeting locus A can be modified with MS2 loops, recruiting MS2-adenosine deaminase, while another sgRNA targeting locus B can be modified with PP7 loops, recruiting PP7-cytidine deaminase. In the same cell, orthogonal, locus-specific modifications are thus realized. This principle can be extended to incorporate other orthogonal RNA-binding proteins. 
     In at least a third design, the Cas-ADAR CRISPR system comprises (a) an adenosine deaminase inserted into an internal loop or unstructured region of a Cas protein, wherein the Cas protein is catalytically inactive or a nickase, and (b) a guide molecule comprising a guide sequence designed to introduce a A-C mismatch in a DNA-RNA or RNA-RNA duplex formed between the guide sequence and the target sequence. 
     C2c1 protein split sites that are suitable for insertion of adenosine deaminase can be identified with the help of a crystal structure. For example, with respect to AacC2c1 mutants, it should be readily apparent what the corresponding position for, for example, a sequence alignment. For other C2c1 protein one can use the crystal structure of an ortholog if a relatively high degree of homology exists between the ortholog and the intended C2c1 protein. Homologous appropriate split sites can be determined in other Cas proteins (e.g. Cas9-like and/or Cas12-like) based on corresponding sites in the other Cas proteins compared to C2c1 protein. Methods of alignment and determining homologous sites are described elsewhere herein. 
     The split position may be located within a region or loop. Preferably, the split position occurs where an interruption of the amino acid sequence does not result in the partial or full destruction of a structural feature (e.g. alpha-helixes or β-sheets). Unstructured regions (regions that did not show up in the crystal structure because these regions are not structured enough to be “frozen” in a crystal) are often preferred options. Splits in all unstructured regions that are exposed on the surface of the Cas protein (e.g. a Cas-like protein (e.g. Cas9-like and/or Cas12-like or C2c1) are envisioned in the practice of the invention. The positions within the unstructured regions or outside loops may not need to be exactly the numbers provided above, but may vary by, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or even 10 amino acids either side of the position given above, depending on the size of the loop, so long as the split position still falls within an unstructured region of outside loop. 
     The Cas-ADAR system described herein can be used to target a specific Adenine within a DNA sequence for deamination. For example, the guide molecule can form a complex with the Cas protein and directs the complex to bind a target sequence at the target locus of interest. Because the guide sequence is designed to have a non-pairing C, the heteroduplex formed between the guide sequence and the target sequence comprises a A-C mismatch, which directs the adenosine deaminase to contact and deaminate the A opposite to the non-pairing C, converting it to a Inosine (I). Since Inosine (I) base pairs with C and functions like G in cellular process, the targeted deamination of A described herein are useful for correction of undesirable G-A and C-T mutations, as well as for obtaining desirable A-G and T-C mutations. 
     Base Excision Repair Inhibitors 
     In some embodiments, the D-functionalized and/or AD-functionalized CRISPR system (i.e. a CRISPR system described herein containing a deaminase (D) or adenosine deaminase (AD)) further comprises a base excision repair (BER) inhibitor. The BER can be configured as an activatable functional domain as described elsewhere herein. In some aspects, the BER is configured in a matched pair of activatable functional domains as a split protein between the two domains in the matched pair. Other configurations within a matched pair of activatable functional domain are also envisioned and as described elsewhere herein. 
     Without wishing to be bound by any particular theory, cellular DNA-repair response to the presence of I:T pairing may be responsible for a decrease in nucleobase editing efficiency in cells. Alkyladenine DNA glycosylase (also known as DNA-3-methyladenine glycosylase, 3-alkyladenine DNA glycosylase, or N-methylpurine DNA glycosylase) catalyzes removal of hypoxanthine from DNA in cells, which may initiate base excision repair, with reversion of the I:T pair to a A:T pair as outcome. 
     In some embodiments, the BER inhibitor is an inhibitor of alkyladenine DNA glycosylase. In some embodiments, the BER inhibitor is an inhibitor of human alkyladenine DNA glycosylase. In some embodiments, the BER inhibitor is a polypeptide inhibitor. In some embodiments, the BER inhibitor is a protein that binds hypoxanthine. In some embodiments, the BER inhibitor is a protein that binds hypoxanthine in DNA. In some embodiments, the BER inhibitor is a catalytically inactive alkyladenine DNA glycosylase protein or binding domain thereof. In some embodiments, the BER inhibitor is a catalytically inactive alkyladenine DNA glycosylase protein or binding domain thereof that does not excise hypoxanthine from the DNA. Other proteins that are capable of inhibiting (e.g., sterically blocking) an alkyladenine DNA glycosylase base-excision repair enzyme are within the scope of this disclosure. Additionally, any proteins that block or inhibit base-excision repair as also within the scope of this disclosure. 
     Without wishing to be bound by any particular theory, base excision repair may be inhibited by molecules that bind the edited strand, block the edited base, inhibit alkyladenine DNA glycosylase, inhibit base excision repair, protect the edited base, and/or promote fixing of the non-edited strand. It is believed that the use of the BER inhibitor described herein can increase the editing efficiency of an adenosine deaminase that is capable of catalyzing a A to I change. 
     Accordingly, in the first design of the AD-functionalized CRISPR system discussed above, the CRISPR-Cas protein or the adenosine deaminase can be fused to or linked to a BER inhibitor (e.g., an inhibitor of alkyladenine DNA glycosylase). 
     In some embodiments, the BER inhibitor can be comprised in one of the following structures (Cas protein=any suitable Cas protein (e.g. C2c1 and variants thereof and Cas-like proteins (e.g. Cas9-like and/or Cas12-like and variants thereof): [AD]-[optional linker]-[Cas protein]-[optional linker]-[BER inhibitor]; [AD]-[optional linker]-[BER inhibitor]-[optional linker]-[Cas protein]; [BER inhibitor]-[optional linker]-[AD]-[optional linker]-[Cas protein]; [BER inhibitor]-[optional linker]-[nC2c1/dC2c1]-[optional linker]-[AD]; [Cas protein]-[optional linker]-[AD]-[optional linker]-[BER inhibitor]; [Cas protein]-[optional linker]-[BER inhibitor]-[optional linker]-[AD]. 
     In some embodiments, the BER inhibitor can be comprised in one of the following structures (nC2c1=C2c1 nickase; dC2c1=dead C2c1): [AD]-[optional linker]-[nC2c1/dC2c1]-[optional linker]-[BER inhibitor]; [AD]-[optional linker]-[BER inhibitor]-[optional linker]-[nC2c1/dC2c1]; [BER inhibitor]-[optional linker]-[AD]-[optional linker]-[nC2c1/dC2c1]; [BER inhibitor]-[optional linker]-[nC2c1/dC2c1]-[optional linker]-[AD]; [nC2c1/dC2c1]-[optional linker]-[AD]-[optional linker]-[BER inhibitor]; [nC2c1/dC2c1]-[optional linker]-[BER inhibitor]-[optional linker]-[AD]. 
     Similarly, in the second design of the AD-functionalized CRISPR system discussed above, the CRISPR-Cas protein, the adenosine deaminase, or the adaptor protein can be fused to or linked to a BER inhibitor (e.g., an inhibitor of alkyladenine DNA glycosylase). 
     In some embodiments, the BER inhibitor can be comprised in one of the following structures (Cas protein=any suitable Cas protein (e.g. C2c1 and variants thereof and Cas-like proteins (e.g. Cas9-like and/or Cas12-like and variants thereof): [Cas Protein]-[optional linker]-[BER inhibitor]; [BER inhibitor]-[optional linker]-[Cas Protein]; [AD]-[optional linker]-[Adaptor]-[optional linker]-[BER inhibitor]; [AD]-[optional linker]-[BER inhibitor]-[optional linker]-[Adaptor]; [BER inhibitor]-[optional linker]-[AD]-[optional linker]-[Adaptor]; [BER inhibitor]-[optional linker]-[Adaptor]-[optional linker]-[AD]; [Adaptor]-[optional linker]-[AD]-[optional linker]-[BER inhibitor]; [Adaptor]-[optional linker]-[BER inhibitor]-[optional linker]-[AD]. 
     In some embodiments, the BER inhibitor can be comprised in one of the following structures (nC2c1=C2c1 nickase; dC2c1=dead C2c1): [nC2c1/dC2c1]-[optional linker]-[BER inhibitor]; [BER inhibitor]-[optional linker]-[nC2c1/dC2c1]; [AD]-[optional linker]-[Adaptor]-[optional linker]-[BER inhibitor]; [AD]-[optional linker]-[BER inhibitor]-[optional linker]-[Adaptor]; [BER inhibitor]-[optional linker]-[AD]-[optional linker]-[Adaptor]; [BER inhibitor]-[optional linker]-[Adaptor]-[optional linker]-[AD]; [Adaptor]-[optional linker]-[AD]-[optional linker]-[BER inhibitor]; [Adaptor]-[optional linker]-[BER inhibitor]-[optional linker]-[AD]. 
     In the third design of the AD-functionalized CRISPR system discussed above, the BER inhibitor can be inserted into an internal loop or unstructured region of a CRISPR-Cas protein. 
     Cytidine Deaminase 
     In some embodiments, the deaminase is a cytidine deaminase. In some aspects, the cytidine deaminase is configured in a matched pair of activatable functional domains as a split protein between the two domains in the matched pair. Other configurations within a matched pair of activatable functional domain are also envisioned and as described elsewhere herein. 
     The term “cytidine deaminase” or “cytidine deaminase protein” or “cytidine deaminase activity” as used herein refers to a protein, a polypeptide, or one or more functional domain(s) of a protein or a polypeptide that is capable of catalyzing a hydrolytic deamination reaction that converts a cytosine (or a cytosine moiety of a molecule) to an uracil (or a uracil moiety of a molecule), as shown below. In some embodiments, the cytosine-containing molecule is a cytidine (C), and the uracil-containing molecule is a uridine (U). The cytosine-containing molecule can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). 
     
       
         
         
             
             
         
       
     
     Cytidine deaminases that can be used in connection with the present disclosure include, but are not limited to, members of the enzyme family known as apolipoprotein B mRNA-editing complex (APOBEC) family deaminase, an activation-induced deaminase (AID), or a cytidine deaminase 1 (CDA1). In particular embodiments, the deaminase in an APOBEC1 deaminase, an APOBEC2 deaminase, an APOBEC3A deaminase, an APOBEC3B deaminase, an APOBEC3C deaminase, and APOBEC3D deaminase, an APOBEC3E deaminase, an APOBEC3F deaminase an APOBEC3G deaminase, an APOBEC3H deaminase, or an APOBEC4 deaminase. 
     In some embodiments, the cytidine deaminase or engineered adenosine deaminase with cytidine deaminase activity is capable of targeting Cytosine in a DNA single strand. In certain example embodiments the cytidine deaminase activity may edit on a single strand present outside of the binding component e.g. bound CRISPR-Cas. In other example embodiments, the cytidine deaminase may edit at a localized bubble, such as a localized bubble formed by a mismatch at the target edit site but the guide sequence. In certain example embodiments the cytidine deaminase may contain mutations that help focus the area of activity such as those disclosed in Kim et al., Nature Biotechnology (2017) 35(4):371-377 (doi:10.1038/nbt.3803. 
     In some embodiments, the cytidine deaminase is derived from one or more metazoa species, including but not limited to, mammals, birds, frogs, squids, fish, flies and worms. In some embodiments, the cytidine deaminase is a human, primate, cow, dog rat or mouse cytidine deaminase. 
     In some embodiments, the cytidine deaminase is a human APOBEC, including hAPOBEC1 or hAPOBEC3. In some embodiments, the cytidine deaminase is a human AID. 
     In some embodiments, the cytidine deaminase protein recognizes and converts one or more target cytosine residue(s) in a single-stranded bubble of a RNA duplex into uracil residues (s). In some embodiments, the cytidine deaminase protein recognizes a binding window on the single-stranded bubble of a RNA duplex. In some embodiments, the binding window contains at least one target cytosine residue(s). In some embodiments, the binding window is in the range of about 3 bp to about 100 bp. In some embodiments, the binding window is in the range of about 5 bp to about 50 bp. In some embodiments, the binding window is in the range of about 10 bp to about 30 bp. In some embodiments, the binding window is about 1 bp, 2 bp, 3 bp, 5 bp, 7 bp, 10 bp, 15 bp, 20 bp, 25 bp, 30 bp, 40 bp, 45 bp, 50 bp, 55 bp, 60 bp, 65 bp, 70 bp, 75 bp, 80 bp, 85 bp, 90 bp, 95 bp, or 100 bp. 
     In some embodiments, the cytidine deaminase protein comprises one or more deaminase domains. Not intended to be bound by theory, it is contemplated that the deaminase domain functions to recognize and convert one or more target cytosine (C) residue(s) contained in a single-stranded bubble of a RNA duplex into (an) uracil (U) residue (s). In some embodiments, the deaminase domain comprises an active center. In some embodiments, the active center comprises a zinc ion. In some embodiments, amino acid residues in or near the active center interact with one or more nucleotide(s) 5′ to a target cytosine residue. In some embodiments, amino acid residues in or near the active center interact with one or more nucleotide(s) 3′ to a target cytosine residue. 
     In some embodiments, the cytidine deaminase comprises human APOBEC1 full protein (hAPOBEC1) or the deaminase domain thereof (hAPOBEC1-D) or a C-terminally truncated version thereof (hAPOBEC-T). In some embodiments, the cytidine deaminase is an APOBEC family member that is homologous to hAPOBEC1, hAPOBEC-D or hAPOBEC-T. In some embodiments, the cytidine deaminase comprises human AID1 full protein (hAID) or the deaminase domain thereof (hAID-D) or a C-terminally truncated version thereof (hAID-T). In some embodiments, the cytidine deaminase is an AID family member that is homologous to hAID, hAID-D or hAID-T. In some embodiments, the hAID-T is a hAID which is C-terminally truncated by about 20 amino acids. 
     In some embodiments, the cytidine deaminase comprises the wild-type amino acid sequence of a cytosine deaminase. In some embodiments, the cytidine deaminase comprises one or more mutations in the cytosine deaminase sequence, such that the editing efficiency, and/or substrate editing preference of the cytosine deaminase is changed according to specific needs. 
     Certain mutations of APOBEC1 and APOBEC3 proteins have been described in Kim et al., Nature Biotechnology (2017) 35(4):371-377 (doi:10.1038/nbt.3803); and Harris et al. Mol. Cell (2002) 10:1247-1253, each of which is incorporated herein by reference in its entirety. 
     In some embodiments, the cytidine deaminase is an APOBEC1 deaminase comprising one or more mutations at amino acid positions corresponding to W90, R118, H121, H122, R126, or R132 in rat APOBEC1, or an APOBEC3G deaminase comprising one or more mutations at amino acid positions corresponding to W285, R313, D316, D317X, R320, or R326 in human APOBEC3 G. 
     In some embodiments, the cytidine deaminase comprises a mutation at tryptophane90 of the rat APOBEC1 amino acid sequence, or a corresponding position in a homologous APOBEC protein, such as tryptophane285 of APOBEC3G. In some embodiments, the tryptophan residue at position 90 is replaced by a tyrosine or phenylalanine residue (W90Y or W90F). 
     In some embodiments, the cytidine deaminase comprises a mutation at Arginine118 of the rat APOBEC1 amino acid sequence, or a corresponding position in a homologous APOBEC protein. In some embodiments, the arginine residue at position 118 is replaced by an alanine residue (R118A). 
     In some embodiments, the cytidine deaminase comprises a mutation at Histidine121 of the rat APOBEC1 amino acid sequence, or a corresponding position in a homologous APOBEC protein. In some embodiments, the histidine residue at position 121 is replaced by an arginine residue (H121R). 
     In some embodiments, the cytidine deaminase comprises a mutation at Histidine122 of the rat APOBEC1 amino acid sequence, or a corresponding position in a homologous APOBEC protein. In some embodiments, the histidine residue at position 122 is replaced by an arginine residue (H122R). 
     In some embodiments, the cytidine deaminase comprises a mutation at Arginine126 of the rat APOBEC1 amino acid sequence, or a corresponding position in a homologous APOBEC protein, such as Arginine320 of APOBEC3G. In some embodiments, the arginine residue at position 126 is replaced by an alanine residue (R126A) or by a glutamic acid (R126E). 
     In some embodiments, the cytidine deaminase comprises a mutation at arginine132 of the APOBEC1 amino acid sequence, or a corresponding position in a homologous APOBEC protein. In some embodiments, the arginine residue at position 132 is replaced by a glutamic acid residue (R132E). 
     In some embodiments, to narrow the width of the editing window, the cytidine deaminase may comprise one or more of the mutations: W90Y, W90F, R126E and R132E, based on amino acid sequence positions of rat APOBEC1, and mutations in a homologous APOBEC protein corresponding to the above. 
     In some embodiments, to reduce editing efficiency, the cytidine deaminase may comprise one or more of the mutations: W90A, R118A, R132E, based on amino acid sequence positions of rat APOBEC1, and mutations in a homologous APOBEC protein corresponding to the above. In particular embodiments, it can be of interest to use a cytidine deaminase enzyme with reduced efficacy to reduce off-target effects. 
     In some embodiments, the cytidine deaminase is wild-type rat APOBEC1 (rAPOBEC1, or a catalytic domain thereof. In some embodiments, the cytidine deaminase comprises one or more mutations in the rAPOBEC1 sequence, such that the editing efficiency, and/or substrate editing preference of rAPOBEC1 is changed according to specific needs. 
     
       
         
           
               
               
            
               
                   
                 rAPOBEC1: 
               
               
                   
                 (SEQ ID NO: 38) 
               
               
                   
                 MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKE 
               
               
                   
               
               
                   
                 TCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKF 
               
               
                   
               
               
                   
                 TTERYFCPNTRCSITWFLSWSPCGECSRAITEFLS 
               
               
                   
               
               
                   
                 RYPHVTLFIYIARLYHEIADPRNRQGLRDLISSGV 
               
               
                   
               
               
                   
                 TIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLW 
               
               
                   
               
               
                   
                 VRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIA 
               
               
                   
               
               
                   
                 LQSCHYQRLPPHILWATGLK 
               
            
           
         
       
     
     In some embodiments, the cytidine deaminase is wild-type human APOBEC1 (hAPOBEC1) or a catalytic domain thereof. In some embodiments, the cytidine deaminase comprises one or more mutations in the hAPOBEC1 sequence, such that the editing efficiency, and/or substrate editing preference of hAPOBEC1 is changed according to specific needs. 
     
       
         
           
               
               
            
               
                   
                 APOBEC1: 
               
               
                   
                 (SEQ ID NO: 39) 
               
               
                   
                 MTSEKGPSTGDPTLRRRIEPWEFDVFYDPRELRKE 
               
               
                   
               
               
                   
                 ACLLYEIKWGMSRKIWRSSGKNTTNHVEVNFIKKF 
               
               
                   
               
               
                   
                 TSERDFHPSMSCSITWFLSWSPCWECSQAIREFLS 
               
               
                   
               
               
                   
                 RHPGVTLVIYVARLFWHMDQQNRQGLRDLVNSGVT 
               
               
                   
               
               
                   
                 IQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWM 
               
               
                   
               
               
                   
                 MLYALELHCIILSLPPCLKISRRWQNHLTFFRLHL 
               
               
                   
               
               
                   
                 QNCHYQTIPPHILLATGLIHPSVAWR 
               
            
           
         
       
     
     In some embodiments, the cytidine deaminase is wild-type human APOBEC3G (hAPOBEC3G) or a catalytic domain thereof. In some embodiments, the cytidine deaminase comprises one or more mutations in the hAPOBEC3G sequence, such that the editing efficiency, and/or substrate editing preference of hAPOBEC3G is changed according to specific needs. 
     
       
         
           
               
               
            
               
                   
                 hAPOBEC3G: 
               
               
                   
                 (SEQ ID NO: 40) 
               
               
                   
                 MELKYHPEMRFFHWFSKWRKLHRDQEYEVTWYISW 
               
               
                   
               
               
                   
                 SPCTKCTRDMATFLAEDPKVTLTIFVARLYYFWDP 
               
               
                   
               
               
                   
                 DYQEALRSLCQKRDGPRATMKIMNYDEFQHCWSKF 
               
               
                   
               
               
                   
                 VYSQRELFEPWNNLPKYYILLHIMLGEILRHSMDP 
               
               
                   
               
               
                   
                 PTFTENENNEPWVRGRHETYLCYEVERMHNDTWVL 
               
               
                   
               
               
                   
                 LNQRRGELCNQAPHKHGELEGRHAELCFLDVIPFW 
               
               
                   
               
               
                   
                 KLDLDQDYRVTCFTSWSPCFScAQEMAKFISKNKH 
               
               
                   
               
               
                   
                 VSLCIFTARIYDDQGRCQEGLRTLAEAGAKISIMT 
               
               
                   
               
               
                   
                 YSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGR 
               
               
                   
               
               
                   
                 LRAILQNQEN 
               
            
           
         
       
     
     In some embodiments, the cytidine deaminase is wild-type  Petromyzon marinus  CDA1 (pmCDA1) or a catalytic domain thereof. In some embodiments, the cytidine deaminase comprises one or more mutations in the pmCDA1 sequence, such that the editing efficiency, and/or substrate editing preference of pmCDA1 is changed according to specific needs. 
     
       
         
           
               
               
            
               
                   
                 pmCDA1: 
               
               
                   
                 (SEQ ID NO: 41) 
               
               
                   
                 MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYV 
               
               
                   
               
               
                   
                 LFELKRRGERRACFWGYAVNKPQSGTERGIHAEIF 
               
               
                   
               
               
                   
                 SIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKIL 
               
               
                   
               
               
                   
                 EWYNQELRGNGHTLKIWACKLYYEKNARNQIGLWN 
               
               
                   
               
               
                   
                 LRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENR 
               
               
                   
               
               
                   
                 WLEKTLKRAEKRRSELSIMIQVKILHTTKSPAV 
               
            
           
         
       
     
     In some embodiments, the cytidine deaminase is wild-type human AID (hAID) or a catalytic domain thereof. In some embodiments, the cytidine deaminase comprises one or more mutations in the pmCDA1 sequence, such that the editing efficiency, and/or substrate editing preference of pmCDA1 is changed according to specific needs. 
     
       
         
           
               
               
            
               
                   
                 hAID: 
               
               
                   
                 (SEQ ID NO: 42) 
               
               
                   
                 MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKR 
               
               
                   
               
               
                   
                 RDSATSFSLDFGYLRNKNGCHVELLFLRYISDWDL 
               
               
                   
               
               
                   
                 DPGRCYRVTWFTSWSPCYDCARHVADFLRGNPYLS 
               
               
                   
               
               
                   
                 LRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMT 
               
               
                   
               
               
                   
                 FKDYFYCWNTFVENHERTFKAWEGLHENSVRLSRQ 
               
               
                   
               
               
                   
                 LRRILLPLYEVDDLRDAFRTLGLLD 
               
            
           
         
       
     
     In some embodiments, the cytidine deaminase is truncated version of hAID (hAID-DC) or a catalytic domain thereof. In some embodiments, the cytidine deaminase comprises one or more mutations in the hAID-DC sequence, such that the editing efficiency, and/or substrate editing preference of hAID-DC is changed according to specific needs. 
     
       
         
           
               
               
            
               
                   
                 hAID-DC: 
               
               
                   
                 (SEQ ID NO: 43) 
               
               
                   
                 MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKR 
               
               
                   
               
               
                   
                 RDSATSFSLDFGYLRNKNGCHVELLFLRYISDWDL 
               
               
                   
               
               
                   
                 DPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLS 
               
               
                   
               
               
                   
                 LRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMT 
               
               
                   
               
               
                   
                 FKDYFYCWNTFVENHERTFKAWEGLHENSVRLSRQ 
               
               
                   
               
               
                   
                 LRRILL 
               
            
           
         
       
     
     Additional embodiments of the cytidine deaminase are disclosed in WO WO2017/070632, titled “Nucleobase Editor and Uses Thereof,” which is incorporated herein by reference in its entirety. 
     In some embodiments, the cytidine deaminase has an efficient deamination window that encloses the nucleotides susceptible to deamination editing. Accordingly, in some embodiments, the “editing window width” refers to the number of nucleotide positions at a given target site for which editing efficiency of the cytidine deaminase exceeds the half-maximal value for that target site. In some embodiments, the cytidine deaminase has an editing window width in the range of about 1 to about 6 nucleotides. In some embodiments, the editing window width of the cytidine deaminase is 1, 2, 3, 4, 5, or 6 nucleotides. 
     Not intended to be bound by theory, it is contemplated that in some embodiments, the length of the linker sequence affects the editing window width. In some embodiments, the editing window width increases (e.g., from about 3 to about 6 nucleotides) as the linker length extends (e.g., from about 3 to about 21 amino acids). In a non-limiting example, a 16-residue linker offers an efficient deamination window of about 5 nucleotides. In some embodiments, the length of the guide RNA affects the editing window width. In some embodiments, shortening the guide RNA leads to a narrowed efficient deamination window of the cytidine deaminase. 
     In some embodiments, mutations to the cytidine deaminase affect the editing window width. In some embodiments, the cytidine deaminase component of the CD-functionalized CRISPR system comprises one or more mutations that reduce the catalytic efficiency of the cytidine deaminase, such that the deaminase is prevented from deamination of multiple cytidines per DNA binding event. In some embodiments, tryptophan at residue 90 (W90) of APOBEC1 or a corresponding tryptophan residue in a homologous sequence is mutated. In some embodiments, the catalytically inactive CRISPR-Cas is fused to or linked to an APOBEC1 mutant that comprises a W90Y or W90F mutation. In some embodiments, tryptophan at residue 285 (W285) of APOBEC3G, or a corresponding tryptophan residue in a homologous sequence is mutated. In some embodiments, the catalytically inactive CRISPR-Cas is fused to or linked to an APOBEC3G mutant that comprises a W285Y or W285F mutation. 
     In some embodiments, the cytidine deaminase component of CD-functionalized CRISPR system comprises one or more mutations that reduce tolerance for non-optimal presentation of a cytidine to the deaminase active site. In some embodiments, the cytidine deaminase comprises one or more mutations that alter substrate binding activity of the deaminase active site. In some embodiments, the cytidine deaminase comprises one or more mutations that alter the conformation of DNA to be recognized and bound by the deaminase active site. In some embodiments, the cytidine deaminase comprises one or more mutations that alter the substrate accessibility to the deaminase active site. In some embodiments, arginine at residue 126 (R126) of APOBEC1 or a corresponding arginine residue in a homologous sequence is mutated. In some embodiments, the catalytically inactive CRISPR-Cas is fused to or linked to an APOBEC1 that comprises a R126A or R126E mutation. In some embodiments, tryptophan at residue 320 (R320) of APOBEC3G, or a corresponding arginine residue in a homologous sequence is mutated. In some embodiments, the catalytically inactive CRISPR-Cas is fused to or linked to an APOBEC3G mutant that comprises a R320A or R320E mutation. In some embodiments, arginine at residue 132 (R132) of APOBEC1 or a corresponding arginine residue in a homologous sequence is mutated. In some embodiments, the catalytically inactive CRISPR-Cas is fused to or linked to an APOBEC1 mutant that comprises a R132E mutation. 
     In some embodiments, the APOBEC1 domain of the CD-functionalized CRISPR system comprises one, two, or three mutations selected from W90Y, W90F, R126A, R126E, and R132E. In some embodiments, the APOBEC1 domain comprises double mutations of W90Y and R126E. In some embodiments, the APOBEC1 domain comprises double mutations of W90Y and R132E. In some embodiments, the APOBEC1 domain comprises double mutations of R126E and R132E. In some embodiments, the APOBEC1 domain comprises three mutations of W90Y, R126E and R132E. 
     In some embodiments, one or more mutations in the cytidine deaminase as disclosed herein reduce the editing window width to about 2 nucleotides. In some embodiments, one or more mutations in the cytidine deaminase as disclosed herein reduce the editing window width to about 1 nucleotide. In some embodiments, one or more mutations in the cytidine deaminase as disclosed herein reduce the editing window width while only minimally or modestly affecting the editing efficiency of the enzyme. In some embodiments, one or more mutations in the cytidine deaminase as disclosed herein reduce the editing window width without reducing the editing efficiency of the enzyme. In some embodiments, one or more mutations in the cytidine deaminase as disclosed herein enable discrimination of neighboring cytidine nucleotides, which would be otherwise edited with similar efficiency by the cytidine deaminase. 
     In some embodiments, the cytidine deaminase protein further comprises or is connected to one or more double-stranded RNA (dsRNA) binding motifs (dsRBMs) or domains (dsRBDs) for recognizing and binding to double-stranded nucleic acid substrates. In some embodiments, the interaction between the cytidine deaminase and the substrate is mediated by one or more additional protein factor(s), including a CRISPR/CAS protein factor. In some embodiments, the interaction between the cytidine deaminase and the substrate is further mediated by one or more nucleic acid component(s), including a guide RNA. 
     According to the present invention, the substrate of the cytidine deaminase is an DNA single strand bubble of a RNA duplex comprising a Cytosine of interest, made accessible to the cytidine deaminase upon binding of the guide molecule to its DNA target which then forms the CRISPR-Cas complex with the CRISPR-Cas enzyme, whereby the cytosine deaminase is fused to or is capable of binding to one or more components of the CRISPR-Cas complex, i.e. the CRISPR-Cas enzyme and/or the guide molecule. The particular features of the guide molecule and CRISPR-Cas enzyme are detailed below. 
     The cytidine deaminase or catalytic domain thereof may be a human, a rat, or a lamprey cytidine deaminase protein or catalytic domain thereof. 
     The cytidine deaminase protein or catalytic domain thereof may be an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase. The cytidine deaminase protein or catalytic domain thereof may be an activation-induced deaminase (AID). The cytidine deaminase protein or catalytic domain thereof may be a cytidine deaminase 1 (CDA1). 
     The cytidine deaminase protein or catalytic domain thereof may be an APOBEC1 deaminase. The APOBEC1 deaminase may comprise one or more mutations corresponding to W90A, W90Y, R118A, H121R, H122R, R126A, R126E, or R132E in rat APOBEC1, or an APOBEC3G deaminase comprising one or more mutations corresponding to W285A, W285Y, R313A, D316R, D317R, R320A, R320E, or R326E in human APOBEC3G. 
     The system may further comprise a uracil glycosylase inhibitor (UGI). Inn some embodiments, the cytidine deaminase protein or catalytic domain thereof is delivered together with a uracil glycosylase inhibitor (UGI). The GI may be linked (e.g., covalently linked) to the cytidine deaminase protein or catalytic domain thereof and/or a catalytically inactive CRISPR-Cas protein. 
     Regulation of Post-Translational Modification of Gene Products 
     In some cases, base editing may be used for regulating post-translational modification of a gene products. In some cases, an amino acid residue that is a post-translational modification site may be mutated by base editing to an amino residue that cannot be modified. Examples of such post-translational modifications include disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, methylation, ubiquitination, sumoylation, or any combinations thereof. 
     In some embodiments, the base editors herein may regulate Stat3/IRF-5 pathway, e.g., for reduction of inflammation. For example, phosphorylation on Tyr705 of Stat3, Thr10, Ser158, Ser309, Ser317, Ser451, and/or Ser462 of IRF-5 may be involved with interleukin signaling. Base editors herein may be used to mutate one or more of these procreation sites for regulating immunity, autoimmunity, and/or inflammation. 
     In some embodiments, the base editors herein may regulate insulin receptor substrate (IRS) pathway. For example, phosphorylation on Ser265, Ser302, Ser325, Ser336, Ser358, Ser407, and/or Ser408 may be involved in regulating (e.g., inhibit) ISR pathway. Alternatively, or additionally, Serine 307 in mouse (or Serine 312 in human) may be mutated so the phosphorylation may be regulated. For example, Serine 307 phosphorylation may lead to degradation of IRS-1 and reduce MAPK signaling. Serine 307 phosphorylation may be induced under insulin insensitivity conditions, such as insulin overstimulation and/or TNFα treatment. In some examples, S307F mutation may be generated for stabilizing the interaction between IRS-1 and other components in the pathway. Base editors herein may be used to mutate one or more of these procreation sites for regulating IRS pathway. 
     Regulation of Stability of Gene Products 
     In some embodiments, base editing may be used for regulating the stability of gene products. For example, one or more amino acid residues that regulate protein degradation rates may be mutated by the base editors herein. In some cases, such amino acid residues may be in a degron. A degron may refer to a portion of a protein involved in regulating the degradation rate of the protein. Degrons may include short amino acid sequences, structural motifs, and exposed amino acids (e.g., lysine or arginine). Some protein may comprise multiple degrons. The degrons be ubiquitin-dependent (e.g., regulating protein degradation based on ubiquitination of the protein) or ubiquitin-independent. 
     In some cases, the based editing may be used to mutate one or more amino acid residues in a signal peptide for protein degradation. In some examples, the signal peptide may be a PEST sequence, which is a peptide sequence that is rich in proline (P), glutamic acid (E), serine (S), and threonine (T). For example, the stability of NANOG, which comprises a PEST sequence, may be increased, e.g., to promote embryonic stem cell pluripotency. 
     In some examples, the base editors may be used for mutating SMN2 (e.g., to generate S270A mutilation) to increase stability of the SMN2 protein, which is involved in spinal muscular atrophy. Other mutations in SMN2 that may be generated by based editors include those described in Cho S. et al., Genes Dev. 2010 Mar. 1; 24(5): 438-442. In certain examples, the base editors may be used for generating mutations on IκBα, as described in Fortmann K T et al., J Mol Biol. 2015 Aug. 28; 427(17): 2748-2756. Target sites in degrons may be identified by computational tools, e.g., the online tools provided on slim.ucd.ie/apc/index.php. Other targets include Cdc25A phosphatase. 
     Examples of Genes that can be Targeted by Base Editors 
     Any desired genes can be targeted by the base editors in the CRISPR-Cas systems described herein. In some examples, the base editors may be used for modifying PCSK9. The base editors may introduce stop codons and/or disease-associated mutations that reduce PCSK9 activity. The base editing may introduce one or more of the following mutations in PCSK9: R46L, R46A, A53V, A53A, E57K, Y142X, L253F, R237W, H391N, N425S, A443T, I474V, I474A, Q554E, Q619P, E670G, E670A, C679X, H417Q, R469W, E482G, F515L, and/or H553R. 
     In some examples, the base editors may be used for modifying ApoE. The base editors may target ApoE in synthetic model and/or patient-derived neurons (e.g., those derived from iPSC). The targeting may be tested by sequencing. 
     In some examples, the base editors may be used for modifying Stat1/3. The base editor may target Y705 and/or S727 for reducing Stat1/3 activation. The base editing may be tested by luciferase-based promoter. Targeting Stat1/3 by base editing may block monocyte to macrophage differentiation, and inflammation in response to ox-LDL stimulation of macrophages. 
     In some examples, the base editors may be used for modifying TFEB (transcription factor for EB). The base editor may target one or more amino acid residues that regulate translocation of the TFEB. In some cases, the base editor may target one or more amino acid residues that regulate autophagy. 
     In some examples, the base editors may be used for modifying Lipin1. The base editor may target one or more serine&#39;s that can be phosphorylated by mTOR. Base editing of Lipin1 may regulate lipid accumulation. The base editors may target Lipin1 in 3T3L1 preadipocyte model. Effects of the base editing may be tested by measuring reduction of lipid accumulation (e.g., via oil red). 
     In some embodiments, the guide sequence is an RNA sequence of between 10 to 50 nt in length, but more particularly of about 20-30 nt advantageously about 20 nt, 23-25 nt or 24 nt. In base editing embodiments, the guide sequence is selected so as to ensure that it hybridizes to the target sequence comprising the adenosine to be deaminated. This is described more in detail below. Selection can encompass further steps which increase efficacy and specificity of deamination. 
     In some embodiments, the guide sequence is about 20 nt to about 30 nt long and hybridizes to the target DNA strand to form an almost perfectly matched duplex, except for having a dA-C mismatch at the target adenosine site. Particularly, in some embodiments, the dA-C mismatch is located close to the center of the target sequence (and thus the center of the duplex upon hybridization of the guide sequence to the target sequence), thereby restricting the adenosine deaminase to a narrow editing window (e.g., about 4 bp wide). In some embodiments, the target sequence may comprise more than one target adenosine to be deaminated. In further embodiments the target sequence may further comprise one or more dA-C mismatch 3′ to the target adenosine site. In some embodiments, to avoid off-target editing at an unintended Adenine site in the target sequence, the guide sequence can be designed to comprise a non-pairing Guanine at a position corresponding to said unintended Adenine to introduce a dA-G mismatch, which is catalytically unfavorable for certain adenosine deaminases such as ADAR1 and ADAR2. See Wong et al., RNA 7:846-858 (2001), which is incorporated herein by reference in its entirety. 
     In some embodiments, a CRISPR-Cas guide sequence having a canonical length (e.g., about 20 nt for AacC2c1) is used to form a heteroduplex with the target DNA. In some embodiments, a CRISPR-Cas guide molecule longer than the canonical length (e.g., &gt;20 nt for AacC2c1) is used to form a heteroduplex with the target DNA including outside of the CRISPR-Cas-guide RNA-target DNA complex. This can be of interest where deamination of more than one adenine within a given stretch of nucleotides is of interest. In alternative embodiments, it is of interest to maintain the limitation of the canonical guide sequence length. In some embodiments, the guide sequence is designed to introduce a dA-C mismatch outside of the canonical length of CRISPR-Cas guide, which may decrease steric hindrance by CRISPR-Cas and increase the frequency of contact between the adenosine deaminase and the dA-C mismatch. 
     In some base editing embodiments, the position of the mismatched nucleobase (e.g., cytidine) is calculated from where the PAM would be on a DNA target. In some embodiments, the mismatched nucleobase is positioned 12-21 nt from the PAM, or 13-21 nt from the PAM, or 14-21 nt from the PAM, or 14-20 nt from the PAM, or 15-20 nt from the PAM, or 16-20 nt from the PAM, or 14-19 nt from the PAM, or 15-19 nt from the PAM, or 16-19 nt from the PAM, or 17-19 nt from the PAM, or about 20 nt from the PAM, or about 19 nt from the PAM, or about 18 nt from the PAM, or about 17 nt from the PAM, or about 16 nt from the PAM, or about 15 nt from the PAM, or about 14 nt from the PAM. In a preferred embodiment, the mismatched nucleobase is positioned 17-19 nt or 18 nt from the PAM. 
     Mismatch distance is the number of bases between the 3′ end of the CRISPR-Cas spacer and the mismatched nucleobase (e.g., cytidine), wherein the mismatched base is included as part of the mismatch distance calculation. In some embodiment, the mismatch distance is 1-10 nt, or 1-9 nt, or 1-8 nt, or 2-8 nt, or 2-7 nt, or 2-6 nt, or 3-8 nt, or 3-7 nt, or 3-6 nt, or 3-5 nt, or about 2 nt, or about 3 nt, or about 4 nt, or about 5 nt, or about 6 nt, or about 7 nt, or about 8 nt. In a preferred embodiment, the mismatch distance is 3-5 nt or 4 nt. 
     In some embodiment, the editing window of a CRISPR-Cas-ADAR system described herein is 12-21 nt from the PAM, or 13-21 nt from the PAM, or 14-21 nt from the PAM, or 14-20 nt from the PAM, or 15-20 nt from the PAM, or 16-20 nt from the PAM, or 14-19 nt from the PAM, or 15-19 nt from the PAM, or 16-19 nt from the PAM, or 17-19 nt from the PAM, or about 20 nt from the PAM, or about 19 nt from the PAM, or about 18 nt from the PAM, or about 17 nt from the PAM, or about 16 nt from the PAM, or about 15 nt from the PAM, or about 14 nt from the PAM. In some embodiment, the editing window of the CRISPR-Cas-ADAR system described herein is 1-10 nt from the 3′ end of the CRISPR-Cas spacer, or 1-9 nt from the 3′ end of the CRISPR-Cas spacer, or 1-8 nt from the 3′ end of the CRISPR-Cas spacer, or 2-8 nt from the 3′ end of the C2c1 spacer, or 2-7 nt from the 3′ end of the CRISPR-Cas spacer, or 2-6 nt from the 3′ end of the CRISPR-Cas spacer, or 3-8 nt from the 3′ end of the CRISPR-Cas spacer, or 3-7 nt from the 3′ end of the CRISPR-Cas spacer, or 3-6 nt from the 3′ end of the CRISPR-Cas spacer, or 3-5 nt from the 3′ end of the CRISPR-Cas spacer, or about 2 nt from the 3′ end of the CRISPR-Cas spacer, or about 3 nt from the 3′ end of the CRISPR-Cas spacer, or about 4 nt from the 3′ end of the CRISPR-Cas spacer, or about 5 nt from the 3′ end of the CRISPR-Cas spacer, or about 6 nt from the 3′ end of the CRISPR-Cas spacer, or about 7 nt from the 3′ end of the CRISPR-Cas spacer, or about 8 nt from the 3′ end of the CRISPR-Cas spacer. 
     Linkers 
     The deaminase herein may be fused to a Cas protein described herein via a linker. It will be appreciated that other methods of incorporating a deaminase into the CRISPR-Cas system or Cas protein described herein are discussed elsewhere herein. It is further envisaged that RNA adenosine methylase (N(6)-methyladenosine) can be fused to the RNA targeting effector proteins of the invention and targeted to a transcript of interest. This methylase causes reversible methylation, has regulatory roles and may affect gene expression and cell fate decisions by modulating multiple RNA-related cellular pathways (Fu et al Nat Rev Genet. 2014; 15(5):293-306). 
     ADAR or other RNA modification enzymes may be linked (e.g., fused) to CRISPR-Cas or a dead CRISPR-Cas protein via a linker, e.g., to the C terminus or the N-terminus of CRISPR-Cas or dead CRISPR-Cas. 
     The term “linker” as used in reference to a fusion protein refers to a molecule which joins the proteins to form a fusion protein. Generally, such molecules have no specific biological activity other than to join or to preserve some minimum distance or other spatial relationship between the proteins. However, in certain embodiments, the linker may be selected to influence some property of the linker and/or the fusion protein such as the folding, net charge, or hydrophobicity of the linker. 
     Suitable linkers for use in the methods of the present invention are well known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. However, as used herein the linker may also be a covalent bond (carbon-carbon bond or carbon-heteroatom bond). In particular embodiments, the linker is used to separate the CRISPR-Cas protein and the nucleotide deaminase by a distance sufficient to ensure that each protein retains its required functional property. Preferred peptide linker sequences adopt a flexible extended conformation and do not exhibit a propensity for developing an ordered secondary structure. In certain embodiments, the linker can be a chemical moiety which can be monomeric, dimeric, multimeric or polymeric. Preferably, the linker comprises amino acids. Typical amino acids in flexible linkers include Gly, Asn and Ser. Accordingly, in particular embodiments, the linker comprises a combination of one or more of Gly, Asn and Ser amino acids. Other near neutral amino acids, such as Thr and Ala, also may be used in the linker sequence. Exemplary linkers are disclosed in Maratea et al. (1985), Gene 40: 39-46; Murphy et al. (1986) Proc. Nat&#39;l. Acad. Sci. USA 83: 8258-62; U.S. Pat. Nos. 4,935,233; and 4,751,180. For example, GlySer linkers GGS, GGGS or GSG can be used. GGS, GSG, GGGS or GGGGS (SEQ ID NO: 7) linkers can be used in repeats of 3 (such as (GGS) 3  (SEQ ID NO: 44), (GGGGS) 3 ) (SEQ ID NO: 10) or 5, 6, 7, 9 (SEQ ID NOS: 11, 12, 16, and 17) or even 12 (SEQ ID NO: 45) and others (see e.g. SEQ ID NOS: 6-20) or more, to provide suitable lengths. In some cases, the linker may be (GGGGS) 3-15  (SEQ ID NOS: 10-20 and 46, 47, 48), For example, in some cases, the linker may be (GGGGS) 3-11 , e.g., GGGGS (SEQ ID NO: 7), (GGGGS) 2  (SEQ ID NO: 14), (GGGGS) 3  (SEQ ID NO: 10), (GGGGS) 4  (SEQ ID NO: 15), (GGGGS) 5  (SEQ ID NO: 16), (GGGGS) 6  (SEQ ID NO: 11), (GGGGS) 7  (SEQ ID NO: 17), (GGGGS) 8  (SEQ ID NO: 18), (GGGGS) 9  (SEQ ID NO: 12), (GGGGS) 10  (SEQ ID NO: 19), or (GGGGS) 11  (SEQ ID NO: 20). 
     In particular embodiments, linkers such as (GGGGS) 3  (SEQ ID NO: 10) are preferably used herein. (GGGGS) 6  (SEQ ID NO: 11), (GGGGS) 9  (SEQ ID NO: 12),or (GGGGS) 12  (SEQ ID NO: 13) may preferably be used as alternatives. Other preferred alternatives are (GGGGS) 1  (SEQ ID NO: 7), (GGGGS) 2  (SEQ ID NO: 14), (GGGGS) 4  (SEQ ID NO: 15), (GGGGS) 5  (SEQ ID NO: 16), (GGGGS) 7  (SEQ ID NO: 17), (GGGGS) 8  (SEQ ID NO: 18), (GGGGS) 10  (SEQ ID NO: 19), or (GGGGS) 11  (SEQ ID NO: 20). In yet a further embodiment, LEPGEKPYKCPECGKSFSQSGALTRHQRTHTR (SEQ ID NO: 49) is used as a linker. In yet an additional embodiment, the linker is an XTEN linker. In particular embodiments, the CRISPR-Cas protein is a CRISPR-Cas protein and is linked to the deaminase protein or its catalytic domain by means of an LEPGEKPYKCPECGKSFSQSGALTRHQRTHTR (SEQ ID NO: 49) linker. In further particular embodiments, the CRISPR-Cas protein is linked C-terminally to the N-terminus of a deaminase protein or its catalytic domain by means of an LEPGEKPYKCPECGKSFSQSGALTRHQRTHTR (SEQ ID NO: 49) linker. In addition, N- and C-terminal NLSs can also function as linker (e.g., PKKKRKVEASSPKKRKVEAS (SEQ ID NOS. 49-50)). Further examples of linkers are shown in the Table 3 below. 
     
       
         
           
               
             
               
                 TABLE 3 
               
               
                   
               
               
                 Example Linkers 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 GGS 
                 GGTGGTAGT 
               
               
                   
               
               
                   
                 GGSx3 (9) 
                 GGTGGTAGTGGAGGGAGCGGCGGTTCA 
               
               
                   
                   
                 (SEQ ID NO: 51) 
               
               
                   
               
               
                   
                 GGSx7 (21) 
                 ggtggaggaggctctggtggaggcggt 
               
               
                   
                   
                 agcggaggcggagggtcgGGTGGTAGT 
               
               
                   
                   
                 GGAGGGAGCGGCGGTTCA 
               
               
                   
                   
                 (SEQ ID NO: 52) 
               
               
                   
               
               
                   
                 XTEN 
                 TCGGGATCTGAGACGCCTGGGACCTC 
               
               
                   
                   
                 GGAATCGGCTACGCCCGAAAGT 
               
               
                   
                   
                 (SEQ ID NO: 53) 
               
               
                   
               
               
                   
                 Z-EGFRShort 
                 gtggataacaaatttaacaaagaaat 
               
               
                   
                   
                 gtgggcggcgtgggaagaaattcgta 
               
               
                   
                   
                 acctgccgaacctgaacggctggcag 
               
               
                   
                   
                 atgaccgcgtttattgcgagcctggt 
               
               
                   
                   
                 ggatgatccgagccagagcgcgaacc 
               
               
                   
                   
                 tgctggcggaagcgaaaaaactgaac 
               
               
                   
                   
                 gatgcgcaggcgccgaaaaccggcgg 
               
               
                   
                   
                 tggttctggt (SEQ ID NO: 54) 
               
               
                   
               
               
                   
                 GSAT 
                 Ggtggttctgccggtggctccggtt 
               
               
                   
                   
                 ctggctccagcggtggcagctctgg 
               
               
                   
                   
                 tgcgtccggcacgggtactgcgggt 
               
               
                   
                   
                 ggcactggcagcggttccggtact 
               
               
                   
                   
                 ggctctggc (SEQ ID NO: 55) 
               
               
                   
               
            
           
         
       
     
     A nucleotide deaminase or other RNA modification enzyme may be linked to CRISPR-Cas or a dead CRISPR-Cas via one or more amino acids. In some cases, the nucleotide deaminase may be linked to the CRISPR-Cas or a dead CRISPR-Cas via one or more amino acids 411-429, 114-124, 197-241, and 607-624. The amino acid position may correspond to a CRISPR-Cas ortholog disclosed herein. In certain examples, the nucleotide deaminase may be is linked to the dead CRISPR-Cas via one or more amino acids corresponding to amino 411-429, 114-124, 197-241, and 607-624 of  Prevotella buccae  CRISPR-Cas. 
     Base Editing Guide Molecule Design Considerations 
     In some embodiments, the guide sequence is an RNA sequence of between 10 to 50 nt in length, but more particularly of about 20-30 nt advantageously about 20 nt, 23-25 nt or 24 nt. In base editing embodiments, the guide sequence is selected so as to ensure that it hybridizes to the target sequence comprising the adenosine to be deaminated. This is described more in detail below. Selection can encompass further steps which increase efficacy and specificity of deamination. 
     In some embodiments, the guide sequence is about 20 nt to about 30 nt long and hybridizes to the target DNA strand to form an almost perfectly matched duplex, except for having a dA-C mismatch at the target adenosine site. Particularly, in some embodiments, the dA-C mismatch is located close to the center of the target sequence (and thus the center of the duplex upon hybridization of the guide sequence to the target sequence), thereby restricting the adenosine deaminase to a narrow editing window (e.g., about 4 bp wide). In some embodiments, the target sequence may comprise more than one target adenosine to be deaminated. In further embodiments the target sequence may further comprise one or more dA-C mismatch 3′ to the target adenosine site. In some embodiments, to avoid off-target editing at an unintended Adenine site in the target sequence, the guide sequence can be designed to comprise a non-pairing Guanine at a position corresponding to said unintended Adenine to introduce a dA-G mismatch, which is catalytically unfavorable for certain adenosine deaminases such as ADAR1 and ADAR2. See Wong et al., RNA 7:846-858 (2001), which is incorporated herein by reference in its entirety. 
     In some embodiments, a CRISPR-Cas guide sequence having a canonical length (e.g., about 20 nt for AacC2c1) is used to form a heteroduplex with the target DNA. In some embodiments, a CRISPR-Cas guide molecule longer than the canonical length (e.g., &gt;20 nt for AacC2c1) is used to form a heteroduplex with the target DNA including outside of the CRISPR-Cas-guide RNA-target DNA complex. This can be of interest where deamination of more than one adenine within a given stretch of nucleotides is of interest. In alternative embodiments, it is of interest to maintain the limitation of the canonical guide sequence length. In some embodiments, the guide sequence is designed to introduce a dA-C mismatch outside of the canonical length of CRISPR-Cas guide, which may decrease steric hindrance by CRISPR-Cas and increase the frequency of contact between the adenosine deaminase and the dA-C mismatch. 
     In some base editing embodiments, the position of the mismatched nucleobase (e.g., cytidine) is calculated from where the PAM would be on a DNA target. In some embodiments, the mismatched nucleobase is positioned 12-21 nt from the PAM, or 13-21 nt from the PAM, or 14-21 nt from the PAM, or 14-20 nt from the PAM, or 15-20 nt from the PAM, or 16-20 nt from the PAM, or 14-19 nt from the PAM, or 15-19 nt from the PAM, or 16-19 nt from the PAM, or 17-19 nt from the PAM, or about 20 nt from the PAM, or about 19 nt from the PAM, or about 18 nt from the PAM, or about 17 nt from the PAM, or about 16 nt from the PAM, or about 15 nt from the PAM, or about 14 nt from the PAM. In a preferred embodiment, the mismatched nucleobase is positioned 17-19 nt or 18 nt from the PAM. 
     Mismatch distance is the number of bases between the 3′ end of the CRISPR-Cas spacer and the mismatched nucleobase (e.g., cytidine), wherein the mismatched base is included as part of the mismatch distance calculation. In some embodiment, the mismatch distance is 1-10 nt, or 1-9 nt, or 1-8 nt, or 2-8 nt, or 2-7 nt, or 2-6 nt, or 3-8 nt, or 3-7 nt, or 3-6 nt, or 3-5 nt, or about 2 nt, or about 3 nt, or about 4 nt, or about 5 nt, or about 6 nt, or about 7 nt, or about 8 nt. In a preferred embodiment, the mismatch distance is 3-5 nt or 4 nt. 
     In some embodiment, the editing window of a CRISPR-Cas-ADAR system described herein is 12-21 nt from the PAM, or 13-21 nt from the PAM, or 14-21 nt from the PAM, or 14-20 nt from the PAM, or 15-20 nt from the PAM, or 16-20 nt from the PAM, or 14-19 nt from the PAM, or 15-19 nt from the PAM, or 16-19 nt from the PAM, or 17-19 nt from the PAM, or about 20 nt from the PAM, or about 19 nt from the PAM, or about 18 nt from the PAM, or about 17 nt from the PAM, or about 16 nt from the PAM, or about 15 nt from the PAM, or about 14 nt from the PAM. In some embodiment, the editing window of the CRISPR-Cas-ADAR system described herein is 1-10 nt from the 3′ end of the CRISPR-Cas spacer, or 1-9 nt from the 3′ end of the CRISPR-Cas spacer, or 1-8 nt from the 3′ end of the CRISPR-Cas spacer, or 2-8 nt from the 3′ end of the C2c1 spacer, or 2-7 nt from the 3′ end of the CRISPR-Cas spacer, or 2-6 nt from the 3′ end of the CRISPR-Cas spacer, or 3-8 nt from the 3′ end of the CRISPR-Cas spacer, or 3-7 nt from the 3′ end of the CRISPR-Cas spacer, or 3-6 nt from the 3′ end of the CRISPR-Cas spacer, or 3-5 nt from the 3′ end of the CRISPR-Cas spacer, or about 2 nt from the 3′ end of the CRISPR-Cas spacer, or about 3 nt from the 3′ end of the CRISPR-Cas spacer, or about 4 nt from the 3′ end of the CRISPR-Cas spacer, or about 5 nt from the 3′ end of the CRISPR-Cas spacer, or about 6 nt from the 3′ end of the CRISPR-Cas spacer, or about 7 nt from the 3′ end of the CRISPR-Cas spacer, or about 8 nt from the 3′ end of the CRISPR-Cas spacer. 
     Prime Editors 
     In some embodiments, the non-Class I engineered CRISPR-Cas systems and/or any component thereof described herein can be configured to carry out prime editing or used with or in a prime editing system. General principles and concepts of prime editing are described in Anzalone et al. 2019. Nature. 576: 149-157, which is incorporated by reference herein and can be adapted for use with the CRISPR-Cas systems described herein. Like base editing systems, prime editing systems can be capable of targeted modification of a polynucleotide without generating double stranded breaks and does not require donor templates. Further prime editing systems can be capable of all 12 possible combination swaps. Prime editing can operate via a “search-and-replace” methodology and can mediate targeted insertions, deletions, all 12 possible base-to-base conversion and combinations thereof. Generally, a prime editing system, as exemplified by PE1, PE2, and PE3 (Id.), can include a reverse transcriptase fused or otherwise coupled or associated with an RNA-programmable nickase and a prime-editing extended guide RNA (pegRNA) to facility direct copying of genetic information from the extension on the pegRNA into the target polynucleotide. Embodiments that can be used with the present invention include these and variants thereof. Prime editing can have the advantage of lower off-target activity than traditional CRIPSR-Cas systems along with few byproducts and greater or similar efficiency as compared to traditional CRISPR-Cas systems. 
     In some embodiments, the prime editing guide molecule can specify both the target polynucleotide information (e.g., sequence) and contain a new polynucleotide cargo that replaces target polynucleotides. To initiate transfer from the guide molecule to the target polynucleotide, the PE system can nick the target polynucleotide at a target side to expose a 3′hydroxyl group, which can prime reverse transcription of an edit-encoding extension region of the guide molecule (e.g. a prime editing guide molecule or peg guide molecule) directly into the target site in the target polynucleotide. See e.g. Anzalone et al. 2019. Nature. 576: 149-157, particularly at FIGS. 1 b , 1 c , related discussion, and Supplementary discussion. 
     In some embodiments, a prime editing system can be composed of a Cas polypeptide (such as any of the Cas effectors described herein, including a Cas-like effector), having nickase activity, a reverse transcriptase, and a guide molecule. The Cas polypeptide can lack nuclease activity. The guide molecule can include a target binding sequence as well as a primer binding sequence and a template containing the edited polynucleotide sequence. The guide molecule, Cas polypeptide, and/or reverse transcriptase can be coupled together or otherwise associate with each other to form an effector complex and edit a target sequence. In some embodiments, the Cas polypeptide is a Class 2, Type V Cas polypeptide. In some embodiments, the Cas polypeptide is a Cas9 polypeptide (e.g. is a Cas9 nickase). In some embodiments, the Cas polypeptide is fused to the reverse transcriptase. In some embodiments, the Cas polypeptide is linked to the reverse transcriptase. 
     In some embodiments, the prime editing system can be a PE1 system or variant thereof, a PE2 system or variant thereof, or a PE3 (e.g. PE3, PE3b) system. See e.g., Anzalone et al. 2019. Nature. 576: 149-157, particularly at pgs. 2-3, FIGS. 2 a , 3 a -3 f , 4 a -4 b , Extended data FIGS. 3 a -3 b , and 4. 
     The peg guide molecule can be about 10 to about 200 or more nucleotides in length, such as 10 to/or 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 or more nucleotides in length. Optimization of the peg guide molecule can be accomplished as described in Anzalone et al. 2019. Nature. 576: 149-157, particularly at pg. 3, FIG. 2 a -2 b , and Extended Data FIGS. 5 a - c.    
     CRISPR Associated Transposase (CAST) Systems 
     In some embodiments, the non-Class I engineered CRISPR-Cas systems and/or any component thereof described herein can be configured as or be used in a CRISPR Associated Transposase (“CAST”) system. CAST system can include a Cas protein that is catalytically inactive, or engineered to be catalytically active, and further comprises a transposase (or subunits thereof) that catalyze RNA-guided DNA transposition. Such systems are able to insert DNA sequences at a target site in a DNA molecule without relying on host cell repair machinery. CAST systems can be Class 1 or Class 2 CAST systems. An example Class 1 system is described in Klompe et al. Nature, doi:10.1038/s41586-019-1323, which is in incorporated herein by reference. An example Class 2 system is described in Strecker et al. Science. 10/1126/science. aax9181 (2019), and PCT/US2019/066835 which are incorporated herein by reference. 
     In one aspect, the present disclosure includes systems comprising transposase(s) associated with, linked to, bound to, or otherwise capable of forming a complex with a CRISPR-Cas system. The system may further comprise one or more transposon components. In certain example embodiments, the one or more transposases, and the CRISPR-Cas system are associated by co-regulation or expression. In other example embodiments, the transposase(s) and CRISPR-Cas system are associated by the ability of the sequence-specific nucleotide-binding domain to direct or recruit the transposase(s) to an insertion site where the transposase(s) direct insertion of a donor polynucleotide into a target polynucleotide sequence. For ease of reference, further example embodiments will be discussed in the context of example CRISPR-associated transposase (CAST) systems. 
     In some embodiments, the non-Class I engineered CRISPR-Cas system, when configured as a CAST-system, includes one or more CRISPR-associated transposases or functional fragments thereof; one or more Cas proteins; and a guide molecule capable of complexing with the Cas protein and directing binding of the guide-Cas protein complex to a target polynucleotide. 
     In some examples, the non-Class I engineered CRISPR-Cas system, when configured as a CAST-system, includes one or more CRISPR-associated Tn7 transposases or functional fragments thereof; one or more Type I-B Cas proteins. In some examples, the system may comprise one or more CRISPR-associated Tn7 transposases or functional fragments thereof; one or more Type V-K Cas proteins (e.g., Cas12k). In some examples, the system may comprise one or more CRISPR-associated Tn5 transposases or functional fragments thereof; one or more Type II Cas proteins (e.g., Cas9). Examples of CAST systems include those described in Strecker J et al., RNA-guided DNA insertion with CRISPR-associated transposases, Science. 2019 Jul. 5; 365(6448): 48-53; Klompe S E et al., Transposon-encoded CRISPR-Cas Systems Direct RNA-guided DNA Integration, Nature. 2019 July; 571(7764):219-225; WO2019090173A1; WO2019090174A1; and WO2019090175A1, which are incorporated herein by reference in their entireties. 
     In certain examples, the system may comprise polynucleotides with sequences encoding the one or more transposases, Cas proteins, and guide sequences. 
     Transposons and Transposases 
     The systems herein may comprise one or more components of a transposon and/or one or more transposases. The transposases in the systems herein may be CRISPR-associated transposases (also used interchangeably with Cas-associated transposases, CRISPR-associated transposase proteins herein) or functional fragments thereof. CRISPR-associated transposases may include any transposases that can be directed to or recruited to a region of a target polynucleotide by sequence-specific binding of a CRISPR-Cas complex. CRISPR-associated transposases may include any transposases that associate (e.g., form a complex) with one or more components in a CRISPR-Cas system, e.g., Cas protein, guide molecule etc.). In certain example embodiments, CRISPR-associated transposases may be fused or tethered (e.g. by a linker) to one or more components in a CRISPR-Cas system, e.g., Cas protein, guide molecule etc.). 
     The term “transposon”, as used herein, refers to a polynucleotide (or nucleic acid segment), which may be recognized by a transposase or an integrase enzyme and which is a component of a functional nucleic acid-protein complex (e.g., a transpososome, or transposon complex) capable of transposition. Transposons employ a variety of regulatory mechanisms to maintain transposition at a low frequency and sometimes coordinate transposition with various cell processes. Some prokaryotic transposons can also mobilize functions that benefit the host or otherwise help maintain the element. 
     The term “transposase” as used herein refers to an enzyme, which is a component of a functional nucleic acid-protein complex capable of transposition and which mediates transposition. The transposase may comprise a single protein or comprise multiple protein subunits. A transposase may be an enzyme capable of forming a functional complex with a transposon end or transposon end sequences. The term “transposase” may also refer in certain embodiments to integrases. The expression “transposition reaction” used herein refers to a reaction wherein a transposase inserts a donor polynucleotide sequence in or adjacent to an insertion site on a target polynucleotide. The insertion site may contain a sequence or secondary structure recognized by the transposase and/or an insertion motif sequence where the transposase cuts or creates staggered breaks in the target polynucleotide into which the donor polynucleotide sequence may be inserted. Exemplary components in a transposition reaction include a transposon, comprising the donor polynucleotide sequence to be inserted, and a transposase or an integrase enzyme. The term “transposon end sequence” as used herein refers to the nucleotide sequences at the distal ends of a transposon. The transposon end sequences may be responsible for identifying the donor polynucleotide for transposition. The transposon end sequences may be the DNA sequences the transpose enzyme uses in order to form transpososome complex and to perform a transposition reaction. 
     T7 Transposases 
     In some embodiments, the system comprises one or more Tn7 or Tn7-like transposases. In a particular embodiment, the Tn7-like transposase may be a Tn5053 transposase. For example, the Tn5053 transposases include those described in Minakhina S et al., Tn5053 family transposons are res site hunters sensing plasmidal res sites occupied by cognate resolvases. Mol Microbiol. 1999 September; 33(5):1059-68; and FIG. 4 and related texts in Partridge S R et al., Mobile Genetic Elements Associated with Antimicrobial Resistance, Clin Microbiol Rev. 2018 Aug. 1; 31(4), both of which are incorporated by reference herein in their entirety. In some cases, the one or more Tn5053 transposases may comprise one or more of TniA, TniB, and TniQ. TniA is also known as TnsB. TniB is also known as TnsC. TniQ is also known as TnsD. Accordingly, in certain embodiments these Tn5053 transposase subunits may be referred to as TnsB, TnsC, and TnsD, respectively. In certain cases, the one or more transposases may comprise TnsB, TnsC, and TnsD. In one example, a CAST system comprises TniA, TniB, TniQ, Cas12k, tracrRNA, and guide RNA(s). In another example, a CAST system comprises TnsB, TnsC, TnsD, Cas12k, tracrRNA, and guide RNA(s). 
     In some examples, the one or more CRISPR-associated transposases may comprise: (a) TnsA, TnsB, TnsC, and TniQ, (b) TnsA, TnsB, and TnsC, (c) TnsB and TnsC, (d) TnsB, TnsC, and TniQ, (e) TnsA, TnsB, and TniQ, (f) TnsE, or (g) any combination thereof. In some cases, the TnsE does not bind to DNA. In some cases, CRISPR-associated transposase protein may comprise one or more transposases, e.g., one or more transposase subunits of a Tn7 transposase or Tn7-like transposase, e.g., one or more of TnsA, TnsB, TnsC, and TniQ. In some examples, the one or more transposases comprise TnsB, TnsC, and TniQ. 
     In some embodiments, three transposon-encoded proteins form the core transposition machinery of Tn7: a heteromeric transposase (TnsA and TnsB) and a regulator protein (TnsC). In addition to the core TnsABC transposition proteins, Tn7 elements encode dedicated target site-selection proteins, TnsD and TnsE. In conjunction with TnsABC, the sequence-specific DNA-binding protein TnsD directs transposition into a conserved site referred to as the “Tn7 attachment site,” attTn7. TnsD is a member of a large family of proteins that also includes TniQ, a protein found in other types of bacterial transposons. TniQ has been shown to target transposition into resolution sites of plasmids. As used herein, a TniQ transposase may be a TnsD transposase. Examples of Tn7 or Tn7-like transposases include TnsA, TnsB, TnsC, TniQ, TnsD, and TnsE. In some embodiments, the system comprises TnsA, TnsB, TnsC, and/or TniQ. Two or more of the components in the system may be comprised in a single protein (e.g., fusion protein). For example, TnsA and TnsB may be comprised in a single protein. 
     As used herein, a right end sequence element or a left end sequence element are made in reference to an example Tn7 transposon. The general structure of the left end (LE) and right end (RE) sequence elements of canonical Tn7 is established. Tn7 ends comprise a series of 22-bp TnsB-binding sites. Flanking the most distal TnsB-binding sites is an 8-bp terminal sequence ending with 5′-TGT-3′/3′-ACA-5′. The right end of Tn7 contains four overlapping TnsB-binding sites in the ˜90-bp right end element. The left end contains three TnsB-binding sites dispersed in the ˜150-bp left end of the element. The number and distribution of TnsB-binding sites can vary among Tn7-like elements. End sequences of Tn7-related elements can be determined by identifying the directly repeated 5-bp target site duplication, the terminal 8-bp sequence, and 22-bp TnsB-binding sites (Peters J E et al., 2017). Example Tn7 elements, including right end sequence element and left end sequence element include those described in Parks A R, Plasmid, 2009 January; 61(1):1-14. 
     Tn5 Transposases 
     In certain embodiments, the one or more transposases are one or more Tn5 transposases. In some examples, the transposases may comprise TnpA. The transposase may be a Y1 transposase of the IS200/IS605 family, encoded by the insertion sequence (IS) IS608 from  Helicobacter pylori , e.g., TnpAIS608. Examples of the transposases include those described in Barabas, O., Ronning, D. R., Guynet, C., Hickman, A. B., TonHoang, B., Chandler, M. and Dyda, F. (2008) Mechanism of IS200/IS605 family DNA transposases: activation and transposon-directed target site selection. Cell, 132, 208-220. In certain example embodiments, the transposase is a single stranded DNA transposase. The DNA transposase may be a Cas9 associated transposase. In certain example embodiments, the single stranded DNA transposase is TnpA or a functional fragment thereof. The Cas9 associated transposase systems may comprise a local architecture of Cas9-TnpA, Cas1-Cas2-CRISPR array. The Cas9 may or may not have a tracrRNA associated with it. The Cas9-associated transposase systems may be coded on the same strand or be part of a larger operon. In certain embodiments, the Cas9 may confer target specificity, allowing the TnpA to move a polynucleotide cargo from other target sites in a sequence specific matter. In certain example embodiments, the Cas9-associated transposase are derived from  Flavobacterium granuli  strain DSM-19729 , Salinivirga cyanobacteriivorans  strain L21-Spi-D4 , Flavobactrium aciduliphilum  strain DSM 25663 , Flavobacterium glacii  strain DSM 19728 , Niabella soli  DSM 19437 , Salnivirga cyanobactriivorans  strain L21-Spi-D4 , Alkaliflexus imshenetskii  DSM 150055 strain Z-7010, or  Alkalitala saponilacus.    
     In certain embodiments, the transposase is a single-stranded DNA transposase. The single stranded DNA transposase may be TnpA, a functional fragment thereof, or a variant thereof. In certain embodiments, the transposase is a Himar1 transposase, a fragment thereof, or a variant thereof. In one example, the system comprises a dead Cas9 associated with Himar1. 
     In certain embodiments, the transposases may be one or more  Vibrio cholerae  Tn6677 transposases. In one example, the system may comprise components of variant Type I-F CRISPR-Cas system or polynucleotide(s) encoding thereof. The transposon may include a terminal operon comprising the tnsA, tnsB, and tnsC genes. The transposon may further comprise a tniQ gene. The tniQ gene may be encoded within the cas rather than tns operon. In certain embodiments, the TnsE may be absent in the transposon. 
     Mu Transposases 
     The transposases may be one of the Mu family transposon systems, e.g., transposon of bacteriophage Mu, a bacterial class III transposon of  Escherichia coli . In some cases, this transposon exhibits high transposition frequency. The Mu bacteriophage with its approximately 37 kb genome is relatively large compared to other transposons. The Mu transposon may have left end and right end transposase (e.g., MuA) recognition sequences (designated “L” and “R”, respectively) that flank the Mu transposable cassette, the region of the transposon that is ultimately integrated into the target site. In some examples, these ends are not inverted repeat sequences. The Mu transposable cassette, when necessary, may include a transpositional enhancer sequence (also referred to herein as the internal activating sequence, or “IAS”) located approximately 950 base pairs inward from the left end recognition sequence. 
     In some examples, a Mu transposon may have a 22 bp symmetrical consensus sequence, located near both ends, for recognition by a Mu transposase (MuA). Random transposition of a Mu transposon into a target gene occur through (1) binding of transposase (e.g., MuA) monomers to the Mu transposon recognition sites to form transposome assemblies, (2) tetramerization of the bound transposase (e.g., MuA) monomers to bridge the ends of the Mu transposon and engage the Mu transposon cleavage sites, (3) subsequent self-cleavage of the Mu transposon at the cleavage sites, and (4) accurate occurrence of a 5 bp staggered cut in a host DNA sequence into which the Mu transposon is subsequently incorporated. 
     The transposases may be Mu transposase family. Examples of transposases in the Mu family includes MuA, MuB, and MuC. 
     In some examples, MuA may be a about 75-kDa multidomain protein (about 663 amino acids) and can be divided into structurally and functionally defined major domains (I, II, III) and subdomains (Iα, Iβ, Iγ; IIα, IIβ; IIIα, IIIβ) The N-terminal subdomain Iα promotes transpososome assembly via an initial binding to a specific transpositional enhancer sequence. The specific DNA binding to transposon ends, crucial for the transpososome assembly, is mediated through amino acid residues located in subdomains Iβ and Iγ. Subdomain IIα contains the critical DDE-motif of acidic residues (D269, D336 and E392), which is involved in the metal ion coordination during the catalysis. Subdomains IIβ and IIIα participate in nonspecific DNA binding, and they appear important during structural transitions. Subdomain IIIα also displays a cryptic endonuclease activity, which is required for the removal of the attached host DNA following the integration of infecting Mu. The C-terminal subdomain IIIβ is responsible for the interaction with the phage-encoded MuB protein, important in targeting transposition into distal target sites. This subdomain is also important in interacting with the host-encoded C1pX protein, a factor which remodels the transpososome for disassemble. 
     In some examples, MuA may catalyze the steps of transposition: (i) initial cleavages at the transposon-host boundaries (donor cleavage) and (ii) covalent integration of the transposon into the target DNA (strand transfer). These steps may proceed via sequential structural transitions within a nucleoprotein complex, a transpososome, the core of which contains four MuA molecules and two synapsed transposon ends. In vivo, the critical MuA-catalyzed reaction steps may also involve the phage-encoded MuB targeting protein, host-encoded DNA architectural proteins (HU and IHF), certain DNA cofactors (MuA binding sites and transpositional enhancer sequence), as well as stringent DNA topology. The reaction steps mimicking Mu transposition into external target DNA can be reconstituted in vitro using MuA transposase, 50 bp Mu R-end DNA segments, and target DNA as the only macromolecular components. 
     In some examples, MuA and variants include those disclosed by EBI accession No. UNIPROT:Q58ZD8 which has 36% identity to wild type MuA protein; Naigamwalla et al., 1998, (Journal of Molecular Biology 282:265-274) (mutations in domain IIIa of the Mu transposase protein); Rasila et al., 2012, (Plos One, 7(5):E37922) (functional mapping of MuA transposase family protein structures with scanning mutagenesis); WO 2010/099296 (hyperactive piggyback transposases). 
     In some examples, MuB may be an ATP-dependent DNA binding protein, which is required for efficient transposition in vivo. Bacteriophage Mu transposition may be influenced by the ATP-utilizing protein MuB. In vitro, the MuA transposase may direct insertions into targets that are bound by MuB. In some cases, there is no particular sequence specificity to MuB binding. However, its distribution on DNA may not be random: MuB binding to target molecules that already contain Mu sequences is specifically destabilized through an ATP-dependent mechanism (19). In some examples, MuB also stimulates the DNA-breakage and DNA-joining activities of MuA (Adzuma and Mizuuchi (1988) Cell 53:257-266; Baker et al. (1991) Cell 65:1003-1013; Maxwell et al. (1987) Proc. Natl. Acad. Sci. USA 84:699-703; Surette and Chaconas (1991) J. Biol Chem. 266:17306-17313; Surette et al. (1991) J. Biol. Chem. 266:3118-3124; and Wu and Chaconas (1992) J. Biol. Chem. 267:9552-9558; and Wu and Chaconas, (1994) J. Biol. Chem. 269:28829-28833). 
     Donor Polynucleotides 
     The systems may comprise one or more donor polynucleotides (e.g., for insertion into the target polynucleotide). A donor polynucleotide may be an equivalent of a transposable element that can be inserted or integrated to a target site. For example, the donor polynucleotide may comprise a polynucleotide to be inserted, a left element sequence, and a right element sequence. The donor polynucleotide may be or comprise one or more components of a transposon. A donor polynucleotide may be any type of polynucleotides, including, but not limited to, a gene, a gene fragment, a non-coding polynucleotide, a regulatory polynucleotide, a synthetic polynucleotide, etc. 
     In some embodiments, the donor polynucleotide is linear. In some embodiments, the donor polynucleotide is circular. In some examples, the donor polynucleotide has a single strand break (a nick). In some cases, the single strand break is on or close to the 3′ end of the donor polynucleotide. In some cases, the single strand break is on or close to the 5′ end of the donor polynucleotide. 
     The donor polynucleotides may be inserted to the upstream or downstream of a PAM sequence of a target polynucleotide. For CRISPR-associated transposases, the donor polynucleotide may be inserted at a position between 10 bases and 200 bases, e.g., between 20 bases and 150 bases, between 30 bases and 100 bases, between 45 bases and 70 bases, between 45 bases and 60 bases, between 55 bases and 70 bases, between 49 bases and 56 bases or between 60 bases and 66 bases, from a PAM sequence on the target polynucleotide. In some cases, the insertion is at a position upstream of the PAM sequence. In some cases, the insertion is at a position downstream of the PAM sequence. In some cases, the insertion is at a position from 49 to 56 bases or base pairs downstream from a PAM sequence. In some cases, the insertion is at a position from 60 to 66 bases or base pairs downstream from a PAM sequence. 
     The donor polynucleotide may be used for editing the target polynucleotide. In some cases, the donor polynucleotide comprises one or more mutations to be introduced into the target polynucleotide. Examples of such mutations include substitutions, deletions, insertions, or a combination thereof. The mutations may cause a shift in an open reading frame on the target polynucleotide. In some cases, the donor polynucleotide alters a stop codon in the target polynucleotide. For example, the donor polynucleotide may correct a premature stop codon. The correction may be achieved by deleting the stop codon or introduces one or more mutations to the stop codon. In other example embodiments, the donor polynucleotide addresses loss of function mutations, deletions, or translocations that may occur, for example, in certain disease contexts by inserting or restoring a functional copy of a gene, or functional fragment thereof, or a functional regulatory sequence or functional fragment of a regulatory sequence. A functional fragment refers to less than the entire copy of a gene by providing sufficient nucleotide sequence to restore the functionality of a wild type gene or non-coding regulatory sequence (e.g. sequences encoding long non-coding RNA). In certain example embodiments, the systems disclosed herein may be used to replace a single allele of a defective gene or defective fragment thereof. In another example embodiment, the systems disclosed herein may be used to replace both alleles of a defective gene or defective gene fragment. A “defective gene” or “defective gene fragment” is a gene or portion of a gene that when expressed fails to generate a functioning protein or non-coding RNA with functionality of a the corresponding wild-type gene. In certain example embodiments, these defective genes may be associated with one or more disease phenotypes. In certain example embodiments, the defective gene or gene fragment is not replaced but the systems described herein are used to insert donor polynucleotides that encode gene or gene fragments that compensate for or override defective gene expression such that cell phenotypes associated with defective gene expression are eliminated or changed to a different or desired cellular phenotype. 
     In certain embodiments of the invention, the donor may include, but not be limited to, genes or gene fragments, encoding proteins or RNA transcripts to be expressed, regulatory elements, repair templates, and the like. According to the invention, the donor polynucleotides may comprise may comprise left end and right end sequence elements that function with transposition components that mediate insertion. 
     In certain cases, the donor polynucleotide manipulates a splicing site on the target polynucleotide. In some examples, the donor polynucleotide disrupts a splicing site. The disruption may be achieved by inserting the polynucleotide to a splicing site and/or introducing one or more mutations to the splicing site. In certain examples, the donor polynucleotide may restore a splicing site. For example, the polynucleotide may comprise a splicing site sequence. 
     The donor polynucleotide to be inserted may has a size from 10 bases to 50 kb in length, e.g., from 50 to 40 kb, from 100 and 30 kb, from 100 bases to 300 bases, from 200 bases to 400 bases, from 300 bases to 500 bases, from 400 bases to 600 bases, from 500 bases to 700 bases, from 600 bases to 800 bases, from 700 bases to 900 bases, from 800 bases to 1000 bases, from 900 bases to from 1100 bases, from 1000 bases to 1200 bases, from 1100 bases to 1300 bases, from 1200 bases to 1400 bases, from 1300 bases to 1500 bases, from 1400 bases to 1600 bases, from 1500 bases to 1700 bases, from 600 bases to 1800 bases, from 1700 bases to 1900 bases, from 1800 bases to 2000 bases, from 1900 bases to 2100 bases, from 2000 bases to 2200 bases, from 2100 bases to 2300 bases, from 2200 bases to 2400 bases, from 2300 bases to 2500 bases, from 2400 bases to 2600 bases, from 2500 bases to 2700 bases, from 2600 bases to 2800 bases, from 2700 bases to 2900 bases, or from 2800 bases to 3000 bases in length. 
     Accessory Molecules 
     Additional accessory molecules, such as additional CRISPR effectors and/or other accessory molecules can be included in the non-Class I nucleic acid targeting systems described herein in addition to the Cas-like polypeptides described elsewhere herein. In some aspects, the accessory molecules can be other effector and/or targeting proteins or molecules. Accessory molecules can be or be derived from a Type I, II, III, IV, V, CRISPR-Cas system. 
     In certain embodiments, an accessory molecule can be identified by their proximity to a Cas gene and/or a CRISPR array (e.g. within the region 20 kb from the start of the Cas gene and/or CRISPR array). Non-limiting examples of Cas proteins that can be included as accessory molecules include, but are not limited to, Cas 1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cash, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Cas12 (also known as Cpfl), Cas13, Cas 14, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, C2c2, homologues thereof, orthologues thereof, or modified versions thereof. The terms “orthologue” (also referred to as “ortholog” herein) and “homologue” (also referred to as “homolog” herein) are well known in the art. By means of further guidance, a “homologue” of a protein as used herein is a protein of the same species which performs the same or a similar function as the protein it is a homologue of. Homologous proteins may but need not be structurally related, or are only partially structurally related. An “orthologue” of a protein as used herein is a protein of a different species which performs the same or a similar function as the protein it is an orthologue of. Orthologous proteins may, but need not be structurally related, or are only partially structurally related. 
     In some embodiments, one or more elements of a nucleic acid-targeting system is derived from a particular organism comprising an endogenous RNA-targeting system. In particular embodiments, the Type VI RNA-targeting Cas enzyme is C2c2. In an embodiment of the invention, there is provided a effector protein which comprises an amino acid sequence having at least 80% sequence homology to the wild-type sequence of any of  Leptotrichia shahii  C2c2, Lachnospiraceae bacterium MA2020 C2c2, Lachnospiraceae bacterium NK4A179 C2c2 , Clostridium aminophilum  (DSM 10710) C2c2 , Carnobacterium gallinarum  (DSM 4847) C2c2 , Paludibacter propionicigenes  (WB4) C2c2 , Listeria weihenstephanensis  (FSL R9-0317) C2c2 , Listeriaceae bacterium  (FSL M6-0635) C2c2 , Listeria newyorkensis  (FSL M6-0635) C2c2 , Leptotrichia wadei  (F0279) C2c2 , Rhodobacter capsulatus  (SB 1003) C2c2 , Rhodobacter capsulatus  (R121) C2c2 , Rhodobacter capsulatus  (DE442) C2c2 , Leptotrichia wadei  (Lw2) C2c2, or  Listeria seeligeri  C2c2. 
     Adaptors 
     In certain embodiments, and as is also described elsewhere herein, the non-class I engineered CRISPR-Cas system described herein can include on or more adaptor proteins. In certain embodiments, the adaptor protein can bind to RNA. The adaptor proteins can be capable of recruitment of, for example, effector proteins or fusions that can have one or more functional domains. In some embodiments, the functional domain is a transcriptional activation domain, preferably VP64. In some embodiments, the functional domain is a transcription repression domain, preferably KRAB. In some embodiments, the transcription repression domain is SID, or concatemers of SID (e.g. SID4X). In some embodiments, the functional domain is an epigenetic modifying domain, such that an epigenetic modifying enzyme is provided. In some embodiments, the functional domain is an activation domain, which may be the P65 activation domain. 
     The functional domain can be, for example, one or more domains from the group consisting of methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, and molecular switches (e.g. light inducible). In some embodiments, the functional domain may be selected from the group of: transposase domain, integrase domain, recombinase domain, resolvase domain, invertase domain, protease domain, DNA methyltransferase domain, DNA hydroxylmethylase domain, DNA demethylase domain, histone acetylase domain, histone deacetylases domain, nuclease domain, repressor domain, activator domain, nuclear-localization signal domains, transcription-regulatory protein (or transcription complex recruiting) domain, cellular uptake activity associated domain, nucleic acid binding domain, antibody presentation domain, histone modifying enzymes, recruiter of histone modifying enzymes; inhibitor of histone modifying enzymes, histone methyltransferase, histone demethylase, histone kinase, histone phosphatase, histone ribosylase, histone deribosylase, histone ubiquitinase, histone deubiquitinase, histone biotinase and histone tail protease. 
     Endogenous transcriptional repression is often mediated by chromatin modifying enzymes such as histone methyltransferases (HMTs) and deacetylases (HDACs). Repressive histone effector domains are known and an exemplary list is provided below. In the exemplary table, preference was given to proteins and functional truncations of small size to facilitate efficient viral packaging (for instance via AAV). In general, however, the domains may include HDACs, histone methyltransferases (HMTs), and histone acetyltransferase (HAT) inhibitors, as well as HDAC and HMT recruiting proteins. The functional domain may be or include, in some embodiments, HDAC Effector Domains, HDAC Recruiter Effector Domains, Histone Methyltransferase (HMT) Effector Domains, Histone Methyltransferase (HMT) Recruiter Effector Domains, or Histone Acetyltransferase Inhibitor Effector Domains. Tables 4-9 below show exemplary chromatin modifying enzymes and/or domains. 
                     TABLE 4                  HDAC Effector Domains                                                                         Selected               Subtype/       Substrate   Modification       Full   truncation   Final   Catalytic       Complex   Name   (if known)   (if known)   Organism   size (aa)   (aa)   size (aa)   domain                                                         HDAC I   HDAC8   —   —     X. laevis     325   1-325   325    1-272:                                       HDAC       HDAC I   RPD3   —   —     S.     433   19-340    322   19-331:                         cerevisiae             (Vannier)   HDAC       HDAC IV   MesoLo4   —   —     M. loti     300   1-300   300   —                               (Gregoretti)       HDAC IV   HDAC11   —   —     H.     347   1-347   347   14-326:                         sapiens         (Gao)       HDAC       HD2   HDT1   —   —     A.     245   1-211   211   —                         thaliana         (Wu)       SIRT I   SIRT3   H3K9Ac   —     H.     399   143-399    257   126-382:                H4K16Ac         sapiens         (Scher)       SIRT               H3K56Ac       SIRT I   HST2   —   —     C.     331   1-331   331   —                         albicans         (Hnisz)       SIRT I   CobB   —   —     E. coli     242   1-242   242   —                       (K12)       (Landry)       SIRT I   HST2   —   —     S.     357   8-298   291   —                         cerevisiae         (Wilson)       SIRT III   SIRT5   H4K8Ac   —     H.     310   37-310    274   41-309:               H4K16Ac         sapiens         (Gertz)       SIRT       SIRT III   Sir2A   —   —     P.     273   1-273   273   19-273:                         falciparum         (Zhu)       SIRT       SIRT IV   SIRT6   H3K9Ac   —     H.     355   1-289   289   35-274:               H3K56Ac         sapiens         (Tennen)       SIRT                    
Accordingly, the repressor domains of the present invention may be selected from histone methyltransferases (HMTs), histone deacetylases (HDACs), histone acetyltransferase (HAT) inhibitors, as well as HDAC and HMT recruiting proteins.
 
     The HDAC domain may be any of those in the table above, namely: HDAC8, RPD3, MesoLo4, HDAC11, HDT1, SIRT3, HST2, CobB, HST2, SIRT5, Sir2A, or SIRT6. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 HDAC Recruiter Effector Domains 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                 Selected 
                   
                   
               
               
                 Subtype/ 
                   
                 Substrate 
                 Modification 
                   
                 Full 
                 truncation 
                 Final 
                 Catalytic 
               
               
                 Complex 
                 Name 
                 (if known) 
                 (if known) 
                 Organism 
                 size (aa) 
                 (aa) 
                 size (aa) 
                 domain 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Sin3a 
                 MeCP2 
                 — 
                 — 
                 
                   R. 
                 
                 492 
                 207-492 
                 286 
                 — 
               
               
                   
                   
                   
                   
                 
                   norvegicus 
                 
                   
                 (Nan) 
               
               
                 Sin3a 
                 MBD2b 
                 — 
                 — 
                 
                   H. 
                 
                 262 
                  45-262 
                 218 
                 — 
               
               
                   
                   
                   
                   
                 
                   sapiens 
                 
                   
                 (Boeke) 
               
               
                 Sin3a 
                 Sin3a 
                 — 
                 — 
                 
                   H. 
                 
                 1273 
                 524-851 
                 328 
                 627-829: 
               
               
                   
                   
                   
                   
                 
                   sapiens 
                 
                   
                 (Laherty) 
                   
                 HDAC1 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 interaction 
               
               
                 NcoR 
                 NcoR 
                 — 
                 — 
                 
                   H. 
                 
                 2440 
                 420-488 
                 69 
                 — 
               
               
                   
                   
                   
                   
                 
                   sapiens 
                 
                   
                 (Zhang) 
               
               
                 NuRD 
                 SALL1 
                 — 
                 — 
                 
                   M. 
                 
                 1322 
                  1-93 
                 93 
                 — 
               
               
                   
                   
                   
                   
                 
                   musculus 
                 
                   
                 (Lauberth) 
               
               
                 CoREST 
                 RCOR1 
                 — 
                 — 
                 
                   H. 
                 
                 482 
                  81-300 
                 220 
                 — 
               
               
                   
                   
                   
                   
                 
                   sapiens 
                 
                   
                 (Gu, Ouyang) 
               
               
                   
               
            
           
         
       
     
     In some embodiments, the functional domain may be a HDAC Recruiter Effector Domain. Preferred examples include those in the Table(s) below, namely MeCP2, MBD2b, Sin3a, NcoR, SALL1, RCOR1. NcoR is exemplified in the present Examples and, although preferred, it is envisaged that others in the class will also be useful. 
     In some embodiments, the functional domain may be a Methyltransferase (HMT) Effector Domain. Preferred examples include those in the Table(S) below, namely NUE, vSET, EHMT2/G9A, SUV39H1, dim-5, KYP, SUVR4, SET4, SET1, SETD8, and TgSET8. NUE is exemplified in the present Examples and, although preferred, it is envisaged that others in the class will also be useful. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Histone Methyltransferase (HMT) Effector Domains 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                 Selected 
                   
                   
               
               
                 Subtype/ 
                   
                 Substrate 
                 Modification 
                   
                 Full 
                 truncation 
                 Final 
                 Catalytic 
               
               
                 Complex 
                 Name 
                 (if known) 
                 (if known) 
                 Organism 
                 size (aa) 
                 (aa) 
                 size (aa) 
                 domain 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 SET 
                 NUE 
                 H2B, H3, H4 
                 — 
                 
                   C. 
                 
                 219 
                 1-219 
                 219 
                 — 
               
               
                   
                   
                   
                   
                 
                   trachomatis 
                 
                   
                 (Pennini) 
               
               
                 SET 
                 vSET 
                 — 
                 H3K27me3 
                 
                   P. 
                 
                 119 
                 1-119 
                 119 
                  4-112: 
               
               
                   
                   
                   
                   
                 
                   bursaria 
                 
                   
                 (Mujtaba) 
                   
                 SET2 
               
               
                   
                   
                   
                   
                 chlorella 
               
               
                   
                   
                   
                   
                 virus 
               
               
                 SUV39 
                 EHMT2/ 
                 H1.4K2, H3K9, 
                 H3K9me1/2, 
                 
                   M. 
                 
                 1263 
                 969-1263  
                 295 
                 1025-1233: 
               
               
                 family 
                 G9A 
                 H3K27 
                 H1K25me1 
                 
                   musculus 
                 
                   
                 (Tachibana) 
                   
                 preSET, SET, 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 postSET 
               
               
                 SUV39 
                 SUV39H1 
                 — 
                 H3K9me2/3 
                 
                   H. 
                 
                 412 
                 79-412  
                 334 
                 172-412: 
               
               
                   
                   
                   
                   
                 
                   sapiens 
                 
                   
                 (Snowden) 
                   
                 preSET, SET, 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 postSET 
               
               
                 Suvar3-9 
                 dim-5 
                 — 
                 H3K9me3 
                 
                   N. 
                 
                 331 
                 1-331 
                 331 
                  77-331: 
               
               
                   
                   
                   
                   
                 
                   crassa 
                 
                   
                 (Rathert) 
                   
                 preSET, SET, 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 postSET 
               
               
                 Suvar3-9 
                 KYP 
                 — 
                 H3K9me1/2 
                 
                   A. 
                 
                 624 
                 335-601  
                 267 
                 — 
               
               
                 (SUVH 
                   
                   
                   
                 
                   thaliana 
                 
                   
                   
                 (Jackson) 
               
               
                 subfamily) 
               
               
                 Suvar3-9 
                 SUVR4 
                 H3K9me1 
                 H3K9me2/3 
                 
                   A. 
                 
                 492 
                 180-492  
                 313 
                 192-462: 
               
               
                 (SUVR 
                   
                   
                   
                 
                   thaliana 
                 
                   
                   
                 (Thorstensen) 
                 preSET, SET, 
               
               
                 subfamily) 
                   
                   
                   
                   
                   
                   
                   
                 postSET 
               
               
                 Suvar4-20 
                 SET4 
                 — 
                 H4K20me3 
                 
                   C. 
                 
                 288 
                 1-288 
                 288 
                 — 
               
               
                   
                   
                   
                   
                 
                   elegans 
                 
                   
                 (Vielle) 
               
               
                 SET8 
                 SET1 
                 — 
                 H4K20me1 
                 
                   C. 
                 
                 242 
                 1-242 
                 242 
                 — 
               
               
                   
                   
                   
                   
                 
                   elegans 
                 
                   
                 (Vielle) 
               
               
                 SET8 
                 SETD8 
                 — 
                 H4K20me1 
                 
                   H. 
                 
                 393 
                 185-393  
                 209 
                 256-382: 
               
               
                   
                   
                   
                   
                 
                   sapiens 
                 
                   
                   
                 (Couture) 
                 SET 
               
               
                 SET8 
                 TgSET8 
                 — 
                 H4K20me1/2/3 
                 
                   T. gondii 
                 
                 1893 
                 1590-1893  
                 304 
                 1749-1884: 
               
               
                   
                   
                   
                   
                   
                   
                 (Sautel) 
                   
                 SET 
               
               
                   
               
            
           
         
       
     
     In some embodiments, the functional domain may be a Histone Methyltransferase (HMT) Recruiter Effector Domain. Preferred examples include those in the Table below, namely Hp1a, PHF19, and NIPP1. 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Histone Methyltransferase (HMT) Recruiter Effector Domains 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                 Selected 
                   
                   
               
               
                 Subtype/ 
                   
                 Substrate 
                 Modification 
                   
                 Full 
                 truncation 
                 Final 
                 Catalytic 
               
               
                 Complex 
                 Name 
                 (if known) 
                 (if known) 
                 Organism 
                 size (aa) 
                 (aa) 
                 size (aa) 
                 domain 
               
               
                   
               
               
                 — 
                 Hp1a 
                 — 
                 H3K9me3 
                 
                   M. 
                 
                 191 
                 73-191 
                 119 
                 121-179: 
               
               
                   
                   
                   
                   
                 
                   musculus 
                 
                   
                   
                 (Hathaway) 
                 chromoshadow 
               
               
                 — 
                 PHF19 
                 — 
                 H3K27me3 
                 
                   H. 
                 
                 580 
                 (1-250) + 
                 335 
                 163-250: 
               
               
                   
                   
                   
                   
                 
                   sapiens 
                 
                   
                 GGSG linker 
                 (Ballaré) 
                 PHD2 
               
               
                   
                   
                   
                   
                   
                   
                 (SEQ ID 
               
               
                   
                   
                   
                   
                   
                   
                 NO: 43) + 
               
               
                   
                   
                   
                   
                   
                   
                 (500-580) 
               
               
                 — 
                 NIPP1 
                 — 
                 H3K27me3 
                 
                   H. 
                 
                 351 
                  1-329 
                 329 
                 310-329: 
               
               
                   
                   
                   
                   
                 
                   sapiens 
                 
                   
                 (Jin) 
                   
                 EED 
               
               
                   
               
            
           
         
       
     
     In some embodiments, the functional domain may be Histone Acetyltransferase Inhibitor Effector Domain. Preferred examples include SET/TAF-1β listed in the Table below. 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Histone Acetyltransferase Inhibitor Effector Domains 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                 Selected 
                   
                   
               
               
                 Subtype/ 
                   
                 Substrate 
                 Modification 
                   
                 Full 
                 truncation 
                 Final 
                 Catalytic 
               
               
                 Complex 
                 Name 
                 (if known) 
                 (if known) 
                 Organism 
                 size (aa) 
                 (aa) 
                 size (aa) 
                 domain 
               
               
                   
               
               
                 — 
                 SET/TAF-1β 
                 — 
                 — 
                 
                   M. 
                 
                 289 
                 1-289 
                 289 
                 — 
               
               
                   
                   
                   
                   
                 
                   musculus 
                 
                   
                 (Cervoni) 
               
               
                   
               
            
           
         
       
     
     It is also preferred to target endogenous (regulatory) control elements (such as enhancers and silencers) in addition to a promoter or promoter-proximal elements. Thus, the invention can also be used to target endogenous control elements (including enhancers and silencers) in addition to targeting of the promoter. These control elements can be located upstream and downstream of the transcriptional start site (TSS), starting from 200 bp from the TSS to 100 kb away. Targeting of known control elements can be used to activate or repress the gene of interest. In some cases, a single control element can influence the transcription of multiple target genes. Targeting of a single control element could therefore be used to control the transcription of multiple genes simultaneously. 
     Targeting of putative control elements on the other hand (e.g. by tiling the region of the putative control element as well as 200 bp up to 100 kB around the element) can be used as a means to verify such elements (by measuring the transcription of the gene of interest) or to detect novel control elements (e.g. by tiling 100 kb upstream and downstream of the TSS of the gene of interest). In addition, targeting of putative control elements can be useful in the context of understanding genetic causes of disease. Many mutations and common SNP variants associated with disease phenotypes are located outside coding regions. Targeting of such regions with either the activation or repression systems described herein can be followed by readout of transcription of either a) a set of putative targets (e.g. a set of genes located in closest proximity to the control element) or b) whole-transcriptome readout by e.g. RNAseq or microarray. This would allow for the identification of likely candidate genes involved in the disease phenotype. Such candidate genes could be useful as novel drug targets. 
     Histone acetyltransferase (HAT) inhibitors are mentioned herein. However, an alternative in some embodiments is for the one or more functional domains to comprise an acetyltransferase, preferably a histone acetyltransferase. These are useful in the field of epigenomics, for example in methods of interrogating the epigenome. Methods of interrogating the epigenome may include, for example, targeting epigenomic sequences. Targeting epigenomic sequences may include the guide being directed to an epigenomic target sequence. Epigenomic target sequence may include, in some embodiments, include a promoter, silencer or an enhancer sequence. 
     Histone modifying domains are also preferred in some embodiments. Exemplary histone modifying domains are discussed elsewhere herein. Transposase domains, HR (Homologous Recombination) machinery domains, recombinase domains, and/or integrase domains are also preferred as the present functional domains. In some embodiments, DNA integration activity includes HR machinery domains, integrase domains, recombinase domains and/or transposase domains. Histone acetyltransferases are preferred in some embodiments. 
     In some embodiments, the DNA cleavage activity is due to a nuclease. In some embodiments, the nuclease comprises a Fok1 nuclease. See, “Dimeric CRISPR RNA-guided Fok1 nucleases for highly specific genome editing”, Shengdar Q. Tsai, Nicolas Wyvekens, Cyd Khayter, Jennifer A. Foden, Vishal Thapar, Deepak Reyon, Mathew J. Goodwin, Martin J. Aryee, J. Keith Joung Nature Biotechnology 32(6): 569-77 (2014), relates to dimeric RNA-guided Fok1 Nucleases that recognize extended sequences and can edit endogenous genes with high efficiencies in human cells. 
     In some preferred embodiments, the functional domain is a transcriptional activation domain, such as, without limitation, VP64, p65, MyoD1, HSF1, RTA, SETT/9 or a histone acetyltransferase. In some embodiments, the functional domain is a transcription repression domain, preferably KRAB. In some embodiments, the transcription repression domain is SID, or concatemers of SID (e.g. SID4X). In some embodiments, the functional domain is an epigenetic modifying domain, such that an epigenetic modifying enzyme is provided. In some aspects, it is advantageous that additionally at least one NLS is provided. In some instances, it is advantageous to position the NLS at the N terminus. When more than one functional domain is included, the functional domains may be the same or different. Positioning the functional domain in the Rec1 domain, the Rec2 domain, the HNH domain, or the PI domain of the Cas protein or any ortholog corresponding to these domains is advantageous in an adaptor or accessory protein; and again, it is mentioned that the functional domain can be a DD. Positioning of the functional domains to the Rec1 domain or the Rec2 domain, of the Cas protein or any ortholog corresponding to these domains, in some instances may be preferred. Positioning of the functional domains to the Rec1 domain at position 553, Rec1 domain at 575, the Rec2 domain at any position of 175-306 or replacement thereof, the HNH domain at any position of 715-901 or replacement thereof, or the PI domain at position 1153 a refence SpCas9-like protein or any ortholog corresponding to these domains or corresponding positions, in some instances may be preferred. Fok1 functional domain may be attached at the N terminus. When more than one functional domain is included, the functional domains may be the same or different. 
     The adaptor protein may be any number of proteins that binds to an aptamer or recognition site introduced into a modified nucleic acid component and which allows proper positioning of one or more functional domains, once the nucleic acid component has been incorporated into the CRISPR complex, to affect the target with the attributed function. As explained in detail in this application such may be coat proteins, preferably bacteriophage coat proteins. The functional domains associated with such adaptor proteins (e.g. in the form of fusion protein) may include, for example, one or more domains from the group consisting of methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, and molecular switches (e.g. light inducible). Preferred domains are Fok1, VP64, P65, HSF1, MyoD1. In the event that the functional domain is a transcription activator or transcription repressor it is advantageous that additionally at least an NLS is provided and preferably at the N terminus. When more than one functional domain is included, the functional domains may be the same or different. The adaptor protein may utilize known linkers to attach such functional domains. The adaptor protein may utilize known linkers to attach such functional domains. Such linkers may be used to associate the AAV (e.g., capsid or VP2) with the CRISPR enzyme or have the CRISPR enzyme comprise the AAV (or vice versa). 
     Attachment of a functional domain or fusion protein can be via a linker, e.g., a flexible glycine-serine or a rigid alpha-helical linker such as (Ala(GluAlaAlaAlaLys)Ala). Such linkers are described elsewhere herein (see e.g., SEQ ID NOS: 6-20). Alternative linkers are available, but highly flexible linkers are thought to work best to allow for maximum opportunity for the 2 parts of the Cas to come together and thus reconstitute Cas activity. One alternative is that the NLS of nucleoplasmin can be used as a linker. For example, a linker can also be used between the Cas and any functional domain. Again, a (GGGGS) 3  (SEQ ID NO: 7) linker may be used here (or the 6, 9, or 12 repeat versions therefore) or the NLS of nucleoplasmin can be used as a linker between Cas and the functional domain. 
     Other Accessory Molecules 
     In some aspects and as described in greater detail elsewhere herein, one or more of the polypeptides of the non-class I nucleic acid targeting system described herein can be configured for expression and/or delivery via an AAV. As such one or more of the polypeptides of the non-class I nucleic acid targeting system described herein can be provided as an AAV-CRISPR enzyme. In some aspects, one or more of the AAV-CRISPR enzyme is part of a complexed with one or more polynucleotides (e.g. nucleic acid components described herein, repair templates, etc. described herein). 
     In one aspect, the invention provides an AAV-CRISPR enzyme comprising one or more nuclear localization sequences and/or NES (nuclear export sequences). In some embodiments, said AAV-CRISPR enzyme includes a regulatory element that drives transcription of component(s) of the CRISPR system (e.g., RNA, such as guide RNA and/or HR template nucleic acid molecule) in a eukaryotic cell such that said AAV-CRISPR enzyme delivers the CRISPR system accumulates in a detectable amount in the nucleus of the eukaryotic cell and/or is exported from the nucleus. In some embodiments, the regulatory element is a polymerase II promoter. In some embodiments, the AAV-CRISPR enzyme is a type II AAV-CRISPR system enzyme. In some embodiments, the AAV-CRISPR enzyme is an AAV-Cas enzyme. In some embodiments, the AAV-Cas enzyme is derived from  S. pneumoniae, S. pyogenes, S. thermophdus, F. novicida  or  S. aureus  Cas9, cas9-like and/or cas12-like (e.g., modified to have or be associated with at least one AAV), and may include further alteration or mutation of the Cas9, Cas9-like, cas12, and/or Cas12-like, and can be a chimeric Cas9-like or chimeric Cas12-like. In some embodiments, the AAV-CRISPR enzyme is codon-optimized for expression in a eukaryotic cell. In some embodiments, the AAV-CRISPR enzyme directs cleavage of one or two strands at the location of the target sequence. In some embodiments, the AAV-CRISPR enzyme lacks or substantially DNA strand cleavage activity (e.g., no more than 5% nuclease activity as compared with a wild type enzyme or enzyme not having the mutation or alteration that decreases nuclease activity). In some embodiments, the first regulatory element is a polymerase III promoter. In some embodiments, the second regulatory element is a polymerase II promoter. In some embodiments, the guide sequence is at least 15, 16, 17, 18, 19, 20, 25 nucleotides, or between 10-30, or between 15-25, or between 15-20 nucleotides in length. 
     With respect to the AAV-CRISPR enzyme described herein the CRISPR enzyme component can be a mutant (e.g. a Cas or Cas-like mutant as described elsewhere herein). In some aspects, when the CRISPR enzyme is not SpCas9 (e.g. is Cas-like (e.g. Cas9-like or Cas12-like), mutations may be made at any or all residues corresponding to positions 10, 762, 840, 854, 863 and/or 986 of SpCas9 (which may be ascertained for instance by standard sequence comparison tools). In particular, any or all of the following mutations are preferred in SpCas9-like: D10A, E762A, H840A, N854A, N863A and/or D986A; as well as conservative substitution for any of the replacement amino acids is also envisaged. Corresponding positions in Cas-like) Cas-like (e.g. Cas9-like or Cas12-like) will be appreciated. In an aspect the invention provides as to any or each or all embodiments herein-discussed wherein the AAV-CRISPR enzyme comprises at least one or more, or at least two or more mutations, wherein the at least one or more mutation or the at least two or more mutations is as to D10, E762, H840, N854, N863, or D986 according or corresponding to SpCas9 or SpCas9-like protein, e.g., D10A, E762A, H840A, N854A, N863A and/or D986A as to SpCas9, or N580 according to SaCas9 or SaCas9-like, e.g., N580A as to SaCas9 or SaCas9-like, or any corresponding mutation(s) in a Cas9 or Cas9-like of an ortholog to Sp or Sa, or the CRISPR enzyme comprises at least one mutation wherein at least H840 or N863A as to Sp Cas9 or N580A as to SaCas9 is mutated; e.g., wherein the CRISPR enzyme comprises H840A, or D10A and H840A, or D10A and N863A, according to SpCas9 or SpCas9-like protein, or any corresponding mutation(s) in a Cas9 or Cas9-like of an ortholog to Sp protein or Sa protein. 
     In an embodiment of the invention the AAV-CRISPR enzyme comprises one or two or more mutations in a residue selected from the group comprising, consisting essentially of, or consisting of D10, E762, H840, N854, N863, or D986. In a further embodiment the AAV-CRISPR enzyme comprises one or two or more mutations selected from the group comprising D10A, E762A, H840A, N854A, N863A or D986A. In another embodiment, the functional domain comprises, consist essentially of a transcriptional activation domain, e.g., VP64. In another embodiment, the functional domain comprises, consist essentially of a transcriptional repressor domain, e.g., KRAB domain, SID domain or a SID4X domain. In embodiments of the invention, the one or more heterologous functional domains have one or more activities selected from the group comprising, consisting essentially of, or consisting of methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity. In further embodiments of the invention the cell is a eukaryotic cell or a mammalian cell or a human cell. In further embodiments, the adaptor protein is selected from the group comprising, consisting essentially of, or consisting of MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, 7s, PRR1. In another embodiment, the at least one loop of the sgRNA is tetraloop and/or loop2. 
     Further, the AAV-CRISPR enzyme with diminished nuclease activity is most effective when the nuclease activity is inactivated (e.g., nuclease inactivation of at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% as compared with the wild type enzyme; or to put in another way, a AAV-Cas enzyme or AAV-CRISPR enzyme having advantageously about 0% of the nuclease activity of the non-mutated or wild type Cas enzyme or CRISPR enzyme, or no more than about 3% or about 5% or about 10% of the nuclease activity of the non-mutated or wild type Cas enzyme or CRISPR enzyme). This is possible by introducing mutations into the RuvC and HNH nuclease domains of the SpCas9 or SpCas-like protein (e.g. SpCas9-like or SpCas12-like) and orthologs thereof. For example, utilizing mutations in a residue selected from the group comprising, consisting essentially of, or consisting of D10, E762, H840, N854, N863, or D986 and more preferably introducing one or more of the mutations selected from the group comprising, consisting essentially of, or consisting of D10A, E762A, H840A, N854A, N863A or D986A. A preferable pair of mutations is D10A with H840A, more preferable is D10A with N863A of SpCas9 or SpCas9-like and orthologs thereof. 
     Design of Non-Class I Engineered CRISPR-Cas Systems 
     In a further aspect, the invention involves a computer-assisted method for identifying or designing potential compounds to fit within or bind to CRISPR-Cas system or a functional portion thereof or vice versa (a computer-assisted method for identifying or designing potential CRISPR-Cas systems or a functional portion thereof for binding to desired compounds) or a computer-assisted method for identifying or designing potential CRISPR-Cas systems (e.g., with regard to predicting areas of the CRISPR-Cas system to be able to be manipulated—for instance, based on crystal structure data or based on data of Cas orthologs, or with respect to where a functional group such as an activator or repressor can be attached to the CRISPR-Cas system, or as to Cas-like (e.g. Cas9-like or Cas12-like) truncations or as to designing nickases), said method comprising: 
     using a computer system, e.g., a programmed computer comprising a processor, a data storage system, an input device, and an output device, the steps of: 
     (a) inputting into the programmed computer through said input device data comprising the three-dimensional co-ordinates of a subset of the atoms from or pertaining to the CRISPR-Cas crystal structure (e.g. a CRISPR-Caslike crystal structure, CRISPR-Cas9-like, CRISPR-Cas12-like, CRISPR-Cas-9-like-Cas12-like crystal structure), e.g., in the CRISPR-Cas system binding domain or alternatively or additionally in domains that vary based on variance among Cas orthologs or as to e.g. Cas9s or as to nickases or as to functional groups, optionally with structural information from CRISPR-Cas system complex(es), thereby generating a data set; 
     (b) comparing, using said processor, said data set to a computer database of structures stored in said computer data storage system, e.g., structures of compounds that bind or putatively bind or that are desired to bind to a CRISPR-Cas system or as to Cas orthologs (e.g., as Cas9s or as to domains or regions that vary amongst Cas orthologs) or as to the CRISPR-Cas crystal structure or as to nickases or as to functional groups; 
     (c) selecting from said database, using computer methods, structure(s)—e.g., CRISPR-Cas structures that may bind to desired structures, desired structures that may bind to certain CRISPR-Cas structures, portions of the CRISPR-Cas system that may be manipulated, e.g., based on data from other portions of the CRISPR-Cas crystal structure and/or from Cas orthologs, truncated Cass, novel nickases or particular functional groups, or positions for attaching functional groups or functional-group-CRISPR-Cas systems; 
     (d) constructing, using computer methods, a model of the selected structure(s); and 
     (e) outputting to said output device the selected structure(s); 
     and optionally synthesizing one or more of the selected structure(s);
 
and further optionally testing said synthesized selected structure(s) as or in a CRISPR-Cas system;
 
or, said method comprising: providing the co-ordinates of at least two atoms of the CRISPR-Cas crystal structure, e.g., at least two atoms of the herein Crystal Structure Table of the CRISPR-Cas crystal structure or co-ordinates of at least a sub-domain of the CRISPR-Cas crystal structure (“selected co-ordinates”), providing the structure of a candidate comprising a binding molecule or of portions of the CRISPR-Cas system that may be manipulated, e.g., based on data from other portions of the CRISPR-Cas crystal structure and/or from Cas orthologs, or the structure of functional groups, and fitting the structure of the candidate to the selected co-ordinates, to thereby obtain product data comprising CRISPR-Cas structures that may bind to desired structures, desired structures that may bind to certain CRISPR-Cas structures, portions of the CRISPR-Cas system that may be manipulated, truncated Cas, novel nickases, or particular functional groups, or positions for attaching functional groups or functional-group-CRISPR-Cas systems, with output thereof; and optionally synthesizing compound(s) from said product data and further optionally comprising testing said synthesized compound(s) as or in a CRISPR-Cas system.
 
     The testing can comprise analyzing the CRISPR-Cas system resulting from said synthesized selected structure(s), e.g., with respect to binding, or performing a desired function. 
     The output in the foregoing methods can comprise data transmission, e.g., transmission of information via telecommunication, telephone, video conference, mass communication, e.g., presentation such as a computer presentation (e.g. POWERPOINT), internet, email, documentary communication such as a computer program (e.g. WORD) document and the like. Accordingly, the invention also comprehends computer readable media containing: atomic co-ordinate data according to the herein-referenced Crystal Structure, said data defining the three-dimensional structure of CRISPR-Cas or at least one sub-domain thereof, or structure factor data for CRISPR-Cas, said structure factor data being derivable from the atomic co-ordinate data of herein-referenced Crystal Structure. The computer readable media can also contain any data of the foregoing methods. The invention further comprehends methods a computer system for generating or performing rational design as in the foregoing methods containing either: atomic co-ordinate data according to herein-referenced Crystal Structure, said data defining the three-dimensional structure of CRISPR-Cas or at least one sub-domain thereof, or structure factor data for CRISPR-Cas, said structure factor data being derivable from the atomic co-ordinate data of herein-referenced Crystal Structure. The invention further comprehends a method of doing business comprising providing to a user the computer system or the media or the three-dimensional structure of CRISPR-Cas or at least one sub-domain thereof, or structure factor data for CRISPR-Cas, said structure set forth in and said structure factor data being derivable from the atomic co-ordinate data of herein-referenced Crystal Structure, or the herein computer media or a herein data transmission. 
     A “binding site” or an “active site” comprises or consists essentially of or consists of a site (such as an atom, a functional group of an amino acid residue or a plurality of such atoms and/or groups) in a binding cavity or region, which may bind to a compound such as a nucleic acid molecule, which is/are involved in binding. 
     By “fitting”, is meant determining by automatic, or semi-automatic means, interactions between one or more atoms of a candidate molecule and at least one atom of a structure of the invention, and calculating the extent to which such interactions are stable. Interactions include attraction and repulsion, brought about by charge, steric considerations and the like. Various computer-based methods for fitting are described further 
     By “root mean square (or rms) deviation”, refers to the square root of the arithmetic mean of the squares of the deviations from the mean. 
     By a “computer system”, is meant the hardware means, software means and data storage means used to analyze atomic coordinate data. The minimum hardware means of the computer-based systems of the present invention typically comprises a central processing unit (CPU), input means, output means and data storage means. Desirably a display or monitor is provided to visualize structure data. The data storage means may be RAM or means for accessing computer readable media of the invention. Examples of such systems are computer and tablet devices running Unix, Windows or Apple operating systems. 
     By “computer readable media”, is meant any medium or media, which can be read and accessed directly or indirectly by a computer e.g., so that the media is suitable for use in the above-mentioned computer system. Such media include, but are not limited to: magnetic storage media such as floppy discs, hard disc storage medium and magnetic tape; optical storage media such as optical discs or CD-ROM; electrical storage media such as RAM and ROM; thumb drive devices; cloud storage devices and hybrids of these categories such as magnetic/optical storage media. 
     The invention comprehends the use of the protected guides described herein above in the optimized functional CRISPR-Cas enzyme systems described herein. 
     Optimizing Efficacy of the CRISPR-Cas Systems 
     The CRISPR-Cas systems described herein can be optimized for efficacy. Such design strategies can take into consideration, for example, the Cas effector activity, guide polynucleotide activity, and on/off target activity. 
     Selection of Most Active Enzyme 
     Enzyme Stability 
     The level of expression of a protein is dependent on many factors, including the quantity of mRNA, its stability and rates of ribosome initiation. The stability or degradation of mRNA is an important factor. Several strategies have been described to increase mRNA stability. One aspect is codon-optimization. It has been found that GC-rich genes are expressed several-fold to over a 100-fold more efficiently than their GC-poor counterparts. This effect could be directly attributed to increased steady-state mRNA levels, and more particularly to efficient transcription or mRNA processing (not decreased degradation) (Kudla et al. Plos Biology http://dx.doi.org/10.1371/journal.pbio.0040180). Also, it has been found that ribosomal density has a significant effect on the transcript half-life. More particularly, it was found that an increase in stability can be achieved through the incorporation of nucleotide sequences that are capable of forming secondary structures, which often recruit ribosomes, which impede mRNA degrading enzymes. WO2011/141027 describes that slowly-read codons can be positioned in such a way as to cause high ribosome occupancy across a critical region of the 5′ end of the mRNA can increase the half-life of a message by as much as 25%, and produce a similar uplift in protein production. In contrast, positioning even a single slow-read codon before this critical region can significantly destabilize the mRNA and result in an attenuation of protein expression. This understanding enables the design of mRNAs so as to suit the desired functionality. In addition, chemical modifications such as those described for guide sequences herein can be envisaged to increase mRNA stability. 
     Selection of Most Active Guide 
     Guide Stability 
     Guide stability can be altered to increase or decrease the efficacy or efficiency of the CRISPR-Cas system. Chemical modification of the guide polynucleotides can alter the stability of the guide polynucleotides. The guide polynucleotides can be designed to achieve a desired stability by the incorporation of chemically modified nucleotides. In certain embodiments, the gRNA(s) incorporated in the CRISPR-Cas system can be chemically modified guide RNAs. Examples of guide RNA chemical modifications include, without limitation, incorporation of 2′-O-methyl (M), 2′-O-methyl 3′phosphorothioate (MS), or 2′-O-methyl 3′thioPACE (MSP) at one or more terminal nucleotides. Such chemically modified guide RNAs can comprise increased stability and increased activity as compared to unmodified guide RNAs, though on-target vs. off-target specificity is not predictable. (See, Hendel, 2015, Nat Biotechnol. 33(9):985-9, doi: 10.1038/nbt.3290, published online 29 Jun. 2015). Chemically modified guide RNAs further include, without limitation, RNAs with phosphorothioate linkages and locked nucleic acid (LNA) nucleotides comprising a methylene bridge between the 2′ and 4′ carbons of the ribose ring. 
     Rahdar et al. describe methods to ensure stabilization in the tracer hybridization region (Proc Natl Acad Sci USA. 2015, 22; 112(51):E7110-7. doi: 10.1073). Such methods can be adapted for use in designing a CRISPR-Cas system described herein. 
     Select Best Target Site in Gene 
     Selection within a Target Gene: 
     Studies to date suggest that while sgRNA activity can be quite high, there is significant variability among sgRNAs in their ability to generate the desired target cleavage. Efforts have been made to identify design criteria to maximize guide RNA efficacy. Doench et al. (Nat Biotechnol. 2014 December; 32(12): 1262-1267 and Nat Biotechnol. PubMed PMID: 26780180) describe the development of a quantitative model to optimize sgRNA activity prediction, and a tool to use this model for sgRNA design. Accordingly, in particular embodiments, the methods provided herein can include identifying an optimal guide sequence based on a statistical comparison of active guide RNAs, such as described by Doench et al. (above). In particular embodiments, at least five gRNAs are designed per target and these are tested empirically in cells to generate at least one which has sufficiently high activity. 
     Identification of Suitable Guide Sequence 
     Currently RNA guides are designed using the reference human genome; however, failing to take into account variation in the human population may confound the therapeutic outcome for a given RNA guide. The recently released ExAC dataset, based on 60,706 individuals, contains on average one variant per eight nucleotides in the human exome (Lek, M. et al. Nature 536, 285-291 (2016)). This highlights the potential for genetic variation to impact the efficacy of certain RNA guides across patient populations for CRISPR-based gene therapy, due to the presence of mismatches between the RNA guide and variants present in the target site of specific patients. To assess this impact, the ExAC dataset was used and can be used to catalog variants present in all possible targets in the human reference exome that either (i) disrupt the target PAM sequence or (ii) introduce mismatches between the RNA guide and the genomic DNA, which can collectively be termed target variation. For treatment of a patient population, avoiding target variation for RNA guides administered to individual patients will maximize the consistency of outcomes for a genome editing therapeutic. 
     In some embodiments, the CRISPR-Cas system can include RNA guide(s) for platinum targets. This can, in some embodiments, achieve targeting for 99.99% of patients. In some embodiments, these RNA guides can be further selected to minimize the number of off-target candidates occurring on high frequency haplotypes in the patient population (discussed elsewhere herein). In some embodiments, low frequency variation captured in large scale sequencing datasets can be used to estimate the number of guide RNA-enzyme combinations required to effectively and safely treat different sizes of patient populations. In some embodiments, pre-therapeutic whole genome sequencing of individual patients can be completed and analyzed to select an optimal guide RNA-Cas enzyme combination for treatment of a specific patient or patient population. In some embodiments, the selected guide RNA-Cas enzyme combination can be a perfect match to the patient&#39;s genome. In some embodiments, the selected guide RNA-Cas enzyme combination can be free of patient-specific off-target candidates. This framework can also be used, in some embodiments, in combination with additional human sequencing data, which can further refine these selection criteria and can allow for the design and validation of genome editing therapeutics while minimizing both the number of guide RNA-enzyme combinations necessary for approval and the cost of delivering effective and safe gene therapies to patients. 
     In some embodiments, the methods provided herein comprise one or more of the following steps: (1) identifying platinum targets, (2) selection of the guides to minimize the number of off-target candidates occurring on high frequency haplotypes in the patient population; (3) select guide (and/or effector protein) based low frequency variation captured in large scale sequencing datasets to estimate the number of guide RNA-enzyme combinations required to effectively and safely treat different sizes of patient populations, and (4) confirm or select guide based on pre-therapeutic whole genome sequencing of individual patient. In particular embodiments, a “platinum” target is one that does not contain variants occurring at ≥0.01% allele frequency. 
     Determination of on/Off-Target Activity and Selecting Suitable Target Sequences/Guides 
     In certain example embodiments, parameters such as, but not limited to, off-target candidates, PAM restrictiveness, target cleavage efficiency, or effector protein specific may be determined using sequencing-based double-strand break (DSB) detection assays. Example sequencing-based DSB detection assay sChIP-seq (Szilard et al. Nat. Struct. Mol. Biol. 18, 299-305 (2010); Iacovoni et al. EMBO J. 29, 1446-1457 (2010)), BLESS (Crosetto et al. Nat. Methods 10, 361-365 (2013); Ran et al. Nature 520, 186-191 (2015); Slaymaker et al. Science 351, 84-88 (2016)), GUIDEseq (Tsai et al. Nat. Biotech 33, 187-197 (2015)), Digenome-seq (Kim et al. Nat. Methods 12, 237-43 (2015)), IDLV-mediated DNA break capture (Wang et al. Nat. Biotechnol. 33, 179-186 (2015), HTGTS (Frock et al. Nat. Biotechnol. 33, 179-186 (2015)), End-Seq (Canela et al. Mol. Cell 63, 898-911 (2016), and DSBCapture (Lensing et al. Nat. Methods 13, 855-857 (2016). Additional methods that may be used to assess target cleavage efficiency include SITE-Seq (Cameron et al. Nature Methods, 14, 600-606 (2017), and CIRCLE-seq (Tsai et al. Nature Methods 14, 607-614 (2017)). 
     Methods useful for assessing Cpfl RNase activity include those disclosed in Zhong et al. Nature Chemical Biology Jun. 19, 2017 doi: 10.1038/NCHEMBIO.2410 and may be similarly applied to Cas effectors described herein (including but not limited to the Cas-like effectors described herein). Increased RNase activity and the ability to excise multiple CRISPR RNAs (crRNA) from a single RNA polymerase II-driven RNA transcript can simplify modification of multiple genomic targets and can be used to increase the efficiency of Cas-like (e.g. Cas9-like and/or Cas12-like)-mediated editing. 
     BLISS 
     Other suitable assays include those described in Yan et al. (“BLISS: quantitative and versatile genome-wide profiling of DNA breaks in situ” BioRxiv, Dec. 4, 2016 doi: http://dx.doi.org/10.1101/091629) describe a versatile, sensitive and quantitative method for detecting DSBs applicable to low-input specimens of both cells and tissues that is scalable for high-throughput DSB mapping in multiple samples. Breaks Labeling In Situ and Sequencing (BLISS), features efficient in situ DSB labeling in fixed cells or tissue sections immobilized onto a solid surface, linear amplification of tagged DSBs via T7-mediated in vitro transcription (IVT) for greater sensitivity, and accurate DSB quantification by incorporation of unique molecular identifiers (UMIs). 
     Curtain 
     A further method, referred to herein as “Curtain” has been developed which may also be useful in assessing certain parameters disclosed herein, the method allowing on target and off target cutting of a nuclease to be assessed in a direct and unbiased way using in vitro cutting of immobilized nucleic acid molecules. Further reference is made to WO/2017/218979, which is. Incorporated by reference herein and can be adapted for use in the design and/or characterization of the CRISRP-Cas systems described herein. 
     This method may also be used to select a suitable guide RNA. The method allows the detection of a nucleic acid modification, by performing the following steps: i) contacting one or more nucleic acid molecules immobilized on a solid support (immobilized nucleic acid molecules) with an agent capable of inducing a nucleic acid modification; and ii) sequencing at least part of said one or more immobilized nucleic acid molecules that comprises the nucleic acid modification using a primer specifically binding to a primer binding site. This method further allows the selection of a guide RNA from a plurality of guide RNAs specific for a selected target sequence. In particular embodiments, the method comprises contacting a plurality of nucleic acid molecules immobilized on a solid support (immobilized nucleic acid molecules) with a plurality of RNA-guided nuclease complexes capable of inducing a nucleic acid break, said plurality of RNA-guided nuclease complexes comprising a plurality of different guide RNA&#39;s, thereby inducing one or more nucleic acid breaks; attaching an adapter comprising a primer binding site to said one or more immobilized nucleic acid molecules comprising a nucleic acid break; sequencing at least part of said one or more immobilized nucleic acid molecules comprising a nucleic acid break using a primer specifically binding to said primer binding site; and selecting a guide RNA based on location and/or amount of said one or more breaks. 
     In particular embodiments, the method comprises determining one or more locations in said one or more immobilized nucleic acid molecules comprising a break other than a location comprising said selected target sequence (off-target breaks) and selecting a guide RNA based on said one or more locations. In particular embodiments, step v comprises determining a number of sites in said one or more immobilized nucleic acid molecules comprising off-target breaks and selecting a guide RNA based on said number of sites. In a further embodiment, step iv comprises both determining the location of off-targets breaks and the number of locations of off-target breaks. 
     Optimizing Safety of the CRISPR-Cas Systems 
     Selection of the Cas-Effector(s) with the Shortest Half-Life 
     Half-Life of the Cas Effector(s) 
     The extended presence of an effector protein after having performed its function at the target site is a potential safety concern, both for off-target effects and direct toxicity of the effector protein. It has been reported that upon direct delivery to the cell by LNP, CRISPR effector proteins degrade rapidly within the cell (Kim et al. Genome Res. 2014 June; 24(6): 1012-1019). Where the effector protein is to be expressed from a plasmid, strategies to actively reduce the half-life of the protein can be used in the design of the CRISPR-Cas system. 
     Use of Destabilized Domains 
     In certain embodiments, the methods provided herein involve the use of a Cas effector (e.g., Cas, Cas-like. Cas9-like, and/or Cas12-like) which is associated with or fused to a destabilization domain (DD). The technology relating to the use of destabilizing domains is described in detail in WO2016/106244, which is incorporated by reference herein. 
     Destabilizing domains (DD) are domains which can confer instability to a wide range of proteins; see, e.g., Miyazaki, J Am Chem Soc. Mar. 7, 2012; 134(9): 3942-3945, and Chung H Nature Chemical Biology Vol. 11 Sep. 2015 pp. 713-720, incorporated herein by reference. The DD can be associated with, e.g., fused to, advantageously with a linker, to a CRISPR enzyme, whereby the DD can be stabilized in the presence of a ligand and when there is the absence thereof the DD can become destabilized, whereby the CRISPR enzyme is entirely destabilized, or the DD can be stabilized in the absence of a ligand and when the ligand is present the DD can become destabilized; the DD allows the Cas effector to be regulated or controlled, thereby providing means for regulation or control of the system. For instance, when a protein of interest is expressed as a fusion with the DD tag, it is destabilized and rapidly degraded in the cell, e.g., by proteasomes. Thus, absence of stabilizing ligand leads to a DD-associated Cas effector being degraded. Peak activity of the Cas effector is relevant to reduce off-target effects and for the general safety of the system. Advantages of the DD system include that it can be dosable, orthogonal (e.g., a ligand only affects its cognate DD so two or more systems can operate independently), transportable (e.g., may work in different cell types or cell lines) and allows for temporal control. 
     Suitable DD—stabilizing ligand pairs are known in the art and also described in WO2016/106244. The size of Destabilization Domain varies but is typically approx.-approx. 100-300 amino acids in size. Suitable examples include ER50 and/or DHFR50. A corresponding stabilizing ligand for ER50 is, for example, 4HT or CMP8. In some embodiments, one or two DDs may be fused to the N-terminal end of the CRISPR enzyme with one or two DDs fused to the C-terminal of the CRISPR enzyme. While the DD can be provided directly at N and/or C terminal(s) of the Cas-like (e.g. Cas9-like and/or Cas12-like) effector protein, they can also be fused via a linker, such as a GlySer linker, or an NLS and/or NES. A commercially available DD system is the CloneTech, ProteoTuner™ system; the stabilizing ligand is Shield1. In some embodiments, the stabilizing ligand is a ‘small molecule’, preferably it is cell-permeable and has a high affinity for its corresponding DD. 
     In some embodiments, the CRISPR enzyme is fused to Destabilization Domain (DD). In other words, the DD may be associated with the CRISPR enzyme by fusion with said CRISPR enzyme. The AAV can then, by way of nucleic acid molecule(s) deliver the stabilizing ligand (or such can be otherwise delivered) In some embodiments, the enzyme may be considered to be a modified CRISPR enzyme, wherein the CRISPR enzyme is fused to at least one destabilization domain (DD) and VP2. 
     Selection of the Least Immunogenic RNP 
     When administering an agent to a mammal, there is always the risk of an immune response to the agent and/or its delivery vehicle. Circumventing the immune response is a major challenge for most delivery vehicles. Viral vectors, which express immunogenic epitopes within the organism typically induce an immune response. Nanoparticle and lipid-based vectors to some extent address this problem. Yin et al. demonstrate a therapeutic approach combining viral delivery of the guide RNA with lipid nanoparticle-mediated delivery of the CRISPR effector protein (Nature Biotechnology 34:328-33(2016)). Ziris et al. describes cationic-lipid mediated delivery of Cas9:guideRNA nuclease complexes to cells, which can be applied to the Cas-like CRISPR systems described herein. The Cas effector proteins (e.g., Cas and Cas-like effectors described herein), which can also of bacterial origin, also inherently carry the risk of eliciting an immune response. This may be addressed by humanizing the Cas effector protein. 
     Introduction of Modifications in Guide RNA to Minimize Immunogenicity 
     Chemical modifications of RNAs have been used to avoid reactions of the innate immune system. Judge et al. (2006) demonstrated that immune stimulation by synthetic siRNA can be completely abrogated by selective incorporation of 2′-O-methyl (2′OMe) uridine or guanosine nucleosides into one strand of the siRNA duplex (Mol. Ther., 13 (2006), pp. 494-505). Cekaite et al. (J. Mol. Biol., 365 (2007), pp. 90-108) observed that replacement of only uridine bases of siRNA with either 2′-fluoro or 2′-O-methyl modified counterparts abrogated upregulation of genes involved in the regulation of the immune response. Similarly, Hendel et al. tested sgRNAs with both backbone and sugar modifications that confer nuclease stability and can reduce immunostimulatory effects (Hendel et al., Nat. Biotechnol., 33 (2015), pp. 985-989). 
     In some embodiments, the guide RNA can be designed so as to minimize immunogenicity using one or more of these methods and/or incorporation of one or more chemical modifications. 
     Identifying Optimal Dosages to Minimize Toxicity and Maximize Specificity 
     It is generally accepted that the dosage of CRISPR-Cas system and/or components thereof will be relevant to toxicity and specificity of the system (Pattanayak et al. Nat Biotechnol. 2013 September; 31(9): 839-843). Hsu et al. (Nat Biotechnol. 2013 September; 31(9): 827-832) demonstrated that the dosage of SpCas9 and sgRNA can be titrated to address these issues and can be applied and/or adapted for the CRISPR-Cas systems described herein. In certain example embodiments, toxicity is minimized by saturating complex with guide by either pre-forming complex, putting guide under control of a strong promoter, or via timing of delivery to ensure saturating conditions available during expression of the effector protein. 
     Identification of Appropriate Delivery Method/Vehicle 
     To increase safety, the delivery method and/or vehicle can be optimized. Delivery methods, including but not limited to, polynucleotides, vectors, virus particles, particles etc. are described in greater detail herein. Further, advantages of various delivery compositions, formulations and techniques, with respect to e.g. safety are also discussed elsewhere herein. In some embodiments, multiple delivery techniques can be mixed and utilized to achieve the appropriate effect. Further, administration route can be altered to increase safety. Various administration routes are described elsewhere herein. Delivery timing and regimen can also be modified to increase safety of the CRISPR-Cas systems described herein. Various exemplary and non-limiting delivery regimens are described elsewhere herein. One of ordinary skill in the art will appreciate appropriate delivery compositions and approaches for specific embodiments of the CRISPR-Cas system and methods of using the CRISPR-Cas system in view of this disclosure. 
     Non-Class I Crispr-Cas Complexes 
     Components of the non-Class I engineered CRISPR-Cas enzyme system described herein can be provided individually or complexed with one or more other components of the non-Class I engineered CRISPR-Cas Enzyme systems. In certain embodiments, a complex can include on or more Cas (e.g. Cas-like (e.g. Cas9-like or Cas12-like)) proteins bound to or otherwise associated with one or more nucleic acid components, accessory molecule(s), adaptors, and/or another component described elsewhere herein. In some embodiments, a complex can include one or more Cas (e.g. Cas-like (e.g. Cas9-like or Cas12-like)) proteins bound to or otherwise associated with a guide polynucleotide and optionally one or more other nucleic acid components accessory molecule(s), adaptors, and/or another component described elsewhere herein. The complexes can be provided to a subject, cell, or target polynucleotide as described in greater detail elsewhere herein. 
     In some embodiments, the complex thus forms a ribonucleoprotein or RNP that includes one or more CRISPR-Cas effector proteins complexed with one or more guide polynucleotides. In some embodiments, the CRISPR-Cas RNP complexes can be delivered to a cell. Suitable delivery techniques and vehicles are described elsewhere herein. An important advantage is that both RNP delivery is transient, reducing off-target effects and toxicity issues. Efficient genome editing in different cell types has been observed by Kim et al. (2014, Genome Res. 24(6):1012-9), Paix et al. (2015, Genetics 204(1):47-54), Chu et al. (2016, BMC Biotechnol. 16:4), and Wang et al. (2013, Cell. 9; 153(4):910-8). 
     In particular embodiments, the ribonucleoprotein is delivered by way of a polypeptide-based shuttle agent as described in WO2016161516. WO2016161516 describes efficient transduction of polypeptide cargos using synthetic peptides comprising an endosome leakage domain (ELD) operably linked to a cell penetrating domain (CPD), to a histidine-rich domain and a CPD. Similarly, these polypeptides can be used for the delivery of CRISPR-effector based RNPs in eukaryotic cells. 
     Delivery 
     The present disclosure also provides delivery systems for introducing components of the systems and compositions herein to cells, tissues, organs, or organisms. A delivery system may comprise one or more delivery vehicles and/or cargos. Exemplary delivery systems and methods include those described in paragraphs [00117] to [00278] of Feng Zhang et al., (WO2016106236A1), and pages 1241-1251 and Table 1 of Lino C A et al., Delivering CRISPR: a review of the challenges and approaches, DRUG DELIVERY, 2018, VOL. 25, NO. 1, 1234-1257, which are incorporated by reference herein in their entireties. 
     In some embodiments, the delivery systems may be used to introduce the components of the systems and compositions to plant cells. For example, the components may be delivered to plant using electroporation, microinjection, aerosol beam injection of plant cell protoplasts, biolistic methods, DNA particle bombardment, and/or  Agrobacterium -mediated transformation. Examples of methods and delivery systems for plants include those described in Fu et al., Transgenic Res. 2000 February; 9(1):11-9; Klein R M, et al., Biotechnology. 1992; 24:384-6; Casas A M et al., Proc Natl Acad Sci USA. 1993 Dec. 1; 90(23): 11212-11216; and U.S. Pat. No. 5,563,055, Davey M R et al., Plant Mol Biol. 1989 September; 13(3):273-85, which are incorporated by reference herein in their entireties. 
     Cargos 
     The delivery systems may comprise one or more cargos. The cargos may comprise one or more components of the CRISPR-Cas systems and compositions herein. A cargo may comprise one or more of the following: i) a vector or vector system (viral or non-viral) encoding one or more Cas proteins; ii) a vector or vector system (viral or non-viral) encoding one or more guide RNAs described herein, iii) mRNA of one or more Cas proteins; iv) one or more guide RNAs; v) one or more Cas proteins; vi) one or more polynucleotides encoding one or more Cas proteins; vii) one or more polynucleotides encoding one or more guide RNAs, or viii) any combination thereof. In some examples, a cargo may comprise a plasmid encoding one or more Cas protein and one or more (e.g., a plurality of) guide RNAs. In some embodiments, a cargo may comprise mRNA encoding one or more Cas proteins and one or more guide RNA. 
     In some embodiments, a cargo may comprise one or more Cas proteins described herein and one or more guide RNAs, e.g., in the form of ribonucleoprotein complexes (RNP). The ribonucleoprotein complexes may be delivered by methods and systems herein. In some cases, the ribonucleoprotein may be delivered by way of a polypeptide-based shuttle agent. In one example, the ribonucleoprotein may be delivered using synthetic peptides comprising an endosome leakage domain (ELD) operably linked to a cell penetrating domain (CPD), to a histidine-rich domain and a CPD, e.g., as describe in WO2016161516. RNP may also be used for delivering the compositions and systems to plant cells, e.g., as described in Wu J W, et al., Nat Biotechnol. 2015 November; 33(11):1162-4. 
     In some embodiments, the cargo(s) can be any of the polynucleotide(s), e.g. CRISPR-Cas System polynucleotides described herein. 
     Polynucleotides 
     Any of the polypeptides described herein can be encoded by one or more polynucleotides. The term “encode” is a term of art that refers to the principle that DNA can be transcribed into RNA, which can then be translated into amino acid sequences that can form polypeptide. As used herein, the term “encode” refers to both steps of the process of transcription and translation individually. Thus, as described herein, the polynucleotide that is said to “encode” a polypeptide described herein can be DNA or RNA. As such, also described herein are polynucleotides that can encode one or more of the polypeptides described herein. In certain embodiments the polypeptide can be a polypeptide capable of allosterically interaction with a polypeptide upon sequence-specific recognition of a target sequence that are described elsewhere herein. The polynucleotide can be “naked,” as the term is used in the art. In other aspects, the polynucleotide is not “naked”. In some aspects, one or more of the polynucleotide(s) can be included in a vector or system thereof as described in greater detail elsewhere herein. In aspects, the polypeptide can be encoded by a polynucleotide having 80-100% sequence identity to one or more of the polynucleotides set forth in any one of SEQ ID NOs: 57-108, which are incorporated by reference herein. See also Tables 14-23 of the Working Example(s) herein. In some embodiments, the polypeptide can be encoded by a polynucleotide having 80-100% sequence identity to one or more of the polynucleotides set forth in any one of SEQ ID NOS: 57-100, which are incorporated herein by reference as if expressed in their entireties. In some embodiments, the polypeptide can be encoded by a polynucleotide having 80-100% sequence identity to one or more of the polynucleotides set forth in any one of SEQ ID NOS: 57-87, 
     In one aspect, the invention provides a recombinant polynucleotide comprising multiple guide RNA sequences up- or downstream (whichever applicable) of a direct repeat sequence, wherein each of the guide sequences when expressed directs sequence-specific binding of a CRISPR-Cas complex to its corresponding target sequence present in a eukaryotic cell. In some embodiments, the target sequence is a viral sequence present in a eukaryotic cell. Where applicable, a tracr sequence may also be provided. In some embodiments, the target sequence is a proto-oncogene or an oncogene. 
     Codon Optimized Nucleic Acid Sequences 
     The polynucleotide described herein can be codon optimized for expression in a particular cell type or subject type. Where the effector protein is to be administered as a nucleic acid, the application envisages the use of codon-optimized polynucleotides, including but not limited to, Cas, Cas9-like and/or Cas-12-like sequences. An example of a codon optimized sequence, is in this instance a sequence optimized for expression in a eukaryote, e.g., humans (i.e. being optimized for expression in humans), or for another eukaryote, animal or mammal as herein discussed; see, e.g., SaCas9 or SaCas9-like human codon optimized sequence in WO 2014/093622 (PCT/US2013/074667) as an example of a codon optimized sequence (from knowledge in the art and this disclosure, codon optimizing coding nucleic acid molecule(s), especially as to effector protein (e.g. Cas9-like, Cas-12 like and other CRISPR-Cas enzymes described herein) is within the ambit of the skilled artisan). Whilst this is preferred, it will be appreciated that other examples are possible and codon optimization for a host species other than human, or for codon optimization for specific organs is known. In some embodiments, an enzyme coding sequence encoding a DNA/RNA-targeting Cas or Cas-like protein is codon optimized for expression in particular cells, such as eukaryotic cells. The eukaryotic cells may be those of or derived from a particular organism, such as a plant or a mammal, including but not limited to human, or non-human eukaryote or animal or mammal as herein discussed, e.g., mouse, rat, rabbit, dog, livestock, or non-human mammal or primate. In some embodiments, processes for modifying the germ line genetic identity of human beings and/or processes for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such processes, may be excluded. In general, codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the “Codon Usage Database” available at www.kazusa.orjp/codon/and these tables can be adapted in a number of ways. See Nakamura, Y., et al. “Codon usage tabulated from the international DNA sequence databases: status for the year 2000” Nucl. Acids Res. 28:292 (2000). Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, Pa.), are also available. In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding a DNA/RNA-targeting Cas protein corresponds to the most frequently used codon for a particular amino acid. As to codon usage in yeast, reference is made to the online Yeast Genome database available at http://www.yeastgenome.org/community/codon_usage.shtml, or Codon selection in yeast, Bennetzen and Hall, J Biol Chem. 1982 Mar. 25; 257(6):3026-31. As to codon usage in plants including algae, reference is made to Codon usage in higher plants, green algae, and cyanobacteria, Campbell and Gowri, Plant Physiol. 1990 January; 92(1): 1-11; as well as Codon usage in plant genes, Murray et al, Nucleic Acids Res. 1989 Jan. 25; 17(2):477-98; or Selection on the codon bias of chloroplast and cyanelle genes in different plant and algal lineages, Morton B R, J Mol Evol. 1998 April; 46(4):449-59. 
     Regulatory Elements 
     In certain embodiments, the polynucleotides described herein can include one or more regulatory elements that can be operatively linked to the polynucleotide that can encode a polypeptide capable of allosterically interaction with a polypeptide upon sequence-specific recognition of a target sequence that are described elsewhere herein. Suitable regulatory elements are described in greater detail elsewhere herein, such as in connection with the vectors and vector systems described herein. Any such regulatory elements can be operatively linked to a CRISPR-Cas system polynucleotide described herein. 
     Selectable Markers and Tags 
     One or more of the polypeptides can be operably linked, fused to, or otherwise modified to include (such inserted between two amino acids between the N- and C-terminus of the polypeptide) a selectable marker, affinity, or other protein tag. It will be appreciated that the polynucleotide encoding such selectable markers or tags can be incorporated into a polynucleotide encoding one or more components of the CRISPR-Cas system described herein in an appropriate manner to allow expression of the selectable marker or tag. Such techniques and methods are described elsewhere herein and will be instantly appreciated by one of ordinary skill in the art in view of this disclosure. 
     Physical Delivery 
     In some embodiments, the cargos may be introduced to cells by physical delivery methods. Examples of physical methods include microinjection, electroporation, and hydrodynamic delivery. Both nucleic acid and proteins may be delivered using such methods. For example, Cas protein may be prepared in vitro, isolated, (refolded, purified if needed), and introduced to cells. 
     Microinjection 
     Microinjection of the cargo directly to cells can achieve high efficiency, e.g., above 90% or about 100%. In some embodiments, microinjection may be performed using a microscope and a needle (e.g., with 0.5-5.0 μm in diameter) to pierce a cell membrane and deliver the cargo directly to a target site within the cell. Microinjection may be used for in vitro and ex vivo delivery. 
     Plasmids comprising coding sequences for Cas proteins and/or guide RNAs, mRNAs, and/or guide RNAs, may be microinjected. In some cases, microinjection may be used i) to deliver DNA directly to a cell nucleus, and/or ii) to deliver mRNA (e.g., in vitro transcribed) to a cell nucleus or cytoplasm. In certain examples, microinjection may be used to delivery sgRNA directly to the nucleus and Cas-encoding mRNA to the cytoplasm, e.g., facilitating translation and shuttling of Cas to the nucleus. 
     Microinjection may be used to generate genetically modified animals. For example, gene editing cargos may be injected into zygotes to allow for efficient germline modification. Such approach can yield normal embryos and full-term mouse pups harboring the desired modification(s). Microinjection can also be used to provide transiently up- or down-regulate a specific gene within the genome of a cell, e.g., using CRISPRa and CRISPRi. 
     Electroporation 
     In some embodiments, the cargos and/or delivery vehicles may be delivered by electroporation. Electroporation may use pulsed high-voltage electrical currents to transiently open nanometer-sized pores within the cellular membrane of cells suspended in buffer, allowing for components with hydrodynamic diameters of tens of nanometers to flow into the cell. In some cases, electroporation may be used on various cell types and efficiently transfer cargo into cells. Electroporation may be used for in vitro and ex vivo delivery. 
     Electroporation may also be used to deliver the cargo to into the nuclei of mammalian cells by applying specific voltage and reagents, e.g., by nucleofection. Such approaches include those described in Wu Y, et al. (2015). Cell Res 25:67-79; Ye L, et al. (2014). Proc Natl Acad Sci USA 111:9591-6; Choi P S, Meyerson M. (2014). Nat Commun 5:3728; Wang J, Quake S R. (2014). Proc Natl Acad Sci 111:13157-62. Electroporation may also be used to deliver the cargo in vivo, e.g., with methods described in Zuckermann M, et al. (2015). Nat Commun 6:7391. 
     Hydrodynamic Delivery 
     Hydrodynamic delivery may also be used for delivering the cargos, e.g., for in vivo delivery. In some examples, hydrodynamic delivery may be performed by rapidly pushing a large volume (8-10% body weight) solution containing the gene editing cargo into the bloodstream of a subject (e.g., an animal or human), e.g., for mice, via the tail vein. As blood is incompressible, the large bolus of liquid may result in an increase in hydrodynamic pressure that temporarily enhances permeability into endothelial and parenchymal cells, allowing for cargo not normally capable of crossing a cellular membrane to pass into cells. This approach may be used for delivering naked DNA plasmids and proteins. The delivered cargos may be enriched in liver, kidney, lung, muscle, and/or heart. 
     Transfection 
     The cargos, e.g., nucleic acids and/or polypeptides, may be introduced to cells by transfection methods for introducing nucleic acids into cells. Examples of transfection methods include calcium phosphate-mediated transfection, cationic transfection, liposome transfection, dendrimer transfection, heat shock transfection, magnetofection, lipofection, impalefection, optical transfection, proprietary agent-enhanced uptake of nucleic acid. 
     Transduction 
     The cargos, e.g. nucleic acids and/or polypeptides, can be introduced to cells by transduction by a viral or pseudoviral particle. Methods of packaging the cargos in viral particles can be accomplished using any suitable viral vector or vector systems. Such viral vector and vector systems are described in greater detail elsewhere herein. As used in this context herein “transduction” refers to the process by which foreign nucleic acids and/or proteins are introduced to a cell (prokaryote or eukaryote) by a viral or pseudo viral particle. After packaging in a viral particle or pseudo viral particle, the viral particles can be exposed to cells (e.g. in vitro, ex vivo, or in vivo) where the viral or pseudoviral particle infects the cell and delivers the cargo to the cell via transduction. Viral and pseudoviral particles can be optionally concentrated prior to exposure to target cells. In some embodiments, the virus titer of a composition containing viral and/or pseudoviral particles can be obtained and a specific titer be used to transduce cells. 
     Biolistics 
     The cargos, e.g. nucleic acids and/or polypeptides, can be introduced to cells using a biolistic method or technique. The term of art “biolistic”, as used herein refers to the delivery of nucleic acids to cells by high-speed particle bombardment. In some embodiments, the cargo(s) can be attached, associated with, or otherwise coupled to particles, which than can be delivered to the cell via a gene-gun (see e.g., Liang et al. 2018. Nat. Protocol. 13:413-430; Svitashev et al. 2016. Nat. Comm. 7:13274; Ortega-Escalante et al., 2019. Plant. J. 97:661-672). In some embodiments, the particles can be gold, tungsten, palladium, rhodium, platinum, or iridium particles. 
     Implantable Devices 
     In some embodiments, the delivery system can include an implantable device that incorporates or is coated with a CRISPR-Cas system or component thereof described herein. Various implantable devices are described in the art, and include any device, graft, or other composition that can be implanted into a subject. 
     Delivery Vehicles 
     The delivery systems may comprise one or more delivery vehicles. The delivery vehicles may deliver the cargo into cells, tissues, organs, or organisms (e.g., animals or plants). The cargos may be packaged, carried, or otherwise associated with the delivery vehicles. The delivery vehicles may be selected based on the types of cargo to be delivered, and/or the delivery is in vitro and/or in vivo. Examples of delivery vehicles include vectors, viruses, non-viral vehicles, and other delivery reagents described herein. 
     The delivery vehicles in accordance with the present invention may a greatest dimension (e.g. diameter) of less than 100 microns (μm). In some embodiments, the delivery vehicles have a greatest dimension of less than 10 μm. In some embodiments, the delivery vehicles may have a greatest dimension of less than 2000 nanometers (nm). In some embodiments, the delivery vehicles may have a greatest dimension of less than 1000 nanometers (nm). In some embodiments, the delivery vehicles may have a greatest dimension (e.g., diameter) of less than 900 nm, less than 800 nm, less than 700 nm, less than 600 nm, less than 500 nm, less than 400 nm, less than 300 nm, less than 200 nm, less than 150 nm, or less than 100 nm, less than 50 nm. In some embodiments, the delivery vehicles may have a greatest dimension ranging between 25 nm and 200 nm. 
     In some embodiments, the delivery vehicles may be or comprise particles. For example, the delivery vehicle may be or comprise nanoparticles (e.g., particles with a greatest dimension (e.g., diameter) no greater than 1000 nm. The particles may be provided in different forms, e.g., as solid particles (e.g., metal such as silver, gold, iron, titanium), non-metal, lipid-based solids, polymers), suspensions of particles, or combinations thereof. Metal, dielectric, and semiconductor particles may be prepared, as well as hybrid structures (e.g., core-shell particles). 
     Nanoparticles may also be used to deliver the compositions and systems to plant cells, e.g., as described in WO 2008042156, US 20130185823, and WO2015089419. In general, a “nanoparticle” refers to any particle having a diameter of less than 1000 nm. In certain preferred embodiments, nanoparticles of the invention have a greatest dimension (e.g., diameter) of 500 nm or less. In other preferred embodiments, nanoparticles of the invention have a greatest dimension ranging between 25 nm and 200 nm. In other preferred embodiments, nanoparticles of the invention have a greatest dimension of 100 nm or less. In other preferred embodiments, nanoparticles of the invention have a greatest dimension ranging between 35 nm and 60 nm. It will be appreciated that reference made herein to particles or nanoparticles can be interchangeable, where appropriate. Nanoparticles made of semiconducting material may also be labeled quantum dots if they are small enough (typically sub 10 nm) that quantization of electronic energy levels occurs. Such nanoscale particles are used in biomedical applications as drug carriers or imaging agents and may be adapted for similar purposes in the present invention. Semi-solid and soft nanoparticles have been manufactured, and are within the scope of the present invention. Nanoparticles with one half hydrophilic and the other half hydrophobic are termed Janus particles and are particularly effective for stabilizing emulsions. They can self-assemble at water/oil interfaces and act as solid surfactants. 
     Particle characterization (including e.g., characterizing morphology, dimension, etc.) is done using a variety of different techniques. Common techniques are electron microscopy (TEM, SEM), atomic force microscopy (AFM), dynamic light scattering (DLS), X-ray photoelectron spectroscopy (XPS), powder X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF), ultraviolet-visible spectroscopy, dual polarization interferometry and nuclear magnetic resonance (NMR). Characterization (dimension measurements) may be made as to native particles (i.e., preloading) or after loading of the cargo (herein cargo refers to e.g., one or more components of CRISPR-Cas system e.g., CRISPR enzyme or mRNA or guide RNA, or any combination thereof, and may include additional carriers and/or excipients) to provide particles of an optimal size for delivery for any in vitro, ex vivo and/or in vivo application of the present invention. In certain preferred embodiments, particle dimension (e.g., diameter) characterization is based on measurements using dynamic laser scattering (DLS). Mention is made of U.S. Pat. Nos. 8,709,843; 6,007,845; 5,855,913; 5,985,309; 5,543,158; and the publication by James E. Dahlman and Carmen Barnes et al. Nature Nanotechnology (2014) published online 11 May 2014, doi:10.1038/nnano.2014.84, describing particles, methods of making and using them and measurements thereof. 
     Vectors and Vector Systems 
     Also provided herein are vectors that can contain one or more of the CRISPR-Cas system polynucleotides described herein. In certain embodiments, the vector can contain one or more polynucleotides encoding one or more elements of a CRISPR-Cas system described herein. The vectors can be useful in producing bacterial, fungal, yeast, plant cells, animal cells, and transgenic animals that can express one or more components of the CRISPR-Cas system described herein. Within the scope of this disclosure are vectors containing one or more of the polynucleotide sequences described herein. One or more of the polynucleotides that are part of the CRISPR-Cas system described herein can be included in a vector or vector system. The vectors and/or vector systems can be used, for example, to express one or more of the polynucleotides in a cell, such as a producer cell, to produce CRISPR-Cas system containing virus particles described elsewhere herein. Other uses for the vectors and vector systems described herein are also within the scope of this disclosure. In general, and throughout this specification, the term “vector” refers to a tool that allows or facilitates the transfer of an entity from one environment to another. In some contexts which will be appreciated by those of ordinary skill in the art, “vector” can be a term of art to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. A vector can be a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. Generally, a vector is capable of replication when associated with the proper control elements. 
     Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g. circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, wherein virally-derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g. retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses (AAVs)). Viral vectors also include polynucleotides carried by a virus for transfection into a host cell. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as “expression vectors.” Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. 
     Recombinant expression vectors can be composed of a nucleic acid (e.g. a polynucleotide) of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which can be selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably linked” and “operatively-linked” are used interchangeably herein and further defined elsewhere herein. In the context of a vector, the term “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). Advantageous vectors include lentiviruses and adeno-associated viruses, and types of such vectors can also be selected for targeting particular types of cells. These and other aspects of the vectors and vector systems are described elsewhere herein. 
     In some aspects, the vector can be a bicistronic vector. In some aspects, a bicistronic vector can be used for one or more elements of the CRISPR-Cas system described herein. In some aspects, expression of elements of the CRISPR-Cas system described herein can be driven by the CBh promoter or other ubiquitous promoter. Where the element of the CRISPR-Cas system is an RNA, its expression can be driven by a Pol III promoter, such as a U6 promoter. In some aspects, the two are combined. 
     In some aspects, a vector capable of delivering an effector protein and optionally at least one CRISPR guide RNA to a cell can be composed of or contain a minimal promoter operably linked to a polynucleotide sequence encoding the effector protein and a second minimal promoter operably linked to a polynucleotide sequence encoding at least one guide RNA, wherein the length of the vector sequence comprising the minimal promoters and polynucleotide sequences is less than 4.4 Kb. In an embodiment, the vector can be a viral vector. In certain embodiments, the viral vector is an is an adeno-associated virus (AAV) or an adenovirus vector. In another embodiment, the effector protein is a Cas protein. In a further embodiment, the CRISPR enzyme is Cas9-like and/or Cas12-like protein. 
     In some embodiments, the vector capable of delivering a lentiviral vector for an effector protein and at least one CRISPR guide RNA to a cell can be composed of or contain a promoter operably linked to a polynucleotide sequence encoding Cas and a second promoter operably linked to a polynucleotide sequence encoding at least one guide RNA, wherein the polynucleotide sequences are in reverse orientation. 
     In one aspect, the invention provides a vector system comprising one or more vectors. In some embodiments, the system comprises: (a) a first regulatory element operably linked to a direct repeat sequence and one or more insertion sites for inserting one or more guide sequences up- or downstream (whichever applicable) of the direct repeat sequence, wherein when expressed, the one or more guide sequence(s) direct(s) sequence-specific binding of the CRISPR complex to the one or more target sequence(s) in a eukaryotic cell, wherein the CRISPR complex comprises a Cas enzyme complexed with the one or more guide sequence(s) that is hybridized to the one or more target sequence(s); and (b) a second regulatory element operably linked to an enzyme-coding sequence encoding said Cas enzyme, preferably comprising at least one nuclear localization sequence and/or at least one NES; wherein components (a) and (b) are located on the same or different vectors of the system. Where applicable, a tracr sequence may also be provided. In some embodiments, component (a) further comprises two or more guide sequences operably linked to the first regulatory element, wherein when expressed, each of the two or more guide sequences direct sequence specific binding of a Cas CRISPR complex to a different target sequence in a eukaryotic cell. In some embodiments, the CRISPR complex comprises one or more nuclear localization sequences and/or one or more NES of sufficient strength to drive accumulation of said Cas CRISPR complex in a detectable amount in or out of the nucleus of a eukaryotic cell. In some embodiments, the first regulatory element is a polymerase III promoter. In some embodiments, the second regulatory element is a polymerase II promoter. In some embodiments, each of the guide sequences is at least 16, 17, 18, 19, 20, 25 nucleotides, or between 16-30, or between 16-25, or between 16-20 nucleotides in length. 
     These and others are further detailed and described elsewhere herein. 
     Cell-Based Vector Amplification and Expression 
     Vectors may be introduced and propagated in a prokaryote or prokaryotic cell. In some embodiments, a prokaryote is used to amplify copies of a vector to be introduced into a eukaryotic cell or as an intermediate vector in the production of a vector to be introduced into a eukaryotic cell (e.g. amplifying a plasmid as part of a viral vector packaging system). The vectors can be viral-based or non-viral based. In some embodiments, a prokaryote is used to amplify copies of a vector and express one or more nucleic acids, such as to provide a source of one or more proteins for delivery to a host cell or host organism. 
     Vectors can be designed for expression of one or more elements of the CRISPR-Cas system described herein (e.g. nucleic acid transcripts, proteins, enzymes, and combinations thereof) in a suitable host cell. In some aspects, the suitable host cell is a prokaryotic cell. Suitable host cells include, but are not limited to, bacterial cells, yeast cells, insect cells, and mammalian cells. In some aspects, the suitable host cell is a eukaryotic cell. 
     In some aspects, the suitable host cell is a suitable bacterial cell. Suitable bacterial cells include, but are not limited to bacterial cells from the bacteria of the species  Escherichia coli . Many suitable strains of  E. coli  are known in the art for expression of vectors. These include, but are not limited to Pir1, Stb12, Stb13, Stb14, TOP10, XL1 Blue, and XL10 Gold. In some aspects, the host cell is a suitable insect cell. Suitable insect cells include those from  Spodoptera frugiperda . Suitable strains of  S. frugiperda  cells include, but are not limited to Sf9 and Sf21. In some aspects, the host cell is a suitable yeast cell. In some aspects, the yeast cell can be from  Saccharomyces cerevisiae . In some aspects, the host cell is a suitable mammalian cell. Many types of mammalian cells have been developed to express vectors. Suitable mammalian cells include, but are not limited to, HEK293, Chinese Hamster Ovary Cells (CHOs), mouse myeloma cells, HeLa, U205, A549, HT1080, CAD, P19, NIH 3T3, L929, N2a, MCF-7, Y79, SO-Rb50, HepG G2, DIKX-X11, J558L, Baby hamster kidney cells (BHK), and chicken embryo fibroblasts (CEFs). Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). 
     In some aspects, the vector can be a yeast expression vector. Examples of vectors for expression in yeast  Saccharomyces cerevisiae  include pYepSec1 (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kuij an and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.). As used herein, a “yeast expression vector” refers to a nucleic acid that contains one or more sequences encoding an RNA and/or polypeptide and may further contain any desired elements that control the expression of the nucleic acid(s), as well as any elements that enable the replication and maintenance of the expression vector inside the yeast cell. Many suitable yeast expression vectors and features thereof are known in the art; for example, various vectors and techniques are illustrated in in Yeast Protocols, 2nd edition, Xiao, W., ed. (Humana Press, New York, 2007) and Buckholz, R. G. and Gleeson, M. A. (1991) Biotechnology (NY) 9(11): 1067-72. Yeast vectors can contain, without limitation, a centromeric (CEN) sequence, an autonomous replication sequence (ARS), a promoter, such as an RNA Polymerase III promoter, operably linked to a sequence or gene of interest, a terminator such as an RNA polymerase III terminator, an origin of replication, and a marker gene (e.g., auxotrophic, antibiotic, or other selectable markers). Examples of expression vectors for use in yeast may include plasmids, yeast artificial chromosomes, 2μ plasmids, yeast integrative plasmids, yeast replicative plasmids, shuttle vectors, and episomal plasmids. 
     In some aspects, the vector is a baculovirus vector or expression vector and can be suitable for expression of polynucleotides and/or proteins in insect cells. In some embodiments, the suitable host cell is an insect cell. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39). rAAV (recombinant Adeno-associated viral) vectors are preferably produced in insect cells, e.g.,  Spodoptera frugiperda  Sf9 insect cells, grown in serum-free suspension culture. Serum-free insect cells can be purchased from commercial vendors, e.g., Sigma Aldrich (EX-CELL 405). 
     In some embodiments, the vector is a mammalian expression vector. In some aspects, the mammalian expression vector is capable of expressing one or more polynucleotides and/or polypeptides in a mammalian cell. Examples of mammalian expression vectors include, but are not limited to, pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195). The mammalian expression vector can include one or more suitable regulatory elements capable of controlling expression of the one or more polynucleotides and/or proteins in the mammalian cell. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein and known in the art. More detail on suitable regulatory elements are described elsewhere herein. 
     For other suitable expression vectors and vector systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. 
     In some embodiments, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al., 1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame and Eaton, 1988 . Adv. Immunol.  43: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989 . EMBO J.  8: 729-733) and immunoglobulins (Baneiji, et al., 1983 . Cell  33: 729-740; Queen and Baltimore, 1983 . Cell  33: 741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1989 . Proc. Natl. Acad. Sci. USA  86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985 . Science  230: 912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990 . Science  249: 374-379) and the α-fetoprotein promoter (Campes and Tilghman, 1989 . Genes Dev.  3: 537-546). With regards to these prokaryotic and eukaryotic vectors, mention is made of U.S. Pat. No. 6,750,059, the contents of which are incorporated by reference herein in their entirety. Other aspects can utilize viral vectors, with regards to which mention is made of U.S. patent application Ser. No. 13/092,085, the contents of which are incorporated by reference herein in their entirety. Tissue-specific regulatory elements are known in the art and in this regard, mention is made of U.S. Pat. No. 7,776,321, the contents of which are incorporated by reference herein in their entirety. In some embodiments, a regulatory element can be operably linked to one or more elements of a CRISPR-Cas system so as to drive expression of the one or more elements of the CRISPR-Cas system described herein. 
     In some aspects, the vector can be a fusion vector or fusion expression vector. In some aspects, fusion vectors add a number of amino acids to a protein encoded therein, such as to the amino terminus, carboxy terminus, or both of a recombinant protein. Such fusion vectors can serve one or more purposes, such as: (i) to increase expression of recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. In some aspects, expression of polynucleotides (such as non-coding polynucleotides) and proteins in prokaryotes can be carried out in  Escherichia coli  with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion polynucleotides and/or proteins. In some aspects, the fusion expression vector can include a proteolytic cleavage site, which can be introduced at the junction of the fusion vector backbone or other fusion moiety and the recombinant polynucleotide or protein to enable separation of the recombinant polynucleotide or protein from the fusion vector backbone or other fusion moiety subsequent to purification of the fusion polynucleotide or protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Example fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. Examples of suitable inducible non-fusion  E. coli  expression vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89). 
     In some embodiments, one or more vectors driving expression of one or more elements of a CRISPR-Cas system described herein are introduced into a host cell such that expression of the elements of the engineered delivery system described herein direct formation a CRISPR-Cas complex at one or more target sites. For example, a CRISPR-Cas effector protein describe herein and a nucleic acid component (e.g., a guide polynucleotide) can each be operably linked to separate regulatory elements on separate vectors. RNA(s) of different elements of CRISPR-Cas system described herein can be delivered to an animal, plant, microorganism or cell thereof to produce an animal (e.g., a mammal, reptile, avian, etc.), plant, microorganism or cell thereof that constitutively, inducibly, or conditionally expresses different elements of the CRIPSR-Cas system described herein that incorporates one or more elements of the CRISPR-Cas system described herein or contains one or more cells that incorporates and/or expresses one or more elements of the CRISPR-Cas system described herein. 
     In some aspects, two or more of the elements expressed from the same or different regulatory element(s), can be combined in a single vector, with one or more additional vectors providing any components of the system not included in the first vector. CRISPR-Cas system polynucleotides that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5′ with respect to (“upstream” of) or 3′ with respect to (“downstream” of) a second element. The coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element, and oriented in the same or opposite direction. In some embodiments, a single promoter drives expression of a transcript encoding one or more CRISPR-Cas system proteins, embedded within one or more intron sequences (e.g., each in a different intron, two or more in at least one intron, or all in a single intron). In some embodiments, the CRISPR-Cas system polynucleotides can be operably linked to and expressed from the same promoter. 
     Cell-Free Vector and Polynucleotide Expression 
     In some aspects, the polynucleotide encoding one or more features of the CRISPR-Cas system can be expressed from a vector or suitable polynucleotide in a cell-free in vitro system. In other words, the polynucleotide can be transcribed and optionally translated in vitro. In vitro transcription/translation systems and appropriate vectors are generally known in the art and commercially available. Generally, in vitro transcription and in vitro translation systems replicate the processes of RNA and protein synthesis, respectively, outside of the cellular environment. Vectors and suitable polynucleotides for in vitro transcription can include T7, SP6, T3, promoter regulatory sequences that can be recognized and acted upon by an appropriate polymerase to transcribe the polynucleotide or vector. 
     In vitro translation can be stand-alone (e.g. translation of a purified polyribonucleotide) or linked/coupled to transcription. In some aspects, the cell-free (or in vitro) translation system can include extracts from rabbit reticulocytes, wheat germ, and/or  E. coli . The extracts can include various macromolecular components that are needed for translation of exogenous RNA (e.g. 70S or 80S ribosomes, tRNAs, aminoacyl-tRNA, synthetases, initiation, elongation factors, termination factors, etc.). Other components can be included or added during the translation reaction, including but not limited to, amino acids, energy sources (ATP, GTP), energy regenerating systems (creatine phosphate and creatine phosphokinase (eukaryotic systems)) (phosphoenol pyruvate and pyruvate kinase for bacterial systems), and other co-factors (Mg2+, K+, etc.). As previously mentioned, in vitro translation can be based on RNA or DNA starting material. Some translation systems can utilize an RNA template as starting material (e.g. reticulocyte lysates and wheat germ extracts). Some translation systems can utilize a DNA template as a starting material (e.g.  E coli -based systems). In these systems transcription and translation are coupled and DNA is first transcribed into RNA, which is subsequently translated. Suitable standard and coupled cell-free translation systems are generally known in the art and are commercially available. 
     Vector Features 
     The vectors can include additional features that can confer one or more functionalities to the vector, the polynucleotide to be delivered, a virus particle produced there from, or polypeptide expressed thereof. Such features include, but are not limited to, regulatory elements, selectable markers, molecular identifiers (e.g. molecular barcodes), stabilizing elements, and the like. It will be appreciated by those skilled in the art that the design of the expression vector and additional features included can depend on such factors as the choice of the host cell to be transformed, the level of expression desired, etc. 
     Regulatory Elements 
     In certain embodiments, the polynucleotides and/or vectors thereof described herein (such as the CRISPR-Cas system polynucleotides of the present invention) can include one or more regulatory elements that can be operatively linked to the polynucleotide. The term “regulatory element” is intended to include promoters, enhancers, internal ribosomal entry sites (IRES), other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences) and cellular localization signals (e.g. nuclear localization signals). Such regulatory elements are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). A tissue-specific promoter can direct expression primarily in a desired tissue of interest, such as muscle, neuron, bone, skin, blood, specific organs (e.g., liver, pancreas), or particular cell types (e.g., lymphocytes). Regulatory elements may also direct expression in a temporal-dependent manner, such as in a cell-cycle dependent or developmental stage-dependent manner, which may or may not also be tissue or cell-type specific. In some embodiments, a vector comprises one or more pol III promoter (e.g., 1, 2, 3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof. Examples of pol III promoters include, but are not limited to, U6 and H1 promoters. Examples of pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) (see, e.g., Boshart et al, Cell, 41:521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1α promoter. Also encompassed by the term “regulatory element” are enhancer elements, such as WPRE; CMV enhancers; the R-U5′ segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981). 
     In some aspects, the regulatory sequence can be a regulatory sequence described in U.S. Pat. No. 7,776,321, U.S. Pat. Pub. No. 2011/0027239, and International Patent Publication No. WO 2011/028929, the contents of which are incorporated by reference herein in their entirety. In some aspects, the vector can contain a minimal promoter. In some aspects, the minimal promoter is the Mecp2 promoter, tRNA promoter, or U6. In a further embodiment, the minimal promoter is tissue specific. In some aspects, the length of the vector polynucleotide the minimal promoters and polynucleotide sequences is less than 4.4 Kb. 
     To express a polynucleotide, the vector can include one or more transcriptional and/or translational initiation regulatory sequences, e.g. promoters, that direct the transcription of the gene and/or translation of the encoded protein in a cell. In some aspects a constitutive promoter may be employed. Suitable constitutive promoters for mammalian cells are generally known in the art and include, but are not limited to SV40, CAG, CMV, EF-1α, β-actin, RSV, and PGK. Suitable constitutive promoters for bacterial cells, yeast cells, and fungal cells are generally known in the art, such as a T-7 promoter for bacterial expression and an alcohol dehydrogenase promoter for expression in yeast. 
     In some aspects, the regulatory element can be a regulated promoter. “Regulated promoter” refers to promoters that direct gene expression not constitutively, but in a temporally- and/or spatially-regulated manner, and includes tissue-specific, tissue-preferred and inducible promoters. Regulated promoters include conditional promoters and inducible promoters. In some aspects, conditional promoters can be employed to direct expression of a polynucleotide in a specific cell type, under certain environmental conditions, and/or during a specific state of development. Suitable tissue specific promoters can include, but are not limited to, liver specific promoters (e.g. APOA2, SERPIN A1 (hAAT), CYP3A4, and MIR122), pancreatic cell promoters (e.g. INS, IRS2, Pdx1, Alx3, Ppy), cardiac specific promoters (e.g. Myh6 (alpha MHC), MYL2 (MLC-2v), TNI3 (cTn1), NPPA (ANF), Slc8a1 (Ncx1)), central nervous system cell promoters (SYN1, GFAP, INA, NES, MOBP, MBP, TH, FOXA2 (HNF3 beta)), skin cell specific promoters (e.g. FLG, K14, TGM3), immune cell specific promoters, (e.g. ITGAM, CD43 promoter, CD14 promoter, CD45 promoter, CD68 promoter), urogenital cell specific promoters (e.g. Pbsn, Upk2, Sbp, Fer1l4), endothelial cell specific promoters (e.g. ENG), pluripotent and embryonic germ layer cell specific promoters (e.g. Oct4, NANOG, Synthetic Oct4, T brachyury, NES, SOX17, FOXA2, MIR122), and muscle cell specific promoter (e.g. Desmin). Other tissue and/or cell specific promoters are generally known in the art and are within the scope of this disclosure. 
     Inducible/conditional promoters can be positively inducible/conditional promoters (e.g. a promoter that activates transcription of the polynucleotide upon appropriate interaction with an activated activator, or an inducer (compound, environmental condition, or other stimulus) or a negative/conditional inducible promoter (e.g. a promoter that is repressed (e.g. bound by a repressor) until the repressor condition of the promotor is removed (e.g. inducer binds a repressor bound to the promoter stimulating release of the promoter by the repressor or removal of a chemical repressor from the promoter environment). The inducer can be a compound, environmental condition, or other stimulus. Thus, inducible/conditional promoters can be responsive to any suitable stimuli such as chemical, biological, or other molecular agents, temperature, light, and/or pH. Suitable inducible/conditional promoters include, but are not limited to, Tet-On, Tet-Off, Lac promoter, pBad, AlcA, LexA, Hsp70 promoter, Hsp90 promoter, pDawn, XVE/OlexA, GVG, and pOp/LhGR. 
     Where expression in a plant cell is desired, the components of the CRISPR-Cas system described herein are typically placed under control of a plant promoter, i.e. a promoter operable in plant cells. The use of different types of promoters is envisaged. 
     A constitutive plant promoter is a promoter that is able to express the open reading frame (ORF) that it controls in all or nearly all of the plant tissues during all or nearly all developmental stages of the plant (referred to as “constitutive expression”). One non-limiting example of a constitutive promoter is the cauliflower mosaic virus 35S promoter. Different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. In particular embodiments, one or more of the CRISPR-Cas system components are expressed under the control of a constitutive promoter, such as the cauliflower mosaic virus 35S promoter issue-preferred promoters can be utilized to target enhanced expression in certain cell types within a particular plant tissue, for instance vascular cells in leaves or roots or in specific cells of the seed. Examples of particular promoters for use in the CRISPR-Cas system are found in Kawamata et al., (1997) Plant Cell Physiol 38:792-803; Yamamoto et al., (1997) Plant J 12:255-65; Hire et al, (1992) Plant Mol Biol 20:207-18, Kuster et al, (1995) Plant Mol Biol 29:759-72, and Capana et al., (1994) Plant Mol Biol 25:681-91. 
     Examples of promoters that are inducible and that can allow for spatiotemporal control of gene editing or gene expression may use a form of energy. The form of energy may include but is not limited to sound energy, electromagnetic radiation, chemical energy and/or thermal energy. Examples of inducible systems include tetracycline inducible promoters (Tet-On or Tet-Off), small molecule two-hybrid transcription activations systems (FKBP, ABA, etc.), or light inducible systems (Phytochrome, LOV domains, or cryptochrome), such as a Light Inducible Transcriptional Effector (LITE) that direct changes in transcriptional activity in a sequence-specific manner. The components of a light inducible system may include one or more elements of the CRISPR-Cas system described herein, a light-responsive cytochrome heterodimer (e.g. from  Arabidopsis thaliana ), and a transcriptional activation/repression domain. In some aspects, the vector can include one or more of the inducible DNA binding proteins provided in International Patent Publication No. WO 2014/018423 and US Patent Publication Nos., 2015/0291966, 2017/0166903, 2019/0203212, which describe e.g. aspects of inducible DNA binding proteins and methods of use and can be adapted for use with the present invention. 
     In some aspects, transient or inducible expression can be achieved by including, for example, chemical-regulated promotors, i.e. whereby the application of an exogenous chemical induces gene expression. Modulation of gene expression can also be obtained by including a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters include, but are not limited to, the maize 1n2-2 promoter, activated by benzene sulfonamide herbicide safeners (De Veylder et al., (1997) Plant Cell Physiol 38:568-77), the maize GST promoter (GST-11-27, WO93/01294), activated by hydrophobic electrophilic compounds used as pre-emergent herbicides, and the tobacco PR-1 a promoter (Ono et al., (2004) Biosci Biotechnol Biochem 68:803-7) activated by salicylic acid. Promoters which are regulated by antibiotics, such as tetracycline-inducible and tetracycline-repressible promoters (Gatz et al., (1991) Mol Gen Genet 227:229-37; U.S. Pat. Nos. 5,814,618 and 5,789,156) can also be used herein. 
     In some aspects, the polynucleotide, vector or system thereof can include one or more elements capable of translocating and/or expressing a CRISPR-Cas polynucleotide to/in a specific cell component or organelle. Such organelles can include, but are not limited to, nucleus, ribosome, endoplasmic reticulum, Golgi apparatus, chloroplast, mitochondria, vacuole, lysosome, cytoskeleton, plasma membrane, cell wall, peroxisome, centrioles, etc. Such regulatory elements can include, but are not limited to, nuclear localization signals (examples of which are described in greater detail elsewhere herein), any such as those that are annotated in the LocSigDB database (see e.g. http://genome.unmc.edu/LocSigDB/andNegi et al., 2015. Database. 2015: bav003; doi: 10.1093/database/bav003), nuclear export signals (e.g. LXXXLXXLXL and others described elsewhere herein), endoplasmic reticulum localization/retention signals (e.g. KDEL, KDXX, KKXX, KXX, and others described elsewhere herein; and see e.g. Liu et al. 2007 Mol. Biol. Cell. 18(3):1073-1082 and Gorleku et al., 2011. J. Biol. Chem. 286:39573-39584), mitochondria (see e.g. Cell Reports. 22:2818-2826, particularly at FIG. 2; Doyle et al. 2013. PLoS ONE 8, e67938; Funes et al. 2002. J. Biol. Chem. 277:6051-6058; Matouschek et al. 1997. PNAS USA 85:2091-2095; Oca-Cossio et al., 2003. 165:707-720; Waltner et al., 1996. J. Biol. Chem. 271:21226-21230; Wilcox et al., 2005. PNAS USA 102:15435-15440; Galanis et al., 1991. FEBS Lett 282:425-430, peroxisome (e.g. (S/A/C)-(K/R/H)-(L/A), SLK, (R/K)-(L/V/I)-XXXXX-(H/Q)-(L/A/F). Suitable protein targeting motifs can also be designed or identified using any suitable database or prediction tool, including but not limited to Minimotif Miner (http:minimotifminer.org, http://mitominer.mrc-mbu.cam.ac.uk/release-4.0/aspect.do?name=Protein%20MTS), LocDB (see above), PTSs predictor ( ) TargetP-2.0 (http://www.cbs.dtu.dk/services/TargetP/), ChloroP (http://www.cbs.dtu.dk/services/ChloroP/); NetNES (http://www.cbs.dtu.dk/services/NetNES/), Predotar (https://urgi.versailles.inra.fr/predotar/), and SignalP (http://www.cbs.dtu.dk/services/SignalP/). 
     Selectable Markers and Tags 
     One or more of the CRISPR-Cas system polynucleotides can be can be operably linked, fused to, or otherwise modified to include a polynucleotide that encodes or is a selectable marker or tag, which can be a polynucleotide or polypeptide. In some aspects, the polypeptide encoding a polypeptide selectable marker can be incorporated in the CRISPR-Cas system polynucleotide such that the selectable marker polypeptide, when translated, is inserted between two amino acids between the N- and C-terminus of the CRISPR-Cas system polypeptide or at the N- and/or C-terminus of the CRISPR-Cas system polypeptide. In some aspects, the selectable marker or tag is a polynucleotide barcode or unique molecular identifier (UMI). 
     It will be appreciated that the polynucleotide encoding such selectable markers or tags can be incorporated into a polynucleotide encoding one or more components of the CRISPR-Cas system described herein in an appropriate manner to allow expression of the selectable marker or tag. Such techniques and methods are described elsewhere herein and will be instantly appreciated by one of ordinary skill in the art in view of this disclosure. Many such selectable markers and tags are generally known in the art and are intended to be within the scope of this disclosure. 
     Suitable selectable markers and tags include, but are not limited to, affinity tags, such as chitin binding protein (CBP), maltose binding protein (MBP), glutathione-S-transferase (GST), poly(His) tag; solubilization tags such as thioredoxin (TRX) and poly(NANP), MBP, and GST; chromatography tags such as those consisting of polyanionic amino acids, such as FLAG-tag; epitope tags such as V5-tag, Myc-tag, HA-tag and NE-tag; protein tags that can allow specific enzymatic modification (such as biotinylation by biotin ligase) or chemical modification (such as reaction with FlAsH-EDT2 for fluorescence imaging), DNA and/or RNA segments that contain restriction enzyme or other enzyme cleavage sites; DNA segments that encode products that provide resistance against otherwise toxic compounds including antibiotics, such as, spectinomycin, ampicillin, kanamycin, tetracycline, Basta, neomycin phosphotransferase II (NEO), hygromycin phosphotransferase (HPT)) and the like; DNA and/or RNA segments that encode products that are otherwise lacking in the recipient cell (e.g., tRNA genes, auxotrophic markers); DNA and/or RNA segments that encode products which can be readily identified (e.g., phenotypic markers such as β-galactosidase, GUS; fluorescent proteins such as green fluorescent protein (GFP), cyan (CFP), yellow (YFP), red (RFP), luciferase, and cell surface proteins); polynucleotides that can generate one or more new primer sites for PCR (e.g., the juxtaposition of two DNA sequences not previously juxtaposed), DNA sequences not acted upon or acted upon by a restriction endonuclease or other DNA modifying enzyme, chemical, etc.; epitope tags (e.g. GFP, FLAG- and His-tags), and, DNA sequences that make a molecular barcode or unique molecular identifier (UMI), DNA sequences required for a specific modification (e.g., methylation) that allows its identification. Other suitable markers will be appreciated by those of skill in the art. Selectable markers and tags can be operably linked to one or more components of the CRISPR-Cas system described herein via suitable linker, such as a glycine or glycine serine linkers as short as GS or GG up to (GGGGG) 3  (SEQ ID NO: 56) or (GGGGS) 3  (SEQ ID NO: 10). Other suitable linkers are described elsewhere herein. 
     The vector or vector system can include one or more polynucleotides encoding one or more targeting moieties. In some aspects, the targeting moiety encoding polynucleotides can be included in the vector or vector system, such as a viral vector system, such that they are expressed within and/or on the virus particle(s) produced such that the virus particles can be targeted to specific cells, tissues, organs, etc. In some aspects, the targeting moiety encoding polynucleotides can be included in the vector or vector system such that the CRISPR-Cas system polynucleotide(s) and/or products expressed therefrom include the targeting moiety and can be targeted to specific cells, tissues, organs, etc. In some aspects, such as non-viral carriers, the targeting moiety can be attached to the carrier (e.g. polymer, lipid, inorganic molecule etc.) and can be capable of targeting the carrier and any attached or associated CRISPR-Cas system polynucleotide(s) to specific cells, tissues, organs, etc. 
     Codon Optimization of Vector Polynucleotides 
     As described elsewhere herein, the polynucleotide encoding one or more aspects of the CRISPR-Cas system described herein can be codon optimized. In some aspects, one or more polynucleotides contained in a vector (“vector polynucleotides”) described herein that are in addition to an optionally codon optimized polynucleotide encoding aspects of the CRISPR-Cas system described herein can be codon optimized. In general, codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the “Codon Usage Database” available at www.kazusa.orjp/codon/and these tables can be adapted in a number of ways. See Nakamura, Y., et al. “Codon usage tabulated from the international DNA sequence databases: status for the year 2000” Nucl. Acids Res. 28:292 (2000). Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, Pa.), are also available. In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding a DNA/RNA-targeting Cas protein corresponds to the most frequently used codon for a particular amino acid. As to codon usage in yeast, reference is made to the online Yeast Genome database available at http://www.yeastgenome.org/community/codon_usage.shtml, or Codon selection in yeast, Bennetzen and Hall, J Biol Chem. 1982 Mar. 25; 257(6):3026-31. As to codon usage in plants including algae, reference is made to  Codon usage in higher plants, green algae, and cyanobacteria , Campbell and Gowri, Plant Physiol. 1990 January; 92(1): 1-11; as well as  Codon usage in plant genes , Murray et al, Nucleic Acids Res. 1989 Jan. 25; 17(2):477-98; or  Selection on the codon bias of chloroplast and cyanelle genes in different plant and algal lineages , Morton B R, J Mol Evol. 1998 April; 46(4):449-59. 
     The vector polynucleotide can be codon optimized for expression in a specific cell-type, tissue type, organ type, and/or subject type. In some aspects, a codon optimized sequence is a sequence optimized for expression in a eukaryote, e.g., humans (i.e. being optimized for expression in a human or human cell), or for another eukaryote, such as another animal (e.g. a mammal or avian) as is described elsewhere herein. Such codon optimized sequences are within the ambit of the ordinary skilled artisan in view of the description herein. In some aspects, the polynucleotide is codon optimized for a specific cell type. Such cell types can include, but are not limited to, epithelial cells (including skin cells, cells lining the gastrointestinal tract, cells lining other hollow organs), nerve cells (nerves, brain cells, spinal column cells, nerve support cells (e.g. astrocytes, glial cells, Schwann cells etc.), muscle cells (e.g. cardiac muscle, smooth muscle cells, and skeletal muscle cells), connective tissue cells (fat and other soft tissue padding cells, bone cells, tendon cells, cartilage cells), blood cells, stem cells and other progenitor cells, immune system cells, germ cells, and combinations thereof. Such codon optimized sequences are within the ambit of the ordinary skilled artisan in view of the description herein. In some aspects, the polynucleotide is codon optimized for a specific tissue type. Such tissue types can include, but are not limited to, muscle tissue, connective tissue, connective tissue, nervous tissue, and epithelial tissue. Such codon optimized sequences are within the ambit of the ordinary skilled artisan in view of the description herein. In some aspects, the polynucleotide is codon optimized for a specific organ. Such organs include, but are not limited to, muscles, skin, intestines, liver, spleen, brain, lungs, stomach, heart, kidneys, gallbladder, pancreas, bladder, thyroid, bone, blood vessels, blood, and combinations thereof. Such codon optimized sequences are within the ambit of the ordinary skilled artisan in view of the description herein. 
     In some embodiments, a vector polynucleotide is codon optimized for expression in particular cells, such as prokaryotic or eukaryotic cells. The eukaryotic cells may be those of or derived from a particular organism, such as a plant or a mammal, including but not limited to human, or non-human eukaryote or animal or mammal as discussed herein, e.g., mouse, rat, rabbit, dog, livestock, or non-human mammal or primate. 
     Vector Construction 
     The vectors described herein can be constructed using any suitable process or technique. In some aspects, one or more suitable recombination and/or cloning methods or techniques can be used to the vector(s) described herein. Suitable recombination and/or cloning techniques and/or methods can include, but not limited to, those described in U.S. Patent Publication No. US 2004/0171156 A1. Other suitable methods and techniques are described elsewhere herein. 
     Construction of recombinant AAV vectors are described in a number of publications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat &amp; Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol. 63:03822-3828 (1989). Any of the techniques and/or methods can be used and/or adapted for constructing an AAV or other vector described herein. nAAV vectors are discussed elsewhere herein. 
     In some embodiments, a vector comprises one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a “cloning site”). In some embodiments, one or more insertion sites (e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertion sites) are located upstream and/or downstream of one or more sequence elements of one or more vectors. When multiple different guide polynucleotides are used, a single expression construct may be used to target nucleic acid-targeting activity to multiple different, corresponding target sequences within a cell. For example, a single vector may comprise about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more guide s polynucleotides. In some embodiments, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more such guide-polynucleotide-containing vectors may be provided, and optionally delivered to a cell. 
     Delivery vehicles, vectors, particles, nanoparticles, formulations and components thereof for expression of one or more elements of a CRISPR-Cas system described herein are as used in the foregoing documents, such as International Patent Publication No. WO 2014/093622 (PCT/US2013/074667) and are discussed in greater detail herein. 
     Viral Vectors 
     In some aspects, the vector is a viral vector. The term of art “viral vector” and as used herein in this context refers to polynucleotide based vectors that contain one or more elements from or based upon one or more elements of a virus that can be capable of expressing and packaging a polynucleotide, such as a CRISPR-Cas system polynucleotide of the present invention, into a virus particle and producing said virus particle when used alone or with one or more other viral vectors (such as in a viral vector system). Viral vectors and systems thereof can be used for producing viral particles for delivery of and/or expression of one or more components of the CRISPR-Cas system described herein. The viral vector can be part of a viral vector system involving multiple vectors. In some aspects, systems incorporating multiple viral vectors can increase the safety of these systems. Suitable viral vectors can include retroviral-based vectors, lentiviral-based vectors, adenoviral-based vectors, adeno associated vectors, helper-dependent adenoviral (HdAd) vectors, hybrid adenoviral vectors, herpes simplex virus-based vectors, poxvirus-based vectors, and Epstein-Barr virus-based vectors. Other aspects of viral vectors and viral particles produce therefrom are described elsewhere herein. In some aspects, the viral vectors are configured to produce replication incompetent viral particles for improved safety of these systems. 
     In certain embodiments, the virus structural component, which can be encoded by one or more polynucleotides in a viral vector or vector system, comprises one or more capsid proteins including an entire capsid. In certain embodiments, such as wherein a viral capsid comprises multiple copies of different proteins, the delivery system can provide one or more of the same protein or a mixture of such proteins. For example, AAV comprises 3 capsid proteins, VP1, VP2, and VP3, thus delivery systems of the invention can comprise one or more of VP1, and/or one or more of VP2, and/or one or more of VP3. Accordingly, the present invention is applicable to a virus within the family Adenoviridae, such as Atadenovirus, e.g., Ovine atadenovirus D, Aviadenovirus, e.g., Fowl aviadenovirus A, Ichtadenovirus, e.g., Sturgeon ichtadenovirus A, Mastadenovirus (which includes adenoviruses such as all human adenoviruses), e.g., Human mastadenovirus C, and Siadenovirus, e.g., Frog siadenovirus A. Thus, a virus of within the family Adenoviridae is contemplated as within the invention with discussion herein as to adenovirus applicable to other family members. Target-specific AAV capsid variants can be used or selected. Non-limiting examples include capsid variants selected to bind to chronic myelogenous leukemia cells, human CD34 PBPC cells, breast cancer cells, cells of lung, heart, dermal fibroblasts, melanoma cells, stem cell, glioblastoma cells, coronary artery endothelial cells and keratinocytes. See, e.g., Buning et al, 2015, Current Opinion in Pharmacology 24, 94-104. From teachings herein and knowledge in the art as to modifications of adenovirus (see, e.g., U.S. Pat. Nos. 9,410,129, 7,344,872, 7,256,036, 6,911,199, 6,740,525; Matthews, “Capsid-Incorporation of Antigens into Adenovirus Capsid Proteins for a Vaccine Approach,” Mol Pharm, 8(1): 3-11 (2011)), as well as regarding modifications of AAV, the skilled person can readily obtain a modified adenovirus that has a large payload protein or a CRISPR-protein, despite that heretofore it was not expected that such a large protein could be provided on an adenovirus. And as to the viruses related to adenovirus mentioned herein, as well as to the viruses related to AAV mentioned elsewhere herein, the teachings herein as to modifying adenovirus and AAV, respectively, can be applied to those viruses without undue experimentation from this disclosure and the knowledge in the art. 
     In some embodiments, the viral vector is configured such that when the cargo is packaged the cargo(s) (e.g. one or more components of the CRISPR-Cas system, including but not limited to a Cas effector, is external to the capsid or virus particle. In the sense that it is not inside the capsid (enveloped or encompassed with the capsid), but is externally exposed so that it can contact the target genomic DNA. In some embodiments, the viral vector is configured such that all the carog(s) are contained within the capsid after packaging. 
     Split Viral Vector Systems 
     When the CRISPR-Cas system viral vector or vector system (be it a retroviral (e.g. AAV) or lentiviral vector) is designed so as to position the cargo(s) (e.g., one or more CRISPR-Cas system components) at the internal surface of the capsid once formed, the cargo(s) will fill most or all of internal volume of the capsid. In other aspects, the CRISPR protein may be modified or divided so as to occupy a less of the capsid internal volume. Accordingly, in certain embodiments, the CRISPR-Cas system or component thereof (e.g. a Cas effector protein) can be divided in two portions, one portion comprises in one viral particle or capsid and the second portion comprised in a second viral particle or capsid. In certain embodiments, by splitting the CRISPR-Cas system or component thereof in two portions, space is made available to link one or more heterologous domains to one or both CRISPR-Cas system component (e.g., Cas protein) portions. Such systems can be referred to as “split vector systems” or in the context of the present disclosure a “split CRISPR-Cas system” a “split CRISPR protein”, a “split Cas protein” and the like. This split protein approach is also described elsewhere herein. When the concept is applied to a vector system, it thus describes putting pieces of the split proteins on different vectors thus reducing the payload of any one vector. This approach can facilitate delivery of systems where the total system size is close to or exceeds the packaging capacity of the vector. This is independent of any regulation of the CRISPR-Cas system that can be achieved with a split system or split protein design. 
     Split CRISPR proteins that can be incorporated into the AAV or other vectors described herein are set forth elsewhere herein and in documents incorporated herein by reference in further detail herein. In certain embodiments, each part of a split CRISPR proteins are attached to a member of a specific binding pair, and when bound with each other, the members of the specific binding pair maintain the parts of the CRISPR protein in proximity. In certain embodiments, each part of a split CRISPR protein is associated with an inducible binding pair. An inducible binding pair is one which is capable of being switched “on” or “off” by a protein or small molecule that binds to both members of the inducible binding pair. In general, according to the invention, CRISPR proteins may preferably split between domains, leaving domains intact. Preferred, non-limiting examples of such CRISPR proteins include, without limitation, Cas-like protein, and orthologues. Preferred, non-limiting examples of split points include, with reference to SpCas9: a split position between 202A/203S; a split position between 255F/256D; a split position between 310E/3111; a split position between 534R/535K; a split position between 572E/573C; a split position between 7135/714G; a split position between 1003L/104E; a split position between 1054G/1055E; a split position between 1114N/1115S; a split position between 1152K/1153S; a split position between 1245K/1246G; or a split between 1098 and 1099. Corresponding positions in other Cas proteins can be appreciated in view of these positions made with reference to SpCas9. In some embodiments, any AAV serotype is preferred. In some embodiments, the VP2 domain associated with the CRISPR enzyme is an AAV serotype 2 VP2 domain. In some embodiments, the VP2 domain associated with the CRISPR enzyme is an AAV serotype 8 VP2 domain. The serotype can be a mixed serotype as is known in the art. 
     Retroviral and Lentiviral Vectors 
     Retroviral vectors can be composed of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Suitable retroviral vectors for the CRISPR-Cas systems can include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991); PCT/US94/05700). Selection of a retroviral gene transfer system may therefore depend on the target tissue. 
     The tropism of a retrovirus can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and are described in greater detail elsewhere herein. A retrovirus can also be engineered to allow for conditional expression of the inserted transgene, such that only certain cell types are infected by the lentivirus. 
     Lentiviruses are complex retroviruses that have the ability to infect and express their genes in both mitotic and post-mitotic cells. Advantages of using a lentiviral approach can include the ability to transduce or infect non-dividing cells and their ability to typically produce high viral titers, which can increase efficiency or efficacy of production and delivery. Suitable lentiviral vectors include, but are not limited to, human immunodeficiency virus (HIV)-based lentiviral vectors, feline immunodeficiency virus (FIV)-based lentiviral vectors, simian immunodeficiency virus (SIV)-based lentiviral vectors, Moloney Murine Leukaemia Virus (Mo-MLV), Visna.maedi virus (VMV)-based lentiviral vector, carpine arthritis-encephalitis virus (CAEV)-based lentiviral vector, bovine immune deficiency virus (BIV)-based lentiviral vector, and Equine infectious anemia (EIAV)-based lentiviral vector. In some embodiments, an HIV-based lentiviral vector system can be used. In some embodiments, a FIV-based lentiviral vector system can be used. In some aspects, the lentiviral vector is an EIAV-based lentiviral vector or vector system. EIAV vectors have been used to mediate expression, packaging, and/or delivery in other contexts, such as for ocular gene therapy (see, e.g., Balagaan, J Gene Med 2006; 8: 275-285). In another embodiment, RetinoStat®, (see, e.g., Binley et al., HUMAN GENE THERAPY 23:980-991 (September 2012)), which describes RetinoStat®, an equine infectious anemia virus-based lentiviral gene therapy vector that expresses angiostatic proteins endostatin and angiostatin that is delivered via a subretinal injection for the treatment of the wet form of age-related macular degeneration. Any of these vectors described in these publications can be modified for the elements of the CRISPR-Cas system described herein. 
     In some aspects, the lentiviral vector or vector system thereof can be a first-generation lentiviral vector or vector system thereof. First-generation lentiviral vectors can contain a large portion of the lentivirus genome, including the gag and pol genes, other additional viral proteins (e.g. VSV-G) and other accessory genes (e.g. vif, vprm vpu, nef, and combinations thereof), regulatory genes (e.g. tat and/or rev) as well as the gene of interest between the LTRs. First generation lentiviral vectors can result in the production of virus particles that can be capable of replication in vivo, which may not be appropriate for some instances or applications. 
     In some aspects, the lentiviral vector or vector system thereof can be a second-generation lentiviral vector or vector system thereof. Second-generation lentiviral vectors do not contain one or more accessory virulence factors and do not contain all components necessary for virus particle production on the same lentiviral vector. This can result in the production of a replication-incompetent virus particle and thus increase the safety of these systems over first-generation lentiviral vectors. In some aspects, the second-generation vector lacks one or more accessory virulence factors (e.g. vif, vprm, vpu, nef, and combinations thereof). Unlike the first-generation lentiviral vectors, no single second generation lentiviral vector includes all features necessary to express and package a polynucleotide into a virus particle. In some aspects, the envelope and packaging components are split between two different vectors with the gag, pol, rev, and tat genes being contained on one vector and the envelope protein (e.g. VSV-G) are contained on a second vector. The gene of interest, its promoter, and LTRs can be included on a third vector that can be used in conjunction with the other two vectors (packaging and envelope vectors) to generate a replication-incompetent virus particle. 
     In some aspects, the lentiviral vector or vector system thereof can be a third-generation lentiviral vector or vector system thereof. Third-generation lentiviral vectors and vector systems thereof have increased safety over first- and second-generation lentiviral vectors and systems thereof because, for example, the various components of the viral genome are split between two or more different vectors but used together in vitro to make virus particles, they can lack the tat gene (when a constitutively active promoter is included up-stream of the LTRs), and they can include one or more deletions in the 3′LTR to create self-inactivating (SIN) vectors having disrupted promoter/enhancer activity of the LTR. In some aspects, a third-generation lentiviral vector system can include (i) a vector plasmid that contains the polynucleotide of interest and upstream promoter that are flanked by the 5′ and 3′ LTRs, which can optionally include one or more deletions present in one or both of the LTRs to render the vector self-inactivating; (ii) a “packaging vector(s)” that can contain one or more genes involved in packaging a polynucleotide into a virus particle that is produced by the system (e.g. gag, pol, and rev) and upstream regulatory sequences (e.g. promoter(s)) to drive expression of the features present on the packaging vector, and (iii) an “envelope vector” that contains one or more envelope protein genes and upstream promoters. In certain embodiments, the third-generation lentiviral vector system can include at least two packaging vectors, with the gag-pol being present on a different vector than the rev gene. In some aspects, self-inactivating lentiviral vectors with an siRNA targeting a common exon shared by HIV tat/rev, a nucleolar-localizing TAR decoy, and an anti-CCR5-specific hammerhead ribozyme (see, e.g., DiGiusto et al. (2010) Sci Transl Med 2:36ra43) can be used/and or adapted to the CRISPR-Cas system of the present invention. 
     In some aspects, the pseudotype and infectivity or tropisim of a lentivirus particle can be tuned by altering the type of envelope protein(s) included in the lentiviral vector or system thereof. As used herein, an “envelope protein” or “outer protein” means a protein exposed at the surface of a viral particle that is not a capsid protein. For example, envelope or outer proteins typically comprise proteins embedded in the envelope of the virus. In some aspects, a lentiviral vector or vector system thereof can include a VSV-G envelope protein. VSV-G mediates viral attachment to an LDL receptor (LDLR) or an LDLR family member present on a host cell, which triggers endocytosis of the viral particle by the host cell. Because LDLR is expressed by a wide variety of cells, viral particles expressing the VSV-G envelope protein can infect or transduce a wide variety of cell types. Other suitable envelope proteins can be incorporated based on the host cell that a user desires to be infected by a virus particle produced from a lentiviral vector or system thereof described herein and can include, but are not limited to, feline endogenous virus envelope protein (RD114) (see e.g. Hanawa et al. Molec. Ther. 2002 5(3) 242-251), modified Sindbis virus envelope proteins (see e.g. Morizono et al. 2010. J. Virol. 84(14) 6923-6934; Morizono et al. 2001. J. Virol. 75:8016-8020; Morizono et al. 2009. J. Gene Med. 11:549-558; Morizono et al. 2006 Virology 355:71-81; Morizono et al J. Gene Med. 11:655-663, Morizono et al. 2005 Nat. Med. 11:346-352), baboon retroviral envelope protein (see e.g. Girard-Gagnepain et al. 2014. Blood. 124: 1221-1231); Tupaia paramyxovirus glycoproteins (see e.g. Enkirch T. et al., 2013. Gene Ther. 20:16-23); measles virus glycoproteins (see e.g. Funke et al. 2008. Molec. Ther. 16(8): 1427-1436), rabies virus envelope proteins, MLV envelope proteins, Ebola envelope proteins, baculovirus envelope proteins, filovirus envelope proteins, hepatitis E1 and E2 envelope proteins, gp41 and gp120 of HIV, hemagglutinin, neuraminidase, M2 proteins of influenza virus, and combinations thereof. 
     In some aspects, the tropism of the resulting lentiviral particle can be tuned by incorporating cell targeting peptides into a lentiviral vector such that the cell targeting peptides are expressed on the surface of the resulting lentiviral particle. In some aspects, a lentiviral vector can contain an envelope protein that is fused to a cell targeting protein (see e.g. Buchholz et al. 2015. Trends Biotechnol. 33:777-790; Bender et al. 2016. PLoS Pathog. 12(e1005461); and Friedrich et al. 2013. Mol. Ther. 2013. 21: 849-859. 
     In some aspects, a split-intein-mediated approach to target lentiviral particles to a specific cell type can be used (see e.g. Chamoun-Emaneulli et al. 2015. Biotechnol. Bioeng. 112:2611-2617, Ramirez et al. 2013. Protein. Eng. Des. Sel. 26:215-233. In these aspects, a lentiviral vector can contain one half of a splicing-deficient variant of the naturally split intein from  Nostoc punctiforme  fused to a cell targeting peptide and the same or different lentiviral vector can contain the other half of the split intein fused to an envelope protein, such as a binding-deficient, fusion-competent virus envelope protein. This can result in production of a virus particle from the lentiviral vector or vector system that includes a split intein that can function as a molecular Velcro linker to link the cell-binding protein to the pseudotyped lentivirus particle. This approach can be advantageous for use where surface-incompatibilities can restrict the use of, e.g., cell targeting peptides. 
     In some aspects, a covalent-bond-forming protein-peptide pair can be incorporated into one or more of the lentiviral vectors described herein to conjugate a cell targeting peptide to the virus particle (see e.g. Kasaraneni et al. 2018. Sci. Reports (8) No. 10990). In some aspects, a lentiviral vector can include an N-terminal PDZ domain of InaD protein (PDZ1) and its pentapeptide ligand (TEFCA) from NorpA, which can conjugate the cell targeting peptide to the virus particle via a covalent bond (e.g. a disulfide bond). In some aspects, the PDZ1 protein can be fused to an envelope protein, which can optionally be binding deficient and/or fusion competent virus envelope protein and included in a lentiviral vector. In some aspects, the TEFCA can be fused to a cell targeting peptide and the TEFCA-CPT fusion construct can be incorporated into the same or a different lentiviral vector as the PDZ1-envenlope protein construct. During virus production, specific interaction between the PDZ1 and TEFCA facilitates producing virus particles covalently functionalized with the cell targeting peptide and thus capable of targeting a specific cell-type based upon a specific interaction between the cell targeting peptide and cells expressing its binding partner. This approach can be advantageous for use where surface-incompatibilities can restrict the use of, e.g., cell targeting peptides. 
     Lentiviral vectors have been disclosed as in the treatment for Parkinson&#39;s Disease, see, e.g., US Patent Publication No. 20120295960 and U.S. Pat. Nos. 7,303,910 and 7,351,585. Lentiviral vectors have also been disclosed for the treatment of ocular diseases, see e.g., US Patent Publication Nos. 20060281180, 20090007284, US20110117189; US20090017543; US20070054961, US20100317109. Lentiviral vectors have also been disclosed for delivery to the brain, see, e.g., US Patent Publication Nos. US20110293571; US20110293571, US20040013648, US20070025970, US20090111106 and U.S. Pat. No. 7,259,015. Any of these systems or a variant thereof can be used to deliver an CRISPR-Cas system polynucleotide described herein to a cell. 
     In some aspects, a lentiviral vector system can include one or more transfer plasmids. Transfer plasmids can be generated from various other vector backbones and can include one or more features that can work with other retroviral and/or lentiviral vectors in the system that can, for example, improve safety of the vector and/or vector system, increase virial titers, and/or increase or otherwise enhance expression of the desired insert to be expressed and/or packaged into the viral particle. Suitable features that can be included in a transfer plasmid can include, but are not limited to, 5′LTR, 3′LTR, SIN/LTR, origin of replication (Ori), selectable marker genes (e.g. antibiotic resistance genes), Psi (ψ), RRE (rev response element), cPPT (central polypurine tract), promoters, WPRE (woodchuck hepatitis post-transcriptional regulatory element), SV40 polyadenylation signal, pUC origin, SV40 origin, F1 origin, and combinations thereof. 
     In another embodiment, Cocal vesiculovirus envelope pseudotyped retroviral or lentiviral vector particles are contemplated (see, e.g., US Patent Publication No. 20120164118 assigned to the Fred Hutchinson Cancer Research Center). Cocal virus is in the Vesiculovirus genus, and is a causative agent of vesicular stomatitis in mammals. Cocal virus was originally isolated from mites in Trinidad (Jonkers et al., Am. J. Vet. Res. 25:236-242 (1964)), and infections have been identified in Trinidad, Brazil, and Argentina from insects, cattle, and horses. Many of the vesiculoviruses that infect mammals have been isolated from naturally infected arthropods, suggesting that they are vector-borne. Antibodies to vesiculoviruses are common among people living in rural areas where the viruses are endemic and laboratory-acquired; infections in humans usually result in influenza-like symptoms. The Cocal virus envelope glycoprotein shares 71.5% identity at the amino acid level with VSV-G Indiana, and phylogenetic comparison of the envelope gene of vesiculoviruses shows that Cocal virus is serologically distinct from, but most closely related to, VSV-G Indiana strains among the vesiculoviruses. Jonkers et al., Am. J. Vet. Res. 25:236-242 (1964) and Travassos da Rosa et al., Am. J. Tropical Med. &amp; Hygiene 33:999-1006 (1984). The Cocal vesiculovirus envelope pseudotyped retroviral vector particles may include for example, lentiviral, alpharetroviral, betaretroviral, gammaretroviral, deltaretroviral, and epsilonretroviral vector particles that may comprise retroviral Gag, Pol, and/or one or more accessory protein(s) and a Cocal vesiculovirus envelope protein. In certain embodiments of these embodiments, the Gag, Pol, and accessory proteins are lentiviral and/or gammaretroviral. In some embodiments, a retroviral vector can contain encoding polypeptides for one or more Cocal vesiculovirus envelope proteins such that the resulting viral or pseudoviral particles are Cocal vesiculovirus envelope pseudotyped. 
     Adenoviral Vectors, Helper-Dependent Adenoviral Vectors, and Hybrid Adenoviral Vectors 
     In some aspects, the vector can be an adenoviral vector. In some aspects, the adenoviral vector can include elements such that the virus particle produced using the vector or system thereof can be serotype 2 or serotype 5. In some aspects, the polynucleotide to be delivered via the adenoviral particle can be up to about 8 kb. Thus, in some aspects, an adenoviral vector can include a DNA polynucleotide to be delivered that can range in size from about 0.001 kb to about 8 kb. Adenoviral vectors have been used successfully in several contexts (see e.g. Teramato et al. 2000. Lancet. 355:1911-1912; Lai et al. 2002. DNA Cell. Biol. 21:895-913; Flotte et al., 1996. Hum. Gene. Ther. 7:1145-1159; and Kay et al. 2000. Nat. Genet. 24:257-261. 
     In some aspects the vector can be a helper-dependent adenoviral vector or system thereof. These are also referred to in the art as “gutless” or “gutted” vectors and are a modified generation of adenoviral vectors (see e.g. Thrasher et al. 2006. Nature. 443:E5-7). In certain embodiments of the helper-dependent adenoviral vector system one vector (the helper) can contain all the viral genes required for replication but contains a conditional gene defect in the packaging domain. The second vector of the system can contain only the ends of the viral genome, one or more CRISPR-Cas polynucleotides, and the native packaging recognition signal, which can allow selective packaged release from the cells (see e.g. Cideciyan et al. 2009. N Engl J Med. 361:725-727). Helper-dependent adenoviral vector systems have been successful for gene delivery in several contexts (see e.g. Simonelli et al. 2010. J Am Soc Gene Ther. 18:643-650; Cideciyan et al. 2009. N Engl J Med. 361:725-727; Crane et al. 2012. Gene Ther. 19(4):443-452; Alba et al. 2005. Gene Ther. 12:18-S27; Croyle et al. 2005. Gene Ther. 12:579-587; Amalfitano et al. 1998. J. Virol. 72:926-933; and Morral et al. 1999. PNAS. 96:12816-12821). The techniques and vectors described in these publications can be adapted for inclusion and delivery of the CRISPR-Cas system polynucleotides described herein. In some aspects, the polynucleotide to be delivered via the viral particle produced from a helper-dependent adenoviral vector or system thereof can be up to about 37 kb. Thus, in some aspects, a adenoviral vector can include a DNA polynucleotide to be delivered that can range in size from about 0.001 kb to about 37 kb (see e.g. Rosewell et al. 2011. J. Genet. Syndr. Gene Ther. Suppl. 5:001). 
     In some aspects, the vector is a hybrid-adenoviral vector or system thereof. Hybrid adenoviral vectors are composed of the high transduction efficiency of a gene-deleted adenoviral vector and the long-term genome-integrating potential of adeno-associated, retroviruses, lentivirus, and transposon based-gene transfer. In some aspects, such hybrid vector systems can result in stable transduction and limited integration site. See e.g. Balague et al. 2000. Blood. 95:820-828; Morral et al. 1998. Hum. Gene Ther. 9:2709-2716; Kubo and Mitani. 2003. J. Virol. 77(5): 2964-2971; Zhang et al. 2013. PloS One. 8(10) e76771; and Cooney et al. 2015. Mol. Ther. 23(4):667-674), whose techniques and vectors described therein can be modified and adapted for use in the CRISPR-Cas system of the present invention. In some aspects, a hybrid-adenoviral vector can include one or more features of a retrovirus and/or an adeno-associated virus. In some aspects the hybrid-adenoviral vector can include one or more features of a spuma retrovirus or foamy virus (FV). See e.g. Ehrhardt et al. 2007. Mol. Ther. 15:146-156 and Liu et al. 2007. Mol. Ther. 15:1834-1841, whose techniques and vectors described therein can be modified and adapted for use in the CRISPR-Cas system of the present invention. Advantages of using one or more features from the FVs in the hybrid-adenoviral vector or system thereof can include the ability of the viral particles produced therefrom to infect a broad range of cells, a large packaging capacity as compared to other retroviruses, and the ability to persist in quiescent (non-dividing) cells. See also e.g. Ehrhardt et al. 2007. Mol. Ther. 156:146-156 and Shuji et al. 2011. Mol. Ther. 19:76-82, whose techniques and vectors described therein can be modified and adapted for use in the CRISPR-Cas system of the present invention. 
     Adeno Associated Viral (AAV) Vectors 
     In an embodiment, the vector can be an adeno-associated virus (AAV) vector. See, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); and Muzyczka, J. Clin. Invest. 94:1351 (1994). Although similar to adenoviral vectors in some of their features, AAVs have some deficiency in their replication and/or pathogenicity and thus can be safer that adenoviral vectors. In some aspects the AAV can integrate into a specific site on chromosome 19 of a human cell with no observable side effects. In some aspects, the capacity of the AAV vector, system thereof, and/or AAV particles can be up to about 4.7 kb. In some embodiments, utilizing homologs of the Cas effector protein that are shorter can be utilized, such for example those in Table 9. 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Exemplary shorter Cas effector homologs. 
               
            
           
           
               
               
               
            
               
                   
                 Species 
                 Cas9 Size (nt) 
               
               
                   
                   
               
            
           
           
               
               
               
            
               
                   
                 
                   Corynebacter diphtheriae 
                 
                 3252 
               
               
                   
                 
                   Eubacterium ventriosum 
                 
                 3321 
               
               
                   
                 
                   Streptococcus pasteurianus 
                 
                 3390 
               
               
                   
                 
                   Lactobacillus farciminis 
                 
                 3378 
               
               
                   
                 
                   Sphaerochaeta globus 
                 
                 3537 
               
               
                   
                   Azospirillum  B510 
                 3504 
               
               
                   
                 
                   Gluconacetobacter diazotrophicus 
                 
                 3150 
               
               
                   
                 
                   Neisseria cinerea 
                 
                 3246 
               
               
                   
                 
                   Roseburia intestinalis 
                 
                 3420 
               
               
                   
                 
                   Parvibaculum lavamentivorans 
                 
                 3111 
               
               
                   
                 
                   Staphylococcus aureus 
                 
                 3159 
               
               
                   
                   Nitratifractor salsuginis  DSM 16511 
                 3396 
               
               
                   
                   Campylobacter lari  CF89-12 
                 3009 
               
               
                   
                 
                   Campylobacter jejuni 
                 
                 2952 
               
               
                   
                   Streptococcus thermophilus  LMD-9 
                 3396 
               
               
                   
                   
               
            
           
         
       
     
     The AAV vector or system thereof can include one or more regulatory molecules. In some aspects the regulatory molecules can be promoters, enhancers, repressors and the like, which are described in greater detail elsewhere herein. In some aspects, the AAV vector or system thereof can include one or more polynucleotides that can encode one or more regulatory proteins. In some aspects, the one or more regulatory proteins can be selected from Rep78, Rep68, Rep52, Rep40, variants thereof, and combinations thereof. 
     The AAV vector or system thereof can include one or more polynucleotides that can encode one or more capsid proteins. The capsid proteins can be selected from VP1, VP2, VP3, and combinations thereof. The capsid proteins can be capable of assembling into a protein shell of the AAV virus particle. In some aspects, the AAV capsid can contain 60 capsid proteins. In some aspects, the ratio of VP1:VP2:VP3 in a capsid can be about 1:1:10. 
     In some aspects, the AAV vector or system thereof can include one or more adenovirus helper factors or polynucleotides that can encode one or more adenovirus helper factors. Such adenovirus helper factors can include, but are not limited, E1A, E1B, E2A, E4ORF6, and VA RNAs. In some aspects, a producing host cell line expresses one or more of the adenovirus helper factors. 
     The AAV vector or system thereof can be configured to produce AAV particles having a specific serotype. In some aspects, the serotype can be AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-8, AAV-9 or any combinations thereof. In some aspects, the AAV can be AAV1, AAV-2, AAV-5 or any combination thereof. One can select the AAV of the AAV with regard to the cells to be targeted; e.g., one can select AAV serotypes 1, 2, 5 or a hybrid capsid AAV-1, AAV-2, AAV-5 or any combination thereof for targeting brain and/or neuronal cells; and one can select AAV-4 for targeting cardiac tissue; and one can select AAV8 for delivery to the liver. Thus, in some aspects, an AAV vector or system thereof capable of producing AAV particles capable of targeting the brain and/or neuronal cells can be configured to generate AAV particles having serotypes 1, 2, 5 or a hybrid capsid AAV-1, AAV-2, AAV-5 or any combination thereof. In some aspects, an AAV vector or system thereof capable of producing AAV particles capable of targeting cardiac tissue can be configured to generate an AAV particle having an AAV-4 serotype. In some aspects, an AAV vector or system thereof capable of producing AAV particles capable of targeting the liver can be configured to generate an AAV having an AAV-8 serotype. In some aspects, the AAV vector is a hybrid AAV vector or system thereof. Hybrid AAVs are AAVs that include genomes with elements from one serotype that are packaged into a capsid derived from at least one different serotype. For example, if it is the rAAV2/5 that is to be produced, and if the production method is based on the helper-free, transient transfection method discussed above, the 1st plasmid and the 3rd plasmid (the adeno helper plasmid) will be the same as discussed for rAAV2 production. However, the second plasmid, the pRepCap will be different. In this plasmid, called pRep2/Cap5, the Rep gene is still derived from AAV2, while the Cap gene is derived from AAV5. The production scheme is the same as the above-mentioned approach for AAV2 production. The resulting rAAV is called rAAV2/5, in which the genome is based on recombinant AAV2, while the capsid is based on AAV5. It is assumed the cell or tissue-tropism displayed by this AAV2/5 hybrid virus should be the same as that of AAV5. 
     A tabulation of certain AAV serotypes as to these cells can be found in Grimm, D. et al, J. Virol. 82: 5887-5911 (2008), which is recapitulated in Table 10 below. 
     
       
         
           
               
               
               
               
               
               
               
               
               
             
               
                 TABLE 10 
               
               
                   
               
               
                 Cell Line 
                 AAV-1 
                 AAV-2 
                 AAV-3 
                 AAV-4 
                 AAV-5 
                 AAV-6 
                 AAV-8 
                 AAV-9 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Huh-7 
                 13 
                 100 
                 2.5 
                 0.0 
                 0.1 
                 10 
                 0.7 
                 0.0 
               
               
                 HEK293 
                 25 
                 100 
                 2.5 
                 0.1 
                 0.1 
                 5 
                 0.7 
                 0.1 
               
               
                 HeLa 
                 3 
                 100 
                 2.0 
                 0.1 
                 6.7 
                 1 
                 0.2 
                 0.1 
               
               
                 HepG2 
                 3 
                 100 
                 16.7 
                 0.3 
                 1.7 
                 5 
                 0.3 
                 ND 
               
               
                 Hep1A 
                 20 
                 100 
                 0.2 
                 1.0 
                 0.1 
                 1 
                 0.2 
                 0.0 
               
               
                 911 
                 17 
                 100 
                 11 
                 0.2 
                 0.1 
                 17 
                 0.1 
                 ND 
               
               
                 CHO 
                 100 
                 100 
                 14 
                 1.4 
                 333 
                 50 
                 10 
                 1.0 
               
               
                 COS 
                 33 
                 100 
                 33 
                 3.3 
                 5.0 
                 14 
                 2.0 
                 0.5 
               
               
                 MeWo 
                 10 
                 100 
                 20 
                 0.3 
                 6.7 
                 10 
                 1.0 
                 0.2 
               
               
                 NIH3T3 
                 10 
                 100 
                 2.9 
                 2.9 
                 0.3 
                 10 
                 0.3 
                 ND 
               
               
                 A549 
                 14 
                 100 
                 20 
                 ND 
                 0.5 
                 10 
                 0.5 
                 0.1 
               
               
                 HT1180 
                 20 
                 100 
                 10 
                 0.1 
                 0.3 
                 33 
                 0.5 
                 0.1 
               
               
                 Monocytes 
                 1111 
                 100 
                 ND 
                 ND 
                 125 
                 1429 
                 ND 
                 ND 
               
               
                 Immature DC 
                 2500 
                 100 
                 ND 
                 ND 
                 222 
                 2857 
                 ND 
                 ND 
               
               
                 Mature DC 
                 2222 
                 100 
                 ND 
                 ND 
                 333 
                 3333 
                 ND 
                 ND 
               
               
                   
               
            
           
         
       
     
     In some aspects, the AAV vector or system thereof is configured as a “gutless” vector, similar to that described in connection with a retroviral vector. In some aspects, the “gutless” AAV vector or system thereof can have the cis-acting viral DNA elements involved in genome amplification and packaging in linkage with the heterologous sequences of interest (e.g. the CRISPR-Cas system polynucleotide(s)). 
     In some embodiments, the AAV vectors are produced in in insect cells, e.g.,  Spodoptera frugiperda  Sf9 insect cells, grown in serum-free suspension culture. Serum-free insect cells can be purchased from commercial vendors, e.g., Sigma Aldrich (EX-CELL 405). 
     In some embodiments, an AAV vector or vector system can contain or consists essentially of one or more polynucleotides encoding one or more components of a CRISPR system. In some embodiments, the AAV vector or vector system can contain a plurality of cassettes comprising or consisting a first cassette comprising or consisting essentially of a promoter, a nucleic acid molecule encoding a CRISPR-associated (Cas) protein (putative nuclease or helicase proteins), e.g., Cas-like and a terminator, and a two, or more, advantageously up to the packaging size limit of the vector, e.g., in total (including the first cassette) five, cassettes comprising or consisting essentially of a promoter, nucleic acid molecule encoding guide RNA (gRNA) and a terminator (e.g., each cassette schematically represented as Promoter-gRNA1-terminator, Promoter-gRNA2-terminator . . . Promoter-gRNA(N)-terminator (where N is a number that can be inserted that is at an upper limit of the packaging size limit of the vector), or two or more individual rAAVs, each containing one or more than one cassette of a CRISPR system, e.g., a first rAAV containing the first cassette comprising or consisting essentially of a promoter, a nucleic acid molecule encoding Cas, e.g., Cas-like and a terminator, and a second rAAV containing a plurality, four, cassettes comprising or consisting essentially of a promoter, nucleic acid molecule encoding guide RNA (gRNA) and a terminator (e.g., each cassette schematically represented as Promoter-gRNA1-terminator, Promoter-gRNA2-terminator . . . Promoter-gRNA(N)-terminator (where N is a number that can be inserted that is at an upper limit of the packaging size limit of the vector). As rAAV is a DNA virus, the nucleic acid molecules in the herein discussion concerning AAV or rAAV are advantageously DNA. In some embodiments, the promoter is a tissue specific promoter or another tissue specific regulatory element. Suitable tissue specific regulatory elements, including promoters, are described in greater detail elsewhere herein. 
     In another aspect, the invention provides a non-naturally occurring or engineered CRISPR protein associated with Adeno Associated Virus (AAV), e.g., an AAV comprising a CRISPR protein as a fusion, with or without a linker, to or with an AAV capsid protein such as VP1, VP2, and/or VP3; and, for shorthand purposes, such a non-naturally occurring or engineered CRISPR protein is herein termed a “AAV-CRISPR protein” More in particular, modifying the knowledge in the art, e.g., Rybniker et al., “Incorporation of Antigens into Viral Capsids Augments Immunogenicity of Adeno-Associated Virus Vector-Based Vaccines,” J Virol. December 2012; 86(24): 13800-13804, Lux K, et al. 2005. Green fluorescent protein-tagged adeno-associated virus particles allow the study of cytosolic and nuclear trafficking. J. Virol. 79:11776-11787, Munch R C, et al. 2012. “Displaying high-affinity ligands on adeno-associated viral vectors enables tumor cell-specific and safe gene transfer.” Mol. Ther. [Epub ahead of print.] doi:10.1038/mt.2012.186 and Warrington K H, Jr, et al. 2004. Adeno-associated virus type 2 VP2 capsid protein is nonessential and can tolerate large peptide insertions at its N terminus. J. Virol. 78:6595-6609, each incorporated herein by reference, one can obtain a modified AAV capsid of the invention. It will be understood by those skilled in the art that the modifications described herein if inserted into the AAV cap gene may result in modifications in the VP1, VP2 and/or VP3 capsid subunits. Alternatively, the capsid subunits can be expressed independently to achieve modification in only one or two of the capsid subunits (VP1, VP2, VP3, VP1+VP2, VP1+VP3, or VP2+VP3). One can modify the cap gene to have expressed at a desired location a non-capsid protein advantageously a large payload protein, such as a CRISPR-protein. Likewise, these can be fusions, with the protein, e.g., large payload protein such as a CRISPR-protein fused in a manner analogous to prior art fusions. See, e.g., US Patent Publication 20090215879; Nance et al., “Perspective on Adeno-Associated Virus Capsid Modification for Duchenne Muscular Dystrophy Gene Therapy,” Hum Gene Ther. 26(12):786-800 (2015) and documents cited therein, incorporated herein by reference. The skilled person, from this disclosure and the knowledge in the art can make and use modified AAV or AAV capsid as in the herein invention, and through this disclosure one knows now that large payload proteins can be fused to the AAV capsid. Applicants provide AAV capsid-CRISPR protein (e.g., Cas, Cas9-like (e.g. Cas9-like or Cas12-like), dCas-like (e.g. dCas9-like and/or dCas12-like) fusions and those AAV-capsid CRISPR protein (e.g., Cas, Cas9-like (e.g. Cas9-like or Cas12-like) fusions can be a recombinant AAV that contains nucleic acid molecule(s) encoding or providing CRISPR-Cas or CRISPR system or complex RNA guide(s), whereby the CRISPR protein (e.g., Cas, Cas9-like (e.g. Cas9-like or Cas12-like) fusion delivers a CRISPR-Cas or CRISPR system complex (e.g., the CRISPR protein or Cas-like (e.g. Cas9-like and/or Cas12-like) is provided by the fusion, e.g., VP1, VP2, pr VP3 fusion, and the guide RNA is provided by the coding of the recombinant virus, whereby in vivo, in a cell, the CRISPR-Cas or CRISPR system is assembled from the nucleic acid molecule(s) of the recombinant providing the guide RNA and the outer surface of the virus providing the CRISPR-Enzyme (e.g., Cas, Cas9-like (e.g. Cas9-like or Cas12-like). Such as complex may herein be termed an “AAV-CRISPR system” or an “AAV-CRISPR-Cas” or “AAV-CRISPR complex” or AAV-CRISPR-Cas complex.” Accordingly, the instant invention is also applicable to a virus in the genus  Dependoparvovirus  or in the family Parvoviridae, for instance, AAV, or a virus of Amdoparvovirus, e.g., Carnivore amdoparvovirus 1, a virus of Aveparvovirus, e.g., Galliform aveparvovirus 1, a virus of Bocaparvovirus, e.g., Ungulate bocaparvovirus 1, a virus of Copiparvovirus, e.g., Ungulate copiparvovirus 1, a virus of  Dependoparvovirus , e.g., Adeno-associated dependoparvovirus A, a virus of Erythroparvovirus, e.g., Primate erythroparvovirus 1, a virus of Protoparvovirus, e.g., Rodent protoparvovirus 1, a virus of Tetraparvovirus, e.g., Primate tetraparvovirus 1. Thus, a virus of within the family Parvoviridae or the genus  Dependoparvovirus  or any of the other foregoing genera within Parvoviridae is contemplated as within the invention with discussion herein as to AAV applicable to such other viruses. 
     In some embodiments, the CRISPR enzyme is external to the capsid or virus particle. In the sense that it is not inside the capsid (enveloped or encompassed with the capsid), but is externally exposed so that it can contact the target genomic DNA). In some embodiments, the CRISPR enzyme is associated with the AAV VP2 domain by way of a fusion protein. In some embodiments, the association may be considered to be a modification of the VP2 domain. Where reference is made herein to a modified VP2 domain, then this will be understood to include any association discussed herein of the VP2 domain and the CRISPR enzyme. In some embodiments, the AAV VP2 domain may be associated (or tethered) to the CRISPR enzyme via a connector protein, for example using a system such as the streptavidin-biotin system. In an aspect, the present invention provides a polynucleotide encoding the present CRISPR enzyme and associated AAV VP2 domain. In one aspect, the invention provides a non-naturally occurring modified AAV having a VP2-CRISPR enzyme capsid protein, wherein the CRISPR enzyme is part of or tethered to the VP2 domain. In some preferred embodiments, the CRISPR enzyme is fused to the VP2 domain so that, in another aspect, the invention provides a non-naturally occurring modified AAV having a VP2-CRISPR enzyme fusion capsid protein. Thus, reference herein to a VP2-CRISPR enzyme capsid protein may also include a VP2-CRISPR enzyme fusion capsid protein. In some embodiments, the VP2-CRISPR enzyme capsid protein further comprises a linker, whereby the VP2-CRISPR enzyme is distanced from the remainder of the AAV. In some embodiments, the VP2-CRISPR enzyme capsid protein further comprises at least one protein complex, e.g., CRISPR complex, such as CRISPR-Cas-like complex guide RNA that targets a particular DNA, TALE, etc. A CRISPR complex, such as CRISPR-Cas system comprising the VP2-CRISPR enzyme capsid protein and at least one CRISPR complex, such as CRISPR-Cas-like complex guide RNA that targets a particular DNA, is also provided in one aspect. 
     In one aspect, the invention provides a non-naturally occurring or engineered composition comprising a CRISPR enzyme which is part of or tethered to an AAV capsid domain, i.e., VP1, VP2, or VP3 domain of Adeno-Associated Virus (AAV) capsid. In some embodiments, part of or tethered to an AAV capsid domain includes associated with associated with a AAV capsid domain. In some embodiments, the CRISPR enzyme may be fused to the AAV capsid domain. In some embodiments, the fusion may be to the N-terminal end of the AAV capsid domain. As such, in some embodiments, the C-terminal end of the CRISPR enzyme is fused to the N-terminal end of the AAV capsid domain. In some embodiments, an NLS and/or a linker (such as a GlySer linker) may be positioned between the C-terminal end of the CRISPR enzyme and the N-terminal end of the AAV capsid domain. In some embodiments, the fusion may be to the C-terminal end of the AAV capsid domain. In some embodiments, this is not preferred due to the fact that the VP1, VP2 and VP3 domains of AAV are alternative splices of the same RNA and so a C-terminal fusion may affect all three domains. In some embodiments, the AAV capsid domain is truncated. In some embodiments, some or all of the AAV capsid domain is removed. In some embodiments, some of the AAV capsid domain is removed and replaced with a linker (such as a GlySer linker), typically leaving the N-terminal and C-terminal ends of the AAV capsid domain intact, such as the first 2, 5 or 10 amino acids. In this way, the internal (non-terminal) portion of the VP3 domain may be replaced with a linker. It is particularly preferred that the linker is fused to the CRISPR protein. A branched linker may be used, with the CRISPR protein fused to the end of one of the branches. This allows for some degree of spatial separation between the capsid and the CRISPR protein. In this way, the CRISPR protein is part of (or fused to) the AAV capsid domain. 
     In other aspects, the CRISPR enzyme may be fused in frame within, i.e. internal to, the AAV capsid domain. Thus, in some embodiments, the AAV capsid domain again preferably retains its N-terminal and C-terminal ends. In this case, a linker is preferred, in some embodiments, either at one or both ends of the CRISPR enzyme. In this way, the CRISPR enzyme is again part of (or fused to) the AAV capsid domain. In certain embodiments, the positioning of the CRISPR enzyme is such that the CRISPR enzyme is at the external surface of the viral capsid once formed. In one aspect, the invention provides a non-naturally occurring or engineered composition comprising a CRISPR enzyme associated with a AAV capsid domain of Adeno-Associated Virus (AAV) capsid. Here, associated may mean in some embodiments fused, or in some embodiments bound to, or in some embodiments tethered to. The CRISPR protein may, in some embodiments, be tethered to the VP1, VP2, or VP3 domain. This may be via a connector protein or tethering system such as the biotin-streptavidin system. In one example, a biotinylation sequence (15 amino acids) could therefore be fused to the CRISPR protein. When a fusion of the AAV capsid domain, especially the N-terminus of the AAV AAV capsid domain, with streptavidin is also provided, the two will therefore associate with very high affinity. Thus, in some embodiments, provided is a composition or system comprising a CRISPR protein-biotin fusion and a streptavidin-AAV capsid domain arrangement, such as a fusion. The CRISPR protein-biotin and streptavidin-AAV capsid domain forms a single complex when the two parts are brought together. NLSs may also be incorporated between the CRISPR protein and the biotin; and/or between the streptavidin and the AAV capsid domain. 
     As such, provided is a fusion of a CRISPR enzyme with a connector protein specific for a high affinity ligand for that connector, whereas the AAV VP2 domain is bound to said high affinity ligand. For example, streptavidin may be the connector fused to the CRISPR enzyme, while biotin may be bound to the AAV VP2 domain. Upon co-localization, the streptavidin will bind to the biotin, thus connecting the CRISPR enzyme to the AAV VP2 domain. The reverse arrangement is also possible. In some embodiments, a biotinylation sequence (15 amino acids) could therefore be fused to the AAV VP2 domain, especially the N-terminus of the AAV VP2 domain. A fusion of the CRISPR enzyme with streptavidin is also preferred, in some embodiments. In some embodiments, the biotinylated AAV capsids with streptavidin-CRISPR enzyme are assembled in vitro. This way the AAV capsids should assemble in a straightforward manner and the CRISPR enzyme-streptavidin fusion can be added after assembly of the capsid. In other embodiments a biotinylation sequence (15 amino acids) could therefore be fused to the CRISPR enzyme, together with a fusion of the AAV VP2 domain, especially the N-terminus of the AAV VP2 domain, with streptavidin. For simplicity, a fusion of the CRISPR enzyme and the AAV VP2 domain is preferred in some embodiments. In some embodiments, the fusion may be to the N-terminal end of the CRISPR enzyme. In other words, in some embodiments, the AAV and CRISPR enzyme are associated via fusion. In some embodiments, the AAV and CRISPR enzyme are associated via fusion including a linker. Suitable linkers are discussed herein, but include Gly Ser linkers. Fusion to the N-term of AAV VP2 domain is preferred, in some embodiments. In some embodiments, the CRISPR enzyme comprises at least one Nuclear Localization Signal (NLS). In a further aspect, the present invention provides compositions comprising the CRISPR enzyme and associated AAV VP2 domain or the polynucleotides or vectors described herein. Such compositions and formulations are discussed elsewhere herein. 
     An alternative tether may be to fuse or otherwise associate the AAV capsid domain to an adaptor protein which binds to or recognizes to a corresponding RNA sequence or motif. In some embodiments, the adaptor is or comprises a binding protein which recognizes and binds (or is bound by) an RNA sequence specific for said binding protein. In some embodiments, a preferred example is the MS2 (see Konermann et al. December 2014, cited infra, incorporated herein by reference) binding protein which recognizes and binds (or is bound by) an RNA sequence specific for the MS2 protein. 
     With the AAV capsid domain associated with the adaptor protein, the CRISPR protein may, in some embodiments, be tethered to the adaptor protein of the AAV capsid domain. The CRISPR protein may, in some embodiments, be tethered to the adaptor protein of the AAV capsid domain via the CRISPR enzyme being in a complex with a modified guide, see Konermann et al. The modified guide is, in some embodiments, a sgRNA. In some embodiments, the modified guide comprises a distinct RNA sequence; see, e.g., International Patent Application No. PCT/US14/70175, incorporated herein by reference. 
     In some embodiments, distinct RNA sequence is an aptamer. Thus, corresponding aptamer-adaptor protein systems are preferred. One or more functional domains may also be associated with the adaptor protein. An example of a preferred arrangement would be: [AAV AAV capsid domain-adaptor protein]-[modified guide-CRISPR protein] 
     In certain embodiments, the positioning of the CRISPR protein is such that the CRISPR protein is at the internal surface of the viral capsid once formed. In one aspect, the invention provides a non-naturally occurring or engineered composition comprising a CRISPR protein associated with an internal surface of an AAV capsid domain. Here again, associated may mean in some embodiments fused, or in some embodiments bound to, or in some embodiments tethered to. The CRISPR protein may, in some embodiments, be tethered to the VP1, VP2, or VP3 domain such that it locates to the internal surface of the viral capsid once formed. This may be via a connector protein or tethering system such as the biotin-streptavidin system as described above and/or elsewhere herein. 
     In one aspect, the invention provides an engineered, non-naturally occurring CRISPR-Cas system comprising a AAV-Cas protein and a guide RNA that targets a DNA molecule encoding a gene product in a cell, whereby the guide RNA targets the DNA molecule encoding the gene product and the Cas protein cleaves the DNA molecule encoding the gene product, whereby expression of the gene product is altered; and, wherein the Cas protein and the guide RNA do not naturally occur together. The invention comprehends the guide RNA comprising a guide sequence fused to a tracr sequence. In a preferred embodiment the Cas protein is a Cas-like protein. In some embodiments, the polynucleotide encoding the Cas protein is codon optimized for expression in a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell and in a more preferred embodiment the mammalian cell is a human cell. In a further embodiment, the expression of the gene product is decreased. 
     In another aspect, the invention provides an engineered, non-naturally occurring vector system comprising one or more vectors comprising a first regulatory element operably linked to a CRISPR-Cas system guide RNA that targets a DNA molecule encoding a gene product and a AAV-Cas protein. The components may be located on same or different vectors of the system, or may be the same vector whereby the AAV-Cas protein also delivers the RNA of the CRISPR system. The guide RNA targets the DNA molecule encoding the gene product in a cell and the AAV-Cas protein may cleaves the DNA molecule encoding the gene product (it may cleave one or both strands or have substantially no nuclease activity), whereby expression of the gene product is altered; and, wherein the AAV-Cas protein and the guide RNA do not naturally occur together. The invention comprehends the guide RNA comprising a guide sequence fused to a tracr sequence. In an embodiment of the invention the AAV-Cas protein is a type II AAV-CRISPR-Cas protein and in a preferred embodiment the AAV-Cas protein is an AAV-Cas-like protein. The invention further comprehends the coding for the AAV-Cas protein being codon optimized for expression in a eukaryotic cell. In a preferred embodiment the eukaryotic cell is a mammalian cell and in a more preferred embodiment the mammalian cell is a human cell. In a further embodiment of the invention, the expression of the gene product is decreased. 
     In one aspect, the invention provides a vector system comprising one or more vectors. In some embodiments, the system comprises: (a) a first regulatory element operably linked to a tracr mate sequence and one or more insertion sites for inserting one or more guide sequences upstream of the tracr mate sequence, wherein when expressed, the guide sequence directs sequence-specific binding of a AAV-CRISPR complex to a target sequence in a eukaryotic cell, wherein the CRISPR complex comprises a AAV-CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence; and (b) said AAV-CRISPR enzyme comprising at least one nuclear localization sequence and/or at least one NES; wherein components (a) and (b) are located on or in the same or different vectors of the system. In some embodiments, component (a) further comprises the tracr sequence downstream of the tracr mate sequence under the control of the first regulatory element. In some embodiments, component (a) further comprises two or more guide sequences operably linked to the first regulatory element, wherein when expressed, each of the two or more guide sequences direct sequence specific binding of an AAV-CRISPR complex to a different target sequence in a eukaryotic cell. In some embodiments, the system comprises the tracr sequence under the control of a third regulatory element, such as a polymerase III promoter. In some embodiments, the tracr sequence exhibits at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of sequence complementarity along the length of the tracr mate sequence when optimally aligned. Determining optimal alignment is within the purview of one of skill in the art. For example, there are publicly and commercially available alignment algorithms and programs such as, but not limited to, ClustalW, Smith-Waterman in matlab, Bowtie, Geneious, Biopython and SeqMan. In some embodiments, the AAV-CRISPR complex comprises one or more nuclear localization sequences of sufficient strength to drive accumulation of said CRISPR complex in a detectable amount in the nucleus of a eukaryotic cell. Without wishing to be bound by theory, it is believed that a nuclear localization sequence is not necessary for AAV-CRISPR complex activity in eukaryotes, but that including such sequences enhances activity of the system, especially as to targeting nucleic acid molecules in the nucleus and/or having molecules exit the nucleus. In some embodiments, the AAV-CRISPR enzyme is an AAV-Cas-like enzyme. In some embodiments, the AAV-Cas enzyme is derived from  S. pneumoniae, S. pyogenes, S. thermophiles, F. novicida  or  S. aureus  Cas9 (e.g., a Cas-like of one of these organisms modified to have or be associated with at least one AAV), and may include further mutations or alterations or be a chimeric Cas9. The enzyme may be an AAV-Cas9 homolog or ortholog. In some embodiments, the AAV-CRISPR enzyme is codon-optimized for expression in a eukaryotic cell. In some embodiments, the AAV-CRISPR enzyme directs cleavage of one or two strands at the location of the target sequence. In some embodiments, the AAV-CRISPR enzyme lacks DNA strand cleavage activity. In some embodiments, the first regulatory element is a polymerase III promoter. In some embodiments, the second regulatory element is a polymerase II promoter. In some embodiments, the guide sequence is at least 15, 16, 17, 18, 19, 20, 25 nucleotides, or between 10-30, or between 15-25, or between 15-20 nucleotides in length. 
     In general, in some embodiments, the AAV further comprises a repair template. It will be appreciated that comprises here may mean encompassed within the viral capsid or that the virus encodes the comprised protein. In some embodiments, one or more, preferably two or more guide RNAs, may be comprised/encompassed within the AAV vector. Two may be preferred, in some embodiments, as it allows for multiplexing or dual nickase approaches. Particularly for multiplexing, two or more guides may be used. In fact, in some embodiments, three or more, four or more, five or more, or even six or more guide RNAs may be comprised/encompassed within the AAV. More space has been freed up within the AAV by virtue of the fact that the AAV no longer needs to comprise/encompass the CRISPR enzyme. In each of these instances, a repair template may also be provided comprised/encompassed within the AAV. In some embodiments, the repair template corresponds to or includes the DNA target. 
     Herpes Simplex Viral Vectors 
     In some aspects, the vector can be a Herpes Simplex Viral (HSV)-based vector or system thereof. HSV systems can include the disabled infections single copy (DISC) viruses, which are composed of a glycoprotein H defective mutant HSV genome. When the defective HSV is propagated in complementing cells, virus particles can be generated that are capable of infecting subsequent cells permanently replicating their own genome but are not capable of producing more infectious particles. See e.g. 2009. Trobridge. Exp. Opin. Biol. Ther. 9:1427-1436, whose techniques and vectors described therein can be modified and adapted for use in the CRISPR-Cas system of the present invention. In some aspects where an HSV vector or system thereof is utilized, the host cell can be a complementing cell. In some aspects, HSV vector or system thereof can be capable of producing virus particles capable of delivering a polynucleotide cargo of up to 150 kb. Thus, in some aspect the CRISPR-Cas system polynucleotide(s) included in the HSV-based viral vector or system thereof can sum from about 0.001 to about 150 kb. HSV-based vectors and systems thereof have been successfully used in several contexts including various models of neurologic disorders. See e.g. Cockrell et al. 2007. Mol. Biotechnol. 36:184-204; Kafri T. 2004. Mol. Biol. 246:367-390; Balaggan and Ali. 2012. Gene Ther. 19:145-153; Wong et al. 2006. Hum. Gen. Ther. 2002. 17:1-9; Azzouz et al. J. Neruosci. 22L10302-10312; and Betchen and Kaplitt. 2003. Curr. Opin. Neurol. 16:487-493, whose techniques and vectors described therein can be modified and adapted for use in the CRISPR-Cas system of the present invention. 
     Poxvirus Vectors 
     In some aspects, the vector can be a poxvirus vector or system thereof. In some aspects, the poxvirus vector can result in cytoplasmic expression of one or more CRISPR-Cas system polynucleotides of the present invention. In some aspects the capacity of a poxvirus vector or system thereof can be about 25 kb or more. In some aspects, a poxvirus vector or system thereof can include one or more CRISPR-Cas system polynucleotides described herein. 
     Viral Vectors for Delivery to Plants 
     The systems and compositions may be delivered to plant cells using viral vehicles. In particular embodiments, the compositions and systems may be introduced in the plant cells using a plant viral vector (e.g., as described in Scholthof et al. 1996, Annu Rev Phytopathol. 1996; 34:299-323). Such viral vector may be a vector from a DNA virus, e.g., geminivirus (e.g., cabbage leaf curl virus, bean yellow dwarf virus, wheat dwarf virus, tomato leaf curl virus, maize streak virus, tobacco leaf curl virus, or tomato golden mosaic virus) or nanovirus (e.g.,  Faba  bean necrotic yellow virus). The viral vector may be a vector from an RNA virus, e.g., tobravirus (e.g., tobacco rattle virus, tobacco mosaic virus), potexvirus (e.g., potato virus X), or hordeivirus (e.g., barley stripe mosaic virus). The replicating genomes of plant viruses may be non-integrative vectors. 
     Virus Particle Production from Viral Vectors 
     Retroviral Production 
     In some aspects, one or more viral vectors and/or system thereof can be delivered to a suitable cell line for production of virus particles containing the polynucleotide or other payload to be delivered to a host cell. Suitable host cells for virus production from viral vectors and systems thereof described herein are known in the art and are commercially available. For example, suitable host cells include HEK 293 cells and its variants (HEK 293T and HEK 293TN cells). In some aspects, the suitable host cell for virus production from viral vectors and systems thereof described herein can stably express one or more genes involved in packaging (e.g. pol, gag, and/or VSV-G) and/or other supporting genes. 
     In some aspects, after delivery of one or more viral vectors to the suitable host cells for or virus production from viral vectors and systems thereof, the cells are incubated for an appropriate length of time to allow for viral gene expression from the vectors, packaging of the polynucleotide to be delivered (e.g. an CRISPR-Cas system polynucleotide), and virus particle assembly, and secretion of mature virus particles into the culture media. Various other methods and techniques are generally known to those of ordinary skill in the art. 
     Mature virus particles can be collected from the culture media by a suitable method. In some aspects, this can involve centrifugation to concentrate the virus. The titer of the composition containing the collected virus particles can be obtained using a suitable method. Such methods can include transducing a suitable cell line (e.g. NIH 3T3 cells) and determining transduction efficiency, infectivity in that cell line by a suitable method. Suitable methods include PCR-based methods, flow cytometry, and antibiotic selection-based methods. Various other methods and techniques are generally known to those of ordinary skill in the art. The concentration of virus particle can be adjusted as needed. In some aspects, the resulting composition containing virus particles can contain 1×10 1 -1×10 20  particles/mL. 
     Lentiviruses may be prepared from any lentiviral vector or vector system described herein. In one example embodiment, after cloning pCasES10 (which contains a lentiviral transfer plasmid backbone), HEK293FT at low passage (p=5) can be seeded in a T-75 flask to 50% confluence the day before transfection in DMEM with 10% fetal bovine serum and without antibiotics. After 20 hours, the media can be changed to OptiMEM (serum-free) media and transfection of the lentiviral vectors can done 4 hours later. Cells can be transfected with 10 μg of lentiviral transfer plasmid (pCasES10) and the appropriate packaging plasmids (e.g., 5 μg of pMD2.G (VSV-g pseudotype), and 7.5 ug of psPAX2 (gag/pol/rev/tat)). Transfection can be carried out in 4 mL OptiMEM with a cationic lipid delivery agent (50 uL Lipofectamine 2000 and 100 ul Plus reagent). After 6 hours, the media can be changed to antibiotic-free DMEM with 10% fetal bovine serum. These methods can use serum during cell culture, but serum-free methods are preferred. 
     Following transfection and allowing the producing cells (also referred to as packaging cells) to package and produce virus particles with packaged cargo, the lentiviral particles can be purified. In an exemplary embodiment, virus-containing supernatants can be harvested after 48 hours. Collected virus-containing supernatants can first be cleared of debris and filtered through a 0.45 μm low protein binding (PVDF) filter. They can then be spun in an ultracentrifuge for 2 hours at 24,000 rpm. The resulting virus-containing pellets can be resuspended in 50 ul of DMEM overnight at 4 degrees C. They can be then aliquoted and used immediately or immediately frozen at −80 degrees C. for storage. 
     AAV Particle Production 
     There are two main strategies for producing AAV particles from AAV vectors and systems thereof, such as those described herein, which depend on how the adenovirus helper factors are provided (helper v. helper free). In some aspects, a method of producing AAV particles from AAV vectors and systems thereof can include adenovirus infection into cell lines that stably harbor AAV replication and capsid encoding polynucleotides along with AAV vector containing the polynucleotide to be packaged and delivered by the resulting AAV particle (e.g. the CRISPR-Cas system polynucleotide(s)). In some aspects, a method of producing AAV particles from AAV vectors and systems thereof can be a “helper free” method, which includes co-transfection of an appropriate producing cell line with three vectors (e.g. plasmid vectors): (1) an AAV vector that contains a polynucleotide of interest (e.g. the CRISPR-Cas system polynucleotide(s)) between 2 ITRs; (2) a vector that carries the AAV Rep-Cap encoding polynucleotides; and (helper polynucleotides. One of skill in the art will appreciate various methods and variations thereof that are both helper and −helper free and as well as the different advantages of each system. 
     Non-Viral Vectors 
     In some aspects, the vector is a non-viral vector or vector system. The term of art “Non-viral vector” and as used herein in this context refers to molecules and/or compositions that are vectors but that are not based on one or more component of a virus or virus genome (excluding any nucleotide to be delivered and/or expressed by the non-viral vector) that can be capable of incorporating CRISPR-Cas polynucleotide(s) and delivering said CRISPR-Cas polynucleotide(s) to a cell and/or expressing the polynucleotide in the cell. It will be appreciated that this does not exclude vectors containing a polynucleotide designed to target a virus-based polynucleotide that is to be delivered. For example, if a gRNA to be delivered is directed against a virus component and it is inserted or otherwise coupled to an otherwise non-viral vector or carrier, this would not make said vector a “viral vector”. Non-viral vectors can include, without limitation, naked polynucleotides and polynucleotide (non-viral) based vector and vector systems. 
     Naked Polynucleotides 
     In some aspects one or more CRISPR-Cas system polynucleotides described elsewhere herein can be included in a naked polynucleotide. The term of art “naked polynucleotide” as used herein refers to polynucleotides that are not associated with another molecule (e.g. proteins, lipids, and/or other molecules) that can often help protect it from environmental factors and/or degradation. As used herein, associated with includes, but is not limited to, linked to, adhered to, adsorbed to, enclosed in, enclosed in or within, mixed with, and the like. Naked polynucleotides that include one or more of the CRISPR-Cas system polynucleotides described herein can be delivered directly to a host cell and optionally expressed therein. The naked polynucleotides can have any suitable two- and three-dimensional configurations. By way of non-limiting examples, naked polynucleotides can be single-stranded molecules, double stranded molecules, circular molecules (e.g. plasmids and artificial chromosomes), molecules that contain portions that are single stranded and portions that are double stranded (e.g. ribozymes), and the like. In some aspects, the naked polynucleotide contains only the CRISPR-Cas system polynucleotide(s) of the present invention. In some aspects, the naked polynucleotide can contain other nucleic acids and/or polynucleotides in addition to the CRISPR-Cas system polynucleotide(s) of the present invention. The naked polynucleotides can include one or more elements of a transposon system. Transposons and system thereof are described in greater detail elsewhere herein. 
     Non-Viral Polynucleotide Vectors 
     In some aspects, one or more of the CRISPR-Cas system polynucleotides can be included in a non-viral polynucleotide vector. Suitable non-viral polynucleotide vectors include, but are not limited to, transposon vectors and vector systems, plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, AR (antibiotic resistance)-free plasmids and miniplasmids, circular covalently closed vectors (e.g. minicircles, minivectors, miniknots), linear covalently closed vectors (“dumbbell shaped”), MIDGE (minimalistic immunologically defined gene expression) vectors, MiLV (micro-linear vector) vectors, Ministrings, mini-intronic plasmids, PSK systems (post-segregationally killing systems), ORT (operator repressor titration) plasmids, and the like. See e.g. Hardee et al. 2017. Genes. 8(2):65. 
     In some aspects, the non-viral polynucleotide vector can have a conditional origin of replication. In some aspects, the non-viral polynucleotide vector can be an ORT plasmid. In some aspects, the non-viral polynucleotide vector can have a minimalistic immunologically defined gene expression. In some aspects, the non-viral polynucleotide vector can have one or more post-segregationally killing system genes. In some aspects, the non-viral polynucleotide vector is AR-free. In some aspects, the non-viral polynucleotide vector is a minivector. In some aspects, the non-viral polynucleotide vector includes a nuclear localization signal. In some aspects, the non-viral polynucleotide vector can include one or more CpG motifs. In some aspects, the non-viral polynucleotide vectors can include one or more scaffold/matrix attachment regions (S/MARs). See e.g. Mirkovitch et al. 1984. Cell. 39:223-232, Wong et al. 2015. Adv. Genet. 89:113-152, whose techniques and vectors can be adapted for use in the present invention. S/MARs are AT-rich sequences that play a role in the spatial organization of chromosomes through DNA loop base attachment to the nuclear matrix. S/MARs are often found close to regulatory elements such as promoters, enhancers, and origins of DNA replication. Inclusion of one or S/MARs can facilitate a once-per-cell-cycle replication to maintain the non-viral polynucleotide vector as an episome in daughter cells. In certain embodiments, the S/MAR sequence is located downstream of an actively transcribed polynucleotide (e.g. one or more CRISPR-Cas system polynucleotides of the present invention) included in the non-viral polynucleotide vector. In some aspects, the S/MAR can be a S/MAR from the beta-interferon gene cluster. See e.g. Verghese et al. 2014. Nucleic Acid Res. 42:e53; Xu et al. 2016. Sci. China Life Sci. 59:1024-1033; Jin et al. 2016. 8:702-711; Koirala et al. 2014. Adv. Exp. Med. Biol. 801:703-709; and Nehlsen et al. 2006. Gene Ther. Mol. Biol. 10:233-244, whose techniques and vectors can be adapted for use in the present invention. 
     In some aspects, the non-viral vector is a transposon vector or system thereof. As used herein, “transposon” (also referred to as transposable element) refers to a polynucleotide sequence that is capable of moving form location in a genome to another. There are several classes of transposons. Transposons include retrotransposons and DNA transposons. Retrotransposons require the transcription of the polynucleotide that is moved (or transposed) in order to transpose the polynucleotide to a new genome or polynucleotide. DNA transposons are those that do not require reverse transcription of the polynucleotide that is moved (or transposed) in order to transpose the polynucleotide to a new genome or polynucleotide. In some aspects, the non-viral polynucleotide vector can be a retrotransposon vector. In some aspects, the retrotransposon vector includes long terminal repeats. In some aspects, the retrotransposon vector does not include long terminal repeats. In some aspects, the non-viral polynucleotide vector can be a DNA transposon vector. DNA transposon vectors can include a polynucleotide sequence encoding a transposase. In some aspects, the transposon vector is configured as a non-autonomous transposon vector, meaning that the transposition does not occur spontaneously on its own. In some of these aspects, the transposon vector lacks one or more polynucleotide sequences encoding proteins required for transposition. In some aspects, the non-autonomous transposon vectors lack one or more Ac elements. 
     In some aspects a non-viral polynucleotide transposon vector system can include a first polynucleotide vector that contains the CRISPR-Cas system polynucleotide(s) of the present invention flanked on the 5′ and 3′ ends by transposon terminal inverted repeats (TIRs) and a second polynucleotide vector that includes a polynucleotide capable of encoding a transposase coupled to a promoter to drive expression of the transposase. When both are expressed in the same cell the transposase can be expressed from the second vector and can transpose the material between the TIRs on the first vector (e.g. the CRISPR-Cas system polynucleotide(s) of the present invention) and integrate it into one or more positions in the host cell&#39;s genome. In some aspects the transposon vector or system thereof can be configured as a gene trap. In some aspects, the TIRs can be configured to flank a strong splice acceptor site followed by a reporter and/or other gene (e.g. one or more of the CRISPR-Cas system polynucleotide(s) of the present invention) and a strong poly A tail. When transposition occurs while using this vector or system thereof, the transposon can insert into an intron of a gene and the inserted reporter or other gene can provoke a mis-splicing process and as a result it in activates the trapped gene. 
     Any suitable transposon system can be used. Suitable transposon and systems thereof can include, Sleeping Beauty transposon system (Tc1/mariner superfamily) (see e.g. Ivics et al. 1997. Cell. 91(4): 501-510), piggyBac (piggyBac superfamily) (see e.g. Li et al. 2013 110(25): E2279-E2287 and Yusa et al. 2011. PNAS. 108(4): 1531-1536), To12 (superfamily hAT), Frog Prince (Tc1/mariner superfamily) (see e.g. Miskey et al. 2003 Nucleic Acid Res. 31(23):6873-6881) and variants thereof. 
     Non-Vector Delivery Vehicles 
     The delivery vehicles may comprise non-viral vehicles. In general, methods and vehicles capable of delivering nucleic acids and/or proteins may be used for delivering the systems compositions herein. Examples of non-viral vehicles include lipid nanoparticles, cell-penetrating peptides (CPPs), DNA nanoclews, metal nanoparticles, streptolysin O, multifunctional envelope-type nanodevices (MENDs), lipid-coated mesoporous silica particles, and other inorganic nanoparticles. 
     Lipid Particles 
     The delivery vehicles may comprise lipid particles, e.g., lipid nanoparticles (LNPs) and liposomes. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Felgner, International Patent Publication Nos. WO 91/17424 and WO 91/16024. The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787). 
     Lipid Nanoparticles (LNPs) 
     LNPs may encapsulate nucleic acids within cationic lipid particles (e.g., liposomes), and may be delivered to cells with relative ease. In some examples, lipid nanoparticles do not contain any viral components, which helps minimize safety and immunogenicity concerns. Lipid particles may be used for in vitro, ex vivo, and in vivo deliveries. Lipid particles may be used for various scales of cell populations. 
     In some examples. LNPs may be used for delivering DNA molecules (e.g., those comprising coding sequences of Cas and/or gRNA) and/or RNA molecules (e.g., mRNA of Cas, gRNAs). In certain cases, LNPs may be use for delivering RNP complexes of Cas/gRNA. Components in LNPs may comprise cationic lipids 1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP), 1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxyketo-N,N-dimethyl-3-aminopropane (DLinK-DMA), 1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA), (3-o-[2″-(methoxypolyethyleneglycol 2000) succinoyl]-1,2-dimyristoyl-sn-glycol (PEG-S-DMG), R-3-[(ro-methoxy-poly(ethylene glycol)2000) carbamoyl]-1,2-dimyristyloxlpropyl-3-amine (PEG-C-DOMG, and any combination thereof. Preparation of LNPs and encapsulation may be adapted from Rosin et al, Molecular Therapy, vol. 19, no. 12, pages 1286-2200, December 2011). 
     In some embodiments, an LNP delivery vehicle can be used to deliver a virus particle containing a CRISPR-Cas system and/or component(s) thereof. In some embodiments, the virus particle(s) can be adsorbed to the lipid particle, such as through electrostatic interactions, and/or can be attached to the liposomes via a linker. 
     In some embodiments, the LNP contains a nucleic acid, wherein the charge ratio of nucleic acid backbone phosphates to cationic lipid nitrogen atoms is about 1:1.5-7 or about 1:4. 
     In some embodiments, the LNP also includes a shielding compound, which is removable from the lipid composition under in vivo conditions. In some embodiments, the shielding compound is a biologically inert compound. In some embodiments, the shielding compound does not carry any charge on its surface or on the molecule as such. In some embodiments, the shielding compounds are polyethylenglycoles (PEGs), hydroxyethylglucose (HEG) based polymers, polyhydroxyethyl starch (polyHES) and polypropylene. In some embodiments, the PEG, HEG, polyHES, and a polypropylene weight between about 500 to 10,000 Da or between about 2000 to 5000 Da. In some embodiments, the shielding compound is PEG2000 or PEG5000. 
     In some embodiments, the LNP can include one or more helper lipids. In some embodiments, the helper lipid can be a phosphor lipid or a steroid. In some embodiments, the helper lipid is between about 20 mol % to 80 mol % of the total lipid content of the composition. In some embodiments, the helper lipid component is between about 35 mol % to 65 mol % of the total lipid content of the LNP. In some embodiments, the LNP includes lipids at 50 mol % and the helper lipid at 50 mol % of the total lipid content of the LNP. 
     Other non-limiting, exemplary LNP delivery vehicles are described in U.S. Patent Publication Nos. US 20160174546, US 20140301951, US 20150105538, US 20150250725, Wang et al., J. Control Release, 2017 Jan. 31. pii: 50168-3659(17)30038-X. doi: 10.1016/j.jconrel.2017.01.037. [Epub ahead of print]; Altinoğlu et al., Biomater Sci., 4(12):1773-80, Nov. 15, 2016; Wang et al., PNAS, 113(11):2868-73 Mar. 15, 2016; Wang et al., PloS One, 10(11): e0141860. doi: 10.1371/journal.pone.0141860. eCollection 2015, Nov. 3, 2015; Takeda et al., Neural Regen Res. 10(5):689-90, May 2015; Wang et al., Adv. Healthc Mater., 3(9):1398-403, September 2014; and Wang et al., Agnew Chem Int Ed Engl., 53(11):2893-8, Mar. 10, 2014; James E. Dahlman and Carmen Barnes et al. Nature Nanotechnology (2014) published online 11 May 2014, doi:10.1038/nnano.2014.84; Coelho et al., N Engl J Med 2013; 369:819-29; Aleku et al.,  Cancer Res.,  68(23): 9788-98 (Dec. 1, 2008), Strumberg et al.,  Int. J. Clin. Pharmacol. Ther.,  50(1): 76-8 (January 2012), Schultheis et al.,  J. Clin. Oncol.,  32(36): 4141-48 (Dec. 20, 2014), and Fehring et al.,  Mol. Ther.,  22(4): 811-20 (Apr. 22, 2014); Novobrantseva,  Molecular Therapy - Nucleic Acids  (2012) 1, e4; doi:10.1038/mtna.2011.3; WO2012135025; US 20140348900; US 20140328759; US 20140308304; WO 2005/105152; WO 2006/069782; WO 2007/121947; US 2015/082080; US 20120251618; U.S. Pat. Nos. 7,982,027; 7,799,565; 8,058,069; 8,283,333; 7,901,708; 7,745,651; 7,803,397; 8,101,741; 8,188,263; 7,915,399; 8,236,943 and 7,838,658 and European Pat. Nos 1766035; 1519714; 1781593 and 1664316; 
     Liposomes 
     In some embodiments, a lipid particle may be liposome. Liposomes are spherical vesicle structures composed of a uni- or multilamellar lipid bilayer surrounding internal aqueous compartments and a relatively impermeable outer lipophilic phospholipid bilayer. In some embodiments, liposomes are biocompatible, nontoxic, can deliver both hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their load across biological membranes and the blood brain barrier (BBB). 
     Liposomes can be made from several different types of lipids, e.g., phospholipids. A liposome may comprise natural phospholipids and lipids such as 1,2-distearoryl-sn-glycero-3-phosphatidyl choline (DSPC), sphingomyelin, egg phosphatidylcholines, monosialoganglioside, or any combination thereof. 
     Several other additives may be added to liposomes in order to modify their structure and properties. For instance, liposomes may further comprise cholesterol, sphingomyelin, and/or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), e.g., to increase stability and/or to prevent the leakage of the liposomal inner cargo. 
     In some embodiments, a liposome delivery vehicle can be used to deliver a virus particle containing a CRISPR-Cas system and/or component(s) thereof. In some embodiments, the virus particle(s) can be adsorbed to the liposome, such as through electrostatic interactions, and/or can be attached to the liposomes via a linker. 
     In some embodiments, the liposome can be a Trojan Horse liposome (also known in the art as Molecular Trojan Horses), see e.g. http://cshprotocols.cshlp.org/content/2010/4/pdb.prot5407.long, the teachings of which can be applied and/or adapted to generated and/or deliver the CRISPR-Cas systems described herein. Other non-limiting, exemplary liposomes can be those as set forth in Wang et al., ACS Synthetic Biology, 1, 403-07 (2012); Wang et al., PNAS, 113(11) 2868-2873 (2016); Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679; WO 2008/042973; U.S. Pat. No. 8,071,082; WO 2014/186366; 20160257951; US20160129120; US 20160244761; 20120251618; WO2013/093648; Lipofectin (a combination of DOTMA and DOPE), Lipofectase, LIPOFECTAMINE® (e.g., LIPOFECTAMINE® 2000, LIPOFECTAMINE® 3000, LIPOFECTAMINE® RNAiMAX, LIPOFECTAMINE® LTX), SAINT-RED (Synvolux Therapeutics, Groningen Netherlands), DOPE, Cytofectin (Gilead Sciences, Foster City, Calif.), and Eufectins (JBL, San Luis Obispo, Calif.). 
     Stable Nucleic-Acid-Lipid Particles (SNALPs) 
     In some embodiments, the lipid particles may be stable nucleic acid lipid particles (SNALPs). SNALPs may comprise an ionizable lipid (DLinDMA) (e.g., cationic at low pH), a neutral helper lipid, cholesterol, a diffusible polyethylene glycol (PEG)-lipid, or any combination thereof. In some examples, SNALPs may comprise synthetic cholesterol, dipalmitoylphosphatidylcholine, 3-N-[(w-methoxy polyethylene glycol)2000)carbamoyl]-1,2-dimyrestyloxypropylamine, and cationic 1,2-dilinoleyloxy-3-N,Ndimethylaminopropane. In some examples, SNALPs may comprise synthetic cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine, PEG-cDMA, and 1,2-dilinoleyloxy-3-(N;N-dimethyl)aminopropane (DLinDMAo). 
     Other non-limiting, exemplary SNALPs that can be used to deliver the CRISPR-Cas systems described herein can be any such SNALPs as described in Morrissey et al., Nature Biotechnology, Vol. 23, No. 8, August 2005, Zimmerman et al., Nature Letters, Vol. 441, 4 May 2006; Geisbert et al., Lancet 2010; 375: 1896-905; Judge, J. Clin. Invest. 119:661-673 (2009); and Semple et al., Nature Niotechnology, Volume 28 Number 2 Feb. 2010, pp. 172-177. 
     Other Lipids 
     The lipid particles may also comprise one or more other types of lipids, e.g., cationic lipids, such as amino lipid 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), DLin-KC2-DMA4, C12-200 and colipids disteroylphosphatidyl choline, cholesterol, and PEG-DMG. 
     In some embodiments, the delivery vehicle can be or include a lipidoid, such as any of those set forth in, for example, US 20110293703. 
     In some embodiments, the delivery vehicle can be or include an amino lipid, such as any of those set forth in, for example, Jayaraman, Angew. Chem. Int. Ed. 2012, 51, 8529-8533. In some embodiments, the delivery vehicle can be or include a lipid envelope, such as any of those set forth in, for example, Korman et al., 2011. Nat. Biotech. 29:154-157. 
     Lipoplexes/Polyplexes 
     In some embodiments, the delivery vehicles comprise lipoplexes and/or polyplexes. Lipoplexes may bind to negatively charged cell membrane and induce endocytosis into the cells. Examples of lipoplexes may be complexes comprising lipid(s) and non-lipid components. Examples of lipoplexes and polyplexes include FuGENE-6 reagent, a non-liposomal solution containing lipids and other components, zwitterionic amino lipids (ZALs), Ca2β (e.g., forming DNA/Ca 2+  microcomplexes), polyethenimine (PEI) (e.g., branched PEI), and poly(L-lysine) (PLL). 
     Sugar-Based Particles 
     In some embodiments, the delivery vehicle can be a sugar-based particle. In some embodiments, the sugar-based particles can be or include GalNAc, such as any of those described in WO2014118272; US 20020150626; Nair, J K et al., 2014, Journal of the American Chemical Society 136 (49), 16958-16961; Østergaard et al., Bioconjugate Chem., 2015, 26 (8), pp 1451-1455; 
     Cell Penetrating Peptides 
     In some embodiments, the delivery vehicles comprise cell penetrating peptides (CPPs). CPPs are short peptides that facilitate cellular uptake of various molecular cargo (e.g., from nanosized particles to small chemical molecules and large fragments of DNA). 
     CPPs may be of different sizes, amino acid sequences, and charges. In some examples, CPPs can translocate the plasma membrane and facilitate the delivery of various molecular cargoes to the cytoplasm or an organelle. CPPs may be introduced into cells via different mechanisms, e.g., direct penetration in the membrane, endocytosis-mediated entry, and translocation through the formation of a transitory structure. 
     CPPs may have an amino acid composition that either contains a high relative abundance of positively charged amino acids such as lysine or arginine or has sequences that contain an alternating pattern of polar/charged amino acids and non-polar, hydrophobic amino acids. These two types of structures are referred to as polycationic or amphipathic, respectively. A third class of CPPs are the hydrophobic peptides, containing only apolar residues, with low net charge or have hydrophobic amino acid groups that are crucial for cellular uptake. Another type of CPPs is the trans-activating transcriptional activator (Tat) from Human Immunodeficiency Virus 1 (HIV-1). Examples of CPPs include to Penetratin, Tat (48-60), Transportan, and (R-AhX-R4) (Ahx refers to aminohexanoyl), Kaposi fibroblast growth factor (FGF) signal peptide sequence, integrin β3 signal peptide sequence, polyarginine peptide Args sequence, Guanine rich-molecular transporters, and sweet arrow peptide. Examples of CPPs and related applications also include those described in U.S. Pat. No. 8,372,951. 
     CPPs can be used for in vitro and ex vivo work quite readily, and extensive optimization for each cargo and cell type is usually required. In some examples, CPPs may be covalently attached to the Cas protein directly, which is then complexed with the gRNA and delivered to cells. In some examples, separate delivery of CPP-Cas and CPP-gRNA to multiple cells may be performed. CPP may also be used to delivery RNPs. 
     CPPs may be used to deliver the compositions and systems to plants. In some examples, CPPs may be used to deliver the components to plant protoplasts, which are then regenerated to plant cells and further to plants. 
     DNA Nanoclews 
     In some embodiments, the delivery vehicles comprise DNA nanoclews. A DNA nanoclew refers to a sphere-like structure of DNA (e.g., with a shape of a ball of yarn). The nanoclew may be synthesized by rolling circle amplification with palindromic sequences that aide in the self-assembly of the structure. The sphere may then be loaded with a payload. An example of DNA nanoclew is described in Sun W et al, J Am Chem Soc. 2014 Oct. 22; 136(42):14722-5; and Sun W et al, Angew Chem Int Ed Engl. 2015 Oct. 5; 54(41):12029-33. DNA nanoclew may have a palindromic sequences to be partially complementary to the gRNA within the Cas:gRNA ribonucleoprotein complex. A DNA nanoclew may be coated, e.g., coated with PEI to induce endosomal escape. 
     Metal Nanoparticles 
     In some embodiments, the delivery vehicles comprise gold nanoparticles (also referred to AuNPs or colloidal gold). Gold nanoparticles may form complex with cargos, e.g., Cas:gRNA RNP. Gold nanoparticles may be coated, e.g., coated in a silicate and an endosomal disruptive polymer, PAsp(DET). Examples of gold nanoparticles include AuraSense Therapeutics&#39; Spherical Nucleic Acid (SNA™) constructs, and those described in Mout R, et al. (2017). ACS Nano 11:2452-8; Lee K, et al. (2017). Nat Biomed Eng 1:889-901. Other metal nanoparticles can also be complexed with cargo(s). Such metal particles include, tungsten, palladium, rhodium, platinum, and iridium particles. Other non-limiting, exemplary metal nanoparticles are described in US 20100129793. 
     iTOP 
     In some embodiments, the delivery vehicles comprise iTOP. iTOP refers to a combination of small molecules drives the highly efficient intracellular delivery of native proteins, independent of any transduction peptide. iTOP may be used for induced transduction by osmocytosis and propanebetaine, using NaCl-mediated hyperosmolality together with a transduction compound (propanebetaine) to trigger macropinocytotic uptake into cells of extracellular macromolecules. Examples of iTOP methods and reagents include those described in D&#39;Astolfo D S, Pagliero R J, Pras A, et al. (2015). Cell 161:674-690. 
     Polymer-Based Particles 
     In some embodiments, the delivery vehicles may comprise polymer-based particles (e.g., nanoparticles). In some embodiments, the polymer-based particles may mimic a viral mechanism of membrane fusion. The polymer-based particles may be a synthetic copy of Influenza virus machinery and form transfection complexes with various types of nucleic acids ((siRNA, miRNA, plasmid DNA or shRNA, mRNA) that cells take up via the endocytosis pathway, a process that involves the formation of an acidic compartment. The low pH in late endosomes acts as a chemical switch that renders the particle surface hydrophobic and facilitates membrane crossing. Once in the cytosol, the particle releases its payload for cellular action. This Active Endosome Escape technology is safe and maximizes transfection efficiency as it is using a natural uptake pathway. In some embodiments, the polymer-based particles may comprise alkylated and carboxyalkylated branched polyethylenimine. In some examples, the polymer-based particles are VIROMER, e.g., VIROMER RNAi, VIROMER RED, VIROMER mRNA, VIROMER CRISPR. Example methods of delivering the systems and compositions herein include those described in Bawage S S et al., Synthetic mRNA expressed Cas13a mitigates RNA virus infections, www.biorxiv.org/content/10.1101/370460v1.full doi:doi.org/10.1101/370460, Viromer® RED, a powerful tool for transfection of keratinocytes. doi: 10.13140/RG.2.2.16993.61281, Viromer® Transfection—Factbook 2018: technology, product overview, users&#39; data, doi:10.13140/RG.2.2.23912.16642. Other exemplary and non-limiting polymeric particles are described in US 20170079916, US 20160367686, US 20110212179, US 20130302401, U.S. Pat. Nos. 6,007,845, 5,855,913, 5,985,309, 5,543,158, WO2012135025, US 20130252281, US 20130245107, US 20130244279; US 20050019923, 20080267903; 
     Streptolysin (SLO) 
     The delivery vehicles may be streptolysin O (SLO). SLO is a toxin produced by Group A streptococci that works by creating pores in mammalian cell membranes. SLO may act in a reversible manner, which allows for the delivery of proteins (e.g., up to 100 kDa) to the cytosol of cells without compromising overall viability. Examples of SLO include those described in Sierig G, et al. (2003). Infect Immun 71:446-55; Walev I, et al. (2001). Proc Natl Acad Sci USA 98:3185-90; Teng K W, et al. (2017). Elife 6:e25460. 
     Multifunctional Envelope-Type Nanodevice (MEND) 
     The delivery vehicles may comprise multifunctional envelope-type nanodevice (MENDs). MENDs may comprise condensed plasmid DNA, a PLL core, and a lipid film shell. A MEND may further comprise cell-penetrating peptide (e.g., stearyl octaarginine). The cell penetrating peptide may be in the lipid shell. The lipid envelope may be modified with one or more functional components, e.g., one or more of: polyethylene glycol (e.g., to increase vascular circulation time), ligands for targeting of specific tissues/cells, additional cell-penetrating peptides (e.g., for greater cellular delivery), lipids to enhance endosomal escape, and nuclear delivery tags. In some examples, the MEND may be a tetra-lamellar MEND (T-MEND), which may target the cellular nucleus and mitochondria. In certain examples, a MEND may be a PEG-peptide-DOPE-conjugated MEND (PPD-MEND), which may target bladder cancer cells. Examples of MENDs include those described in Kogure K, et al. (2004). J Control Release 98:317-23; Nakamura T, et al. (2012). Acc Chem Res 45:1113-21. 
     Lipid-Coated Mesoporous Silica Particles 
     The delivery vehicles may comprise lipid-coated mesoporous silica particles. Lipid-coated mesoporous silica particles may comprise a mesoporous silica nanoparticle core and a lipid membrane shell. The silica core may have a large internal surface area, leading to high cargo loading capacities. In some embodiments, pore sizes, pore chemistry, and overall particle sizes may be modified for loading different types of cargos. The lipid coating of the particle may also be modified to maximize cargo loading, increase circulation times, and provide precise targeting and cargo release. Examples of lipid-coated mesoporous silica particles include those described in Du X, et al. (2014). Biomaterials 35:5580-90; Durfee P N, et al. (2016). ACS Nano 10:8325-45. 
     Inorganic Nanoparticles 
     The delivery vehicles may comprise inorganic nanoparticles. Examples of inorganic nanoparticles include carbon nanotubes (CNTs) (e.g., as described in Bates K and Kostarelos K. (2013). Adv Drug Deliv Rev 65:2023-33), bare mesoporous silica nanoparticles (MSNPs) (e.g., as described in Luo G F, et al. (2014). Sci Rep 4:6064), and dense silica nanoparticles (SiNPs) (as described in Luo D and Saltzman W M. (2000). Nat Biotechnol 18:893-5). 
     Exosomes 
     The delivery vehicles may comprise exosomes. Exosomes include membrane bound extracellular vesicles, which can be used to contain and delivery various types of biomolecules, such as proteins, carbohydrates, lipids, and nucleic acids, and complexes thereof (e.g., RNPs). Examples of exosomes include those described in Schroeder A, et al., J Intern Med. 2010 January; 267(1):9-21; E1-Andaloussi S, et al., Nat Protoc. 2012 December; 7(12):2112-26; Uno Y, et al., Hum Gene Ther. 2011 June; 22(6):711-9; Zou W, et al., Hum Gene Ther. 2011 April; 22(4):465-75. 
     In some examples, the exosome may form a complex (e.g., by binding directly or indirectly) to one or more components of the cargo. In certain examples, a molecule of an exosome may be fused with first adapter protein and a component of the cargo may be fused with a second adapter protein. The first and the second adapter protein may specifically bind each other, thus associating the cargo with the exosome. Examples of such exosomes include those described in Ye Y, et al., Biomater Sci. 2020 Apr. 28. doi: 10.1039/d0bm00427h. 
     Other non-limiting, exemplary exosomes include any of those set forth in Alvarez-Erviti et al. 2011, Nat Biotechnol 29: 341; [1401] El-Andaloussi et al. (Nature Protocols 7:2112-2126(2012); and Wahlgren et al. (Nucleic Acids Research, 2012, Vol. 40, No. 17 e130). 
     Spherical Nucleic Acids (SNAs) 
     In some embodiments, the delivery vehicle can be a SNA. SNAs are three dimensional nanostructures that can be composed of densely functionalized and highly oriented nucleic acids that can be covalently attached to the surface of spherical nanoparticle cores. The core of the spherical nucleic acid can impart the conjugate with specific chemical and physical properties, and it can act as a scaffold for assembling and orienting the oligonucleotides into a dense spherical arrangement that gives rise to many of their functional properties, distinguishing them from all other forms of matter. In some embodiments, the core is a crosslinked polymer. Non-limiting, exemplary SNAs can be any of those set forth in Cutler et al., J. Am. Chem. Soc. 2011 133:9254-9257, Hao et al., Small. 2011 7:3158-3162, Zhang et al., ACS Nano. 2011 5:6962-6970, Cutler et al., J. Am. Chem. Soc. 2012 134:1376-1391, Young et al., Nano Lett. 2012 12:3867-71, Zheng et al., Proc. Natl. Acad. Sci. USA. 2012 109:11975-80, Mirkin, Nanomedicine 2012 7:635-638 Zhang et al., J. Am. Chem. Soc. 2012 134:16488-1691, Weintraub, Nature 2013 495:S14-S16, Choi et al., Proc. Natl. Acad. Sci. USA. 2013 110(19):7625-7630, Jensen et al., Sci. Transl. Med. 5, 209ra152 (2013) and Mirkin, et al., and Small, 10:186-192. 
     Self-Assembling Nanoparticles 
     In some embodiments, the delivery vehicle is a self-assembling nanoparticle. The self-assembling nanoparticles can contain one or more polymers. The self-assembling nanoparticles can be PEGylated. Self-assembling nanoparticles are known in the art. Non-limiting, exemplary self-assembling nanoparticles can any as set forth in Schiffelers et al., Nucleic Acids Research, 2004, Vol. 32, No. 19, Bartlett et al. (PNAS, Sep. 25, 2007, vol. 104, no. 39; Davis et al., Nature, Vol 464, 15 Apr. 2010. 
     Supercharged Proteins 
     In some embodiments, the delivery vehicle can be a supercharged protein. As used herein “Supercharged proteins” are a class of engineered or naturally occurring proteins with unusually high positive or negative net theoretical charge. Non-limiting, exemplary supercharged proteins can be any of those set forth in Lawrence et al., 2007, Journal of the American Chemical Society 129, 10110-10112. 
     Targeted Delivery 
     In some embodiments, the delivery vehicle can allow for targeted delivery to a specific cell, tissue, organ, or system. In such embodiments, the delivery vehicle can include one or more targeting moieties that can direct targeted delivery of the cargo(s). In an embodiment, the delivery vehicle comprises a targeting moiety, such as active targeting of a lipid entity of the invention, e.g., lipid particle or nanoparticle or liposome or lipid bilayer of the invention comprising a targeting moiety for active targeting. 
     With regard to targeting moieties, mention is made of Deshpande et al, “Current trends in the use of liposomes for tumor targeting,” Nanomedicine (Lond). 8(9), doi:10.2217/nnm.13.118 (2013), and the documents it cites, all of which are incorporated herein by reference and the teachings of which can be applied and/or adapted for targeted delivery of one or more CRISPR-Cas molecules described herein. Mention is also made of International Patent Publication No. WO 2016/027264, and the documents it cites, all of which are incorporated herein by reference, the teachings of which can be applied and/or adapted for targeted delivery of one or more CRISPR-Cas molecules described herein. And mention is made of Lorenzer et al, “Going beyond the liver: Progress and challenges of targeted delivery of siRNA therapeutics,” Journal of Controlled Release, 203: 1-15 (2015), and the documents it cites, all of which are incorporated herein by reference, the teachings of which can be applied and/or adapted for targeted delivery of one or more CRISPR-Cas molecules described herein. 
     An actively targeting lipid particle or nanoparticle or liposome or lipid bilayer delivery system (generally as to embodiments of the invention, “lipid entity of the invention” delivery systems) are prepared by conjugating targeting moieties, including small molecule ligands, peptides and monoclonal antibodies, on the lipid or liposomal surface; for example, certain receptors, such as folate and transferrin (Tf) receptors (TfR), are overexpressed on many cancer cells and have been used to make liposomes tumor cell specific. Liposomes that accumulate in the tumor microenvironment can be subsequently endocytosed into the cells by interacting with specific cell surface receptors. To efficiently target liposomes to cells, such as cancer cells, it is useful that the targeting moiety have an affinity for a cell surface receptor and to link the targeting moiety in sufficient quantities to have optimum affinity for the cell surface receptors; and determining these aspects are within the ambit of the skilled artisan. In the field of active targeting, there are a number of cell-, e.g., tumor-, specific targeting ligands. 
     Also, as to active targeting, with regard to targeting cell surface receptors such as cancer cell surface receptors, targeting ligands on liposomes can provide attachment of liposomes to cells, e.g., vascular cells, via a noninternalizing epitope; and, this can increase the extracellular concentration of that which is being delivered, thereby increasing the amount delivered to the target cells. A strategy to target cell surface receptors, such as cell surface receptors on cancer cells, such as overexpressed cell surface receptors on cancer cells, is to use receptor-specific ligands or antibodies. Many cancer cell types display upregulation of tumor-specific receptors. For example, TfRs and folate receptors (FRs) are greatly overexpressed by many tumor cell types in response to their increased metabolic demand. Folic acid can be used as a targeting ligand for specialized delivery owing to its ease of conjugation to nanocarriers, its high affinity for FRs and the relatively low frequency of FRs, in normal tissues as compared with their overexpression in activated macrophages and cancer cells, e.g., certain ovarian, breast, lung, colon, kidney and brain tumors. Overexpression of FR on macrophages is an indication of inflammatory diseases, such as psoriasis, Crohn&#39;s disease, rheumatoid arthritis and atherosclerosis; accordingly, folate-mediated targeting of the invention can also be used for studying, addressing or treating inflammatory disorders, as well as cancers. Folate-linked lipid particles or nanoparticles or liposomes or lipid bylayers of the invention (“lipid entity of the invention”) deliver their cargo intracellularly through receptor-mediated endocytosis. Intracellular trafficking can be directed to acidic compartments that facilitate cargo release, and, most importantly, release of the cargo can be altered or delayed until it reaches the cytoplasm or vicinity of target organelles. Delivery of cargo using a lipid entity of the invention having a targeting moiety, such as a folate-linked lipid entity of the invention, can be superior to nontargeted lipid entity of the invention. The attachment of folate directly to the lipid head groups may not be favorable for intracellular delivery of folate-conjugated lipid entity of the invention, since they may not bind as efficiently to cells as folate attached to the lipid entity of the invention surface by a spacer, which may can enter cancer cells more efficiently. A lipid entity of the invention coupled to folate can be used for the delivery of complexes of lipid, e.g., liposome, e.g., anionic liposome and virus or capsid or envelope or virus outer protein, such as those herein discussed such as adenovirous or AAV. Tf is a monomeric serum glycoprotein of approximately 80 KDa involved in the transport of iron throughout the body. Tf binds to the TfR and translocates into cells via receptor-mediated endocytosis. The expression of TfR is can be higher in certain cells, such as tumor cells (as compared with normal cells and is associated with the increased iron demand in rapidly proliferating cancer cells. Accordingly, the invention comprehends a TfR-targeted lipid entity of the invention, e.g., as to liver cells, liver cancer, breast cells such as breast cancer cells, colon such as colon cancer cells, ovarian cells such as ovarian cancer cells, head, neck and lung cells, such as head, neck and non-small-cell lung cancer cells, cells of the mouth such as oral tumor cells. 
     Also, as to active targeting, a lipid entity of the invention can be multifunctional, i.e., employ more than one targeting moiety such as CPP, along with Tf; a bifunctional system; e.g., a combination of Tf and poly-L-arginine which can provide transport across the endothelium of the blood-brain barrier. EGFR, is a tyrosine kinase receptor belonging to the ErbB family of receptors that mediates cell growth, differentiation and repair in cells, especially non-cancerous cells, but EGF is overexpressed in certain cells such as many solid tumors, including colorectal, non-small-cell lung cancer, squamous cell carcinoma of the ovary, kidney, head, pancreas, neck and prostate, and especially breast cancer. The invention comprehends EGFR-targeted monoclonal antibody(ies) linked to a lipid entity of the invention. HER-2 is often overexpressed in patients with breast cancer, and is also associated with lung, bladder, prostate, brain and stomach cancers. HER-2, encoded by the ERBB2 gene. The invention comprehends a HER-2-targeting lipid entity of the invention, e.g., an anti-HER-2-antibody (or binding fragment thereof)-lipid entity of the invention, a HER-2-targeting-PEGylated lipid entity of the invention (e.g., having an anti-HER-2-antibody or binding fragment thereof), a HER-2-targeting-maleimide-PEG polymer-lipid entity of the invention (e.g., having an anti-HER-2-antibody or binding fragment thereof). Upon cellular association, the receptor-antibody complex can be internalized by formation of an endosome for delivery to the cytoplasm. 
     With respect to receptor-mediated targeting, the skilled artisan takes into consideration ligand/target affinity and the quantity of receptors on the cell surface, and that PEGylation can act as a barrier against interaction with receptors. The use of antibody-lipid entity of the invention targeting can be advantageous. Multivalent presentation of targeting moieties can also increase the uptake and signaling properties of antibody fragments. In practice of the invention, the skilled person takes into account ligand density (e.g., high ligand densities on a lipid entity of the invention may be advantageous for increased binding to target cells). Preventing early by macrophages can be addressed with a sterically stabilized lipid entity of the invention and linking ligands to the terminus of molecules such as PEG, which is anchored in the lipid entity of the invention (e.g., lipid particle or nanoparticle or liposome or lipid bilayer). The microenvironment of a cell mass such as a tumor microenvironment can be targeted; for instance, it may be advantageous to target cell mass vasculature, such as the tumor vasculature microenvironment. Thus, the invention comprehends targeting VEGF. VEGF and its receptors are well-known proangiogenic molecules and are well-characterized targets for antiangiogenic therapy. Many small-molecule inhibitors of receptor tyrosine kinases, such as VEGFRs or basic FGFRs, have been developed as anticancer agents and the invention comprehends coupling any one or more of these peptides to a lipid entity of the invention, e.g., phage IVO peptide(s) (e.g., via or with a PEG terminus), tumor-homing peptide APRPG such as APRPG-PEG-modified. VCAM, the vascular endothelium plays a key role in the pathogenesis of inflammation, thrombosis and atherosclerosis. CAMs are involved in inflammatory disorders, including cancer, and are a logical target, E- and P-selectins, VCAM-1 and ICAMs. Can be used to target a lipid entity of the invention, e.g., with PEGylation. 
     Matrix metalloproteases (MMPs) belong to the family of zinc-dependent endopeptidases. They are involved in tissue remodeling, tumor invasiveness, resistance to apoptosis and metastasis. There are four MMP inhibitors called TIMP1-4, which determine the balance between tumor growth inhibition and metastasis; a protein involved in the angiogenesis of tumor vessels is MT1-MMP, expressed on newly formed vessels and tumor tissues. The proteolytic activity of MT1-MMP cleaves proteins, such as fibronectin, elastin, collagen and laminin, at the plasma membrane and activates soluble MMPs, such as MMP-2, which degrades the matrix. An antibody or fragment thereof such as a Fab′ fragment can be used in the practice of the invention such as for an antihuman MT1-MMP monoclonal antibody linked to a lipid entity of the invention, e.g., via a spacer such as a PEG spacer. αβ-integrins or integrins are a group of transmembrane glycoprotein receptors that mediate attachment between a cell and its surrounding tissues or extracellular matrix. 
     Integrins contain two distinct chains (heterodimers) called α- and β-subunits. The tumor tissue-specific expression of integrin receptors can be been utilized for targeted delivery in the invention, e.g., whereby the targeting moiety can be an RGD peptide such as a cyclic RGD. 
     Aptamers are ssDNA or RNA oligonucleotides that impart high affinity and specific recognition of the target molecules by electrostatic interactions, hydrogen bonding and hydro phobic interactions as opposed to the Watson-Crick base pairing, which is typical for the bonding interactions of oligonucleotides. Aptamers as a targeting moiety can have advantages over antibodies: aptamers can demonstrate higher target antigen recognition as compared with antibodies; aptamers can be more stable and smaller in size as compared with antibodies; aptamers can be easily synthesized and chemically modified for molecular conjugation; and aptamers can be changed in sequence for improved selectivity and can be developed to recognize poorly immunogenic targets. Such moieties as a sgc8 aptamer can be used as a targeting moiety (e.g., via covalent linking to the lipid entity of the invention, e.g., via a spacer, such as a PEG spacer). 
     Also, as to active targeting, the invention also comprehends intracellular delivery. Since liposomes follow the endocytic pathway, they are entrapped in the endosomes (pH 6.5-6) and subsequently fuse with lysosomes (pH&lt;5), where they undergo degradation that results in a lower therapeutic potential. The low endosomal pH can be taken advantage of to escape degradation. Fusogenic lipids or peptides, which destabilize the endosomal membrane after the conformational transition/activation at a lowered pH. Amines are protonated at an acidic pH and cause endosomal swelling and rupture by a buffer effect Unsaturated dioleoylphosphatidylethanolamine (DOPE) readily adopts an inverted hexagonal shape at a low pH, which causes fusion of liposomes to the endosomal membrane. This process destabilizes a lipid entity containing DOPE and releases the cargo into the cytoplasm; fusogenic lipid GALA, cholesteryl-GALA and PEG-GALA may show a highly efficient endosomal release; a pore-forming protein listeriolysin O may provide an endosomal escape mechanism; and, histidine-rich peptides have the ability to fuse with the endosomal membrane, resulting in pore formation, and can buffer the proton pump causing membrane lysis. 
     The invention comprehends a lipid entity of the invention modified with CPP(s), for intracellular delivery that may proceed via energy dependent macropinocytosis followed by endosomal escape. The invention further comprehends organelle-specific targeting. A lipid entity of the invention surface-functionalized with the triphenylphosphonium (TPP) moiety or a lipid entity of the invention with a lipophilic cation, rhodamine 123 can be effective in delivery of cargo to mitochondria. DOPE/sphingomyelin/stearyl-octa-arginine can delivers cargos to the mitochondrial interior via membrane fusion. A lipid entity of the invention surface modified with a lysosomotropic ligand, octadecyl rhodamine B can deliver cargo to lysosomes. Ceramides are useful in inducing lysosomal membrane permeabilization; the invention comprehends intracellular delivery of a lipid entity of the invention having a ceramide. The invention further comprehends a lipid entity of the invention targeting the nucleus, e.g., via a DNA-intercalating moiety. The invention also comprehends multifunctional liposomes for targeting, i.e., attaching more than one functional group to the surface of the lipid entity of the invention, for instance to enhances accumulation in a desired site and/or promotes organelle-specific delivery and/or target a particular type of cell and/or respond to the local stimuli such as temperature (e.g., elevated), pH (e.g., decreased), respond to externally applied stimuli such as a magnetic field, light, energy, heat or ultrasound and/or promote intracellular delivery of the cargo. All of these are considered actively targeting moieties. 
     It should be understood that as to each possible targeting or active targeting moiety herein-discussed, there is an aspect of the invention wherein the delivery system comprises such a targeting or active targeting moiety. Likewise, Table 11 provides exemplary targeting moieties that can be used in the practice of the invention an as to each an aspect of the invention provides a delivery system that comprises such a targeting moiety. 
     
       
         
           
               
               
               
             
               
                 TABLE 11 
               
               
                   
               
               
                 Targeting Moiety 
                 Target Molecule 
                 Target Cell or Tissue 
               
               
                   
               
             
            
               
                 folate 
                 folate receptor 
                 cancer cells 
               
               
                 transferrin 
                 transferrin receptor 
                 cancer cells 
               
               
                 Antibody CC52 
                 rat CC531 
                 rat colon adenocarcinoma CC531 
               
               
                 anti- HER2 antibody 
                 HER2 
                 HER2 -overexpressing tumors 
               
               
                 anti-GD2 
                 GD2 
                 neuroblastoma, melanoma 
               
               
                 anti-EGFR 
                 EGFR 
                 tumor cells overexpressing EGFR 
               
               
                 pH-dependent fusogenic 
                   
                 ovarian carcinoma 
               
               
                 peptide diINF-7 
               
               
                 anti-VEGFR 
                 VEGF Receptor 
                 tumor vasculature 
               
               
                 anti-CD19 
                 CD19 (B cell marker) 
                 leukemia, lymphoma 
               
               
                 cell-penetrating peptide 
                   
                 blood-brain barrier 
               
               
                 cyclic arginine-glycine- 
                 avβ3 
                 glioblastoma cells, human umbilical 
               
               
                 aspartic acid-tyrosine- 
                   
                 vein endothelial cells, tumor 
               
               
                 cysteine peptide 
                   
                 angiogenesis 
               
               
                 (c(RGDyC)-LP) 
               
               
                 ASSHN peptide 
                   
                 endothelial progenitor cells; anti- 
               
               
                   
                   
                 cancer 
               
               
                 PR_b peptide 
                 α 5 β 1  integrin 
                 cancer cells 
               
               
                 AG86 peptide 
                 α 6 β 4  integrin 
                 cancer cells 
               
               
                 KCCYSL (P6.1 peptide) 
                 HER-2 receptor 
                 cancer cells 
               
               
                 affinity peptide LN 
                 Aminopeptidase N 
                 APN-positive tumor 
               
               
                 (YEVGHRC) 
                 (APN/CD13) 
               
               
                 synthetic somatostatin 
                 Somatostatin receptor 2 
                 breast cancer 
               
               
                 analogue 
                 (SSTR2) 
               
               
                 anti-CD20 monoclonal 
                 B-lymphocytes 
                 B cell lymphoma 
               
               
                 antibody 
               
               
                   
               
            
           
         
       
     
     Thus, in an embodiment of the delivery system, the targeting moiety comprises a receptor ligand, such as, for example, hyaluronic acid for CD44 receptor, galactose for hepatocytes, or antibody or fragment thereof such as a binding antibody fragment against a desired surface receptor, and as to each of a targeting moiety comprising a receptor ligand, or an antibody or fragment thereof such as a binding fragment thereof, such as against a desired surface receptor, there is an aspect of the invention wherein the delivery system comprises a targeting moiety comprising a receptor ligand, or an antibody or fragment thereof such as a binding fragment thereof, such as against a desired surface receptor, or hyaluronic acid for CD44 receptor, galactose for hepatocytes (see, e.g., Surace et al, “Lipoplexes targeting the CD44 hyaluronic acid receptor for efficient transfection of breast cancer cells,” J. Mol Pharm 6(4):1062-73; doi: 10.1021/mp800215d (2009); Sonoke et al, “Galactose-modified cationic liposomes as a liver-targeting delivery system for small interfering RNA,” Biol Pharm Bull. 34(8):1338-42 (2011); Torchilin, “Antibody-modified liposomes for cancer chemotherapy,” Expert Opin. Drug Deliv. 5 (9), 1003-1025 (2008); Manjappa et al, “Antibody derivatization and conjugation strategies: application in preparation of stealth immunoliposome to target chemotherapeutics to tumor,” J. Control. Release 150 (1), 2-22 (2011); Sofou S “Antibody-targeted liposomes in cancer therapy and imaging,” Expert Opin. Drug Deliv. 5 (2): 189-204 (2008); Gao J et al, “Antibody-targeted immunoliposomes for cancer treatment,” Mini. Rev. Med. Chem. 13(14): 2026-2035 (2013); Molavi et al, “Anti-CD30 antibody conjugated liposomal doxorubicin with significantly improved therapeutic efficacy against anaplastic large cell lymphoma,” Biomaterials 34(34):8718-25 (2013), each of which and the documents cited therein are hereby incorporated herein by reference), the teachings of which can be applied and/or adapted for targeted delivery of one or more CRISPR-Cas molecules described herein. 
     Other exemplary targeting moieties are described elsewhere herein, such as epitope tags and the like. 
     Responsive Delivery 
     In some embodiments, the delivery vehicle can allow for responsive delivery of the cargo(s). Responsive delivery, as used in this context herein, refers to delivery of cargo(s) by the delivery vehicle in response to an external stimuli. Examples of suitable stimuli include, without limitation, an energy (light, heat, cold, and the like), a chemical stimuli (e.g. chemical composition, etc.), and a biologic or physiologic stimuli (e.g. environmental pH, osmolarity, salinity, biologic molecule, etc.). In some embodiments, the targeting moiety can be responsive to an external stimuli and facilitate responsive delivery. In other embodiments, responsiveness is determined by a non-targeting moiety component of the delivery vehicle. 
     The delivery vehicle can be stimuli-sensitive, e.g., sensitive to an externally applied stimuli, such as magnetic fields, ultrasound or light; and pH-triggering can also be used, e.g., a labile linkage can be used between a hydrophilic moiety such as PEG and a hydrophobic moiety such as a lipid entity of the invention, which is cleaved only upon exposure to the relatively acidic conditions characteristic of the a particular environment or microenvironment such as an endocytic vacuole or the acidotic tumor mass. pH-sensitive copolymers can also be incorporated in embodiments of the invention can provide shielding; diortho esters, vinyl esters, cysteine-cleavable lipopolymers, double esters and hydrazones are a few examples of pH-sensitive bonds that are quite stable at pH 7.5, but are hydrolyzed relatively rapidly at pH 6 and below, e.g., a terminally alkylated copolymer of N-isopropylacrylamide and methacrylic acid that copolymer facilitates destabilization of a lipid entity of the invention and release in compartments with decreased pH value; or, the invention comprehends ionic polymers for generation of a pH-responsive lipid entity of the invention (e.g., poly(methacrylic acid), poly(diethylaminoethyl methacrylate), poly(acrylamide) and poly(acrylic acid)). 
     Temperature-triggered delivery is also within the ambit of the invention. Many pathological areas, such as inflamed tissues and tumors, show a distinctive hyperthermia compared with normal tissues. Utilizing this hyperthermia is an attractive strategy in cancer therapy since hyperthermia is associated with increased tumor permeability and enhanced uptake. This technique involves local heating of the site to increase microvascular pore size and blood flow, which, in turn, can result in an increased extravasation of embodiments of the invention. Temperature-sensitive lipid entity of the invention can be prepared from thermosensitive lipids or polymers with a low critical solution temperature. Above the low critical solution temperature (e.g., at site such as tumor site or inflamed tissue site), the polymer precipitates, disrupting the liposomes to release. Lipids with a specific gel-to-liquid phase transition temperature are used to prepare these lipid entities of the invention; and a lipid for a thermosensitive embodiment can be dipalmitoylphosphatidylcholine. Thermosensitive polymers can also facilitate destabilization followed by release, and a useful thermosensitive polymer is poly (N-isopropylacrylamide). Another temperature triggered system can employ lysolipid temperature-sensitive liposomes. 
     The invention also comprehends redox-triggered delivery. The difference in redox potential between normal and inflamed or tumor tissues, and between the intra- and extra-cellular environments has been exploited for delivery, e.g., GSH is a reducing agent abundant in cells, especially in the cytosol, mitochondria and nucleus. The GSH concentrations in blood and extracellular matrix are just one out of 100 to one out of 1000 of the intracellular concentration, respectively. This high redox potential difference caused by GSH, cysteine and other reducing agents can break the reducible bonds, destabilize a lipid entity of the invention and result in release of payload. The disulfide bond can be used as the cleavable/reversible linker in a lipid entity of the invention, because it causes sensitivity to redox owing to the disulfideto-thiol reduction reaction; a lipid entity of the invention can be made reduction sensitive by using two (e.g., two forms of a disulfide-conjugated multifunctional lipid as cleavage of the disulfide bond (e.g., via tris(2-carboxyethyl)phosphine, dithiothreitol, L-cysteine or GSH), can cause removal of the hydrophilic head group of the conjugate and alter the membrane organization leading to release of payload. Calcein release from reduction-sensitive lipid entity of the invention containing a disulfide conjugate can be more useful than a reduction-insensitive embodiment. 
     Enzymes can also be used as a trigger to release payload. Enzymes, including MMPs (e.g. MMP2), phospholipase A2, alkaline phosphatase, transglutaminase or phosphatidylinositol-specific phospholipase C, have been found to be overexpressed in certain tissues, e.g., tumor tissues. In the presence of these enzymes, specially engineered enzyme-sensitive lipid entity of the invention can be disrupted and release the payload. an MMP2-cleavable octapeptide (Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln) can be incorporated into a linker, and can have antibody targeting, e.g., antibody 2C5. 
     The invention also comprehends light- or energy-triggered delivery, e.g., the lipid entity of the invention can be light-sensitive, such that light or energy can facilitate structural and conformational changes, which lead to direct interaction of the lipid entity of the invention with the target cells via membrane fusion, photo-isomerism, photofragmentation or photopolymerization; such a moiety therefor can be benzoporphyrin photosensitizer. Ultrasound can be a form of energy to trigger delivery; a lipid entity of the invention with a small quantity of particular gas, including air or perfluorated hydrocarbon can be triggered to release with ultrasound, e.g., low-frequency ultrasound (LFUS). Magnetic delivery: A lipid entity of the invention can be magnetized by incorporation of magnetites, such as Fe3O4 or γ-Fe2O3, e.g., those that are less than 10 nm in size. Targeted delivery can be then by exposure to a magnetic field. 
     Modified Cells and Organisms 
     General Discussion 
     One or more component of the non-Class I engineered CRISPR-Cas system described herein, polynucleotides and/or vectors encoding one or more components of the non-Class I engineered CRISPR-Cas system described herein, and/or one or more viral particles carrying a polynucleotide encoding one or more components of the non-Class I engineered CRISPR-Cas system described herein can be delivered to one or more cells. In some aspects, the cells can be ex vivo. In some aspects the cells are in vivo. As such, also described herein are cells that can include and/or express one or more components of the non-Class I engineered CRISPR-Cas system described herein. Thus, also contemplated herein are organisms that can express in one or more cells one or more component of the non-Class I engineered CRISPR-Cas system described herein. In some instances, the organism is a mosaic. In some instances, the organism can express one or more components of the non-Class I engineered CRISPR-Cas system described herein in all cells. The polypeptides, polynucleotides, and vectors described herein can be used to modify one or more cells and/or be used to generate organisms to contain one or more modified cells. 
     As used herein, the term “Cas transgenic cell” refers to a cell, such as a eukaryotic cell, in which a Cas gene has been genomically integrated. The nature, type, or origin of the cell are not particularly limiting according to the present invention. Also, the way the Cas transgene is introduced in the cell may vary and can be any method as is known in the art. In certain embodiments, the Cas transgenic cell is obtained by introducing the Cas transgene in an isolated cell. In certain other embodiments, the Cas transgenic cell is obtained by isolating cells from a Cas transgenic organism. 
     Applications, uses, and actions of the non-Class I engineered CRISPR-Cas system described herein and components thereof, such as genome modification of a cell, screening methods, animal model generation, treatment of a diseases are described elsewhere herein. 
     Modified Cells 
     In some aspects, the modified cell can be a prokaryotic cell. The prokaryotic cells can be bacterial cells. The bacterial cell can be any suitable strain of bacterial cell. 
     In some aspects, the modified cell can be a eukaryotic cell. The eukaryotic cells may be those of or derived from a particular organism, such as a plant or a mammal, including but not limited to human, or non-human eukaryote or animal or mammal as herein discussed, e.g., mouse, rat, rabbit, dog, livestock, or non-human mammal or primate. In some embodiments, processes for modifying the germ line genetic identity of human beings and/or processes for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such processes, may be excluded. 
     In certain embodiments, the methods as described herein may comprise providing a Cas transgenic cell in which one or more nucleic acids encoding one or more guide RNAs are provided or introduced operably connected in the cell with a regulatory element comprising a promoter of one or more gene of interest. By means of example, and without limitation, the Cas transgenic cell as referred to herein may be derived from a Cas transgenic eukaryote, such as a Cas knock-in eukaryote. Reference is made to WO 2014/093622 (PCT/US13/74667), incorporated herein by reference. Methods of US Patent Publication Nos. 20120017290 and 20110265198 assigned to Sangamo BioSciences, Inc. directed to targeting the Rosa locus may be modified to utilize the CRISPR Cas system of the present invention. Methods of US Patent Publication No. 20130236946 assigned to Cellect is directed to targeting the Rosa locus may also be modified to utilize the CRISPR Cas system of the present invention. By means of further example reference is made to Platt et. al. (Cell; 159(2):440-455 (2014)), describing a Cas9 knock-in mouse, which is incorporated herein by reference. The Cas transgene can further comprise a Lox-Stop-polyA-Lox(LSL) cassette thereby rendering Cas expression inducible by Cre recombinase. Alternatively, the Cas transgenic cell may be obtained by introducing the Cas transgene in an isolated cell. Delivery systems for transgenes are well known in the art. By means of example, the Cas transgene may be delivered in for instance eukaryotic cell by means of vector (e.g., AAV, adenovirus, lentivirus) and/or particle and/or nanoparticle delivery, as also described herein elsewhere. 
     It will be understood by the skilled person that the cell, such as the Cas transgenic cell, as referred to herein may comprise further genomic alterations besides having an integrated Cas gene or the mutations arising from the sequence specific action of Cas when complexed with RNA capable of guiding Cas to a target locus. 
     In some aspects, the cell is a cell obtained from a subject to be treated with a CRISPR-based therapy described herein or a cell line made therefrom. In some aspects, the cell is a cell not obtained or derived from the subject to be treated with a CRISPR-based therapy described herein. A wide variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huh1, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panc1, PC-3, TF1, CTLL-2, C1R, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calul, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55, Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRCS, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelial, BALB/3T3 mouse embryo fibroblast, 3T3 Swiss, 3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A172, A20, A253, A431, A-549, ALC, B16, B35, BCP-1 cells, BEAS-2B, bEnd.3, BHK-21, BR 293, BxPC3, C3H-10T1/2, C6/36, Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr−/−, COR-L23, COR-L23/CPR, COR-L23/5010, COR-L23/R23, COS-7, COV-434, CML T1, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6/AR1, EMT6/AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa, Hepa1c1c7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku812, KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCK II, MDCK II, MOR/0.2R, MONO-MAC 6, MTD-1A, MyEnd, NCI-H69/CPR, NCI-H69/LX10, NCI-H69/LX20, NCI-H69/LX4, NIH-3T3, NALM-1, NW-145, OPCN/OPCT cell lines, Peer, PNT-1A/PNT 2, RenCa, RIN-5F, RMA/RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell line, U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, and transgenic varieties thereof. Cell lines are available from a variety of sources known to those with skill in the art (see, e.g., the American Type Culture Collection (ATCC) (Manassas, Va.)). 
     In some embodiments, a cell transfected with one or more vectors described herein is used to establish a new cell line comprising one or more vector-derived sequences. In some embodiments, a cell transiently transfected with the components of a CRISPR system as described herein (such as by transient transfection of one or more vectors, or transfection with RNA), and modified through the activity of a CRISPR complex, is used to establish a new cell line comprising cells containing the modification but lacking any other exogenous sequence. In some embodiments, cells transiently or non-transiently transfected with one or more vectors described herein, or cell lines derived from such cells are used in assessing one or more test compounds. 
     In one aspect, the invention provides a eukaryotic host cell comprising (a) a first regulatory element operably linked to a tracr mate sequence and one or more insertion sites for inserting one or more guide sequences upstream of the tracr mate sequence, wherein when expressed, the guide sequence directs sequence-specific binding of a AAV-CRISPR complex to a target sequence in a eukaryotic cell, wherein the AAV-CRISPR complex comprises a AAV-CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence; and/or (b) a said AAV-CRISPR enzyme optionally comprising at least one nuclear localization sequence and/or NES. In some embodiments, the host cell comprises components (a) and (b). In some embodiments, component (a), component (b), or components (a) and (b) are stably integrated into a genome of the host eukaryotic cell. In some embodiments, component (b) includes or contains component (a). In some embodiments, component (a) further comprises the tracr sequence downstream of the tracr mate sequence under the control of the first regulatory element. In some embodiments, component (a) further comprises two or more guide sequences operably linked to the first regulatory element, wherein when expressed, each of the two or more guide sequences direct sequence specific binding of a AAV-CRISPR complex to a different target sequence in a eukaryotic cell. In some embodiments, the eukaryotic host cell further comprises a third regulatory element, such as a polymerase III promoter, operably linked to said tracr sequence. In some embodiments, the tracr sequence exhibits at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of sequence complementarity along the length of the tracr mate sequence when optimally aligned. 
     In one aspect, the invention provides a eukaryotic host cell comprising (a) a first regulatory element operably linked to a direct repeat sequence and one or more insertion sites for inserting one or more guide RNA sequences up- or downstream (whichever applicable) of the direct repeat sequence, wherein when expressed, the guide sequence(s) direct(s) sequence-specific binding of the Cas CRISPR complex to the respective target sequence(s) in a eukaryotic cell, wherein the Cas CRISPR complex comprises a Cas enzyme complexed with the one or more guide sequence(s) that is hybridized to the respective target sequence(s); and/or (b) a second regulatory element operably linked to an enzyme-coding sequence encoding said Cas-like (e.g. Cas9-like or Cas12-like) enzyme comprising preferably at least one nuclear localization sequence and/or NES. In some embodiments, the host cell comprises components (a) and (b). Where applicable, a tracr sequence may also be provided. In some embodiments, component (a), component (b), or components (a) and (b) are stably integrated into a genome of the host eukaryotic cell. In some embodiments, component (a) further comprises two or more guide sequences operably linked to the first regulatory element, and optionally separated by a direct repeat, wherein when expressed, each of the two or more guide sequences direct sequence specific binding of a Cas CRISPR complex to a different target sequence in a eukaryotic cell. In some embodiments, the Cas enzyme comprises one or more nuclear localization sequences and/or nuclear export sequences or NES of sufficient strength to drive accumulation of said CRISPR enzyme in a detectable amount in and/or out of the nucleus of a eukaryotic cell. 
     Modified Organisms 
     A wide variety of animals, plants, algae, fungi, yeast, etc. and animal, plant, algae, fungus, yeast cell or tissue systems can be engineered for the desired physiological and agronomic characteristics described herein using the nucleic acid constructs of the present disclosure (e.g. the CRISRP-Cas systems described herein) and the various transformation methods mentioned elsewhere herein. In certain embodiments, one or more cells of a plant, animal, algae, fungus, yeast contain one or more polynucleotides, vectors encoding one or more components of the non-class I engineered CRISPR-Cas system described herein. In some aspects, the polynucleotide(s) encoding one or more components of the non-class I engineered CRISPR-Cas system described here can be stably or transiently incorporated into one or more cells of a plant, animal, algae, fungus, and/or yeast or tissue system. In some aspects, one or more of the non-class I engineered CRISPR-Cas system polynucleotides are genomically incorporated into one or more cells of a plant, animal, algae, fungus, and/or yeast or tissue system. Further aspects of the modified organisms and systems are described elsewhere herein. 
     In some aspects, one or more components of the non-class I engineered CRISPR-Cas system described herein are expressed in one or more cells of the plant, animal, algae, fungus, yeast, or tissue systems. In some aspects, the non-class I engineered CRISPR-Cas system described herein can act on a target polynucleotide within the one or more cells of the plant, animal, algae, fungus, yeast, or tissue systems to result in sequence modification of the target polynucleotide. The target polynucleotide can be a genomic polynucleotide. The target polynucleotide can be a non-genomic polynucleotide. Additional methods of polynucleotide modification using the non-class I engineered CRISPR-Cas system described herein are provided elsewhere herein. 
     In an aspect, the invention provides a non-human eukaryotic organism; preferably a multicellular eukaryotic organism, comprising a eukaryotic host cell containing one or more components of a non-class I engineered CRISPR-Cas system described herein according to any of the described embodiments. In other aspects, the invention provides a eukaryotic organism; preferably a multicellular eukaryotic organism, comprising a eukaryotic host cell containing one or more components of a non-class I engineered CRISPR-Cas system described herein according to any of the described embodiments. Advantageously the organism is a host of AAV. 
     The methods for genome editing also described elsewhere herein using the Cas system as described herein can be used to confer desired traits on essentially any animal plant, algae, fungus, yeast, etc. A wide variety of animals, plants, algae, fungus, yeast, etc. and plant algae, fungus, yeast cell or tissue systems may be engineered for the desired physiological and agronomic characteristics described herein using the nucleic acid constructs of the present disclosure and the various transformation and/or delivery methods described elsewhere herein. Various methods (e.g. delivery and transformation methods) described elsewhere herein can result in the generation of “improved animals, plants, algae, fungi, yeast, etc.” in that they have one or more desirable traits compared to the wildtype animal, plant, algae, fungi, yeast, etc. In particular embodiments, the plants, algae, fungi, yeast, etc., cells or parts obtained are transgenic plants, comprising an exogenous DNA sequence incorporated into the genome of all or part of the cells. In particular embodiments, non-transgenic genetically modified animals, plants, algae, fungi, yeast, etc., parts or cells are obtained, in that no exogenous DNA sequence is incorporated into the genome of any of the cells of the modified animals, plants, algae, fungi, yeast, etc. In such embodiments, the improved animals, plants, algae, fungi, yeast, etc. are non-transgenic. Accordingly, as used herein, a “non-transgenic” animal, plant, algae, fungi, yeast, etc. or cell thereof is an animal, plant, algae, fungi, yeast, etc. or cell thereof which does not contain a foreign DNA stably integrated into its genome. 
     Thus, the invention provides a plant, animal or cell, produced by any one or more of the methods described herein, or a progeny thereof. The progeny may be a clone of the produced plant or animal, or may result from sexual reproduction by crossing with other individuals of the same species to introgress further desirable traits into their offspring. The cell may be in vivo or ex vivo in the cases of multicellular organisms, particularly animals or plants. 
     Where only the modification of an endogenous gene is ensured and no foreign genes are introduced or maintained in the animal, plant, algae, fungi, yeast, etc. genome, the resulting genetically modified crops contain no foreign genes and can thus basically be considered non-transgenic. The different applications of the CRISPR-Cas system for animal, plant, algae, fungi, yeast, etc. genome editing include, but are not limited to: introduction of one or more foreign genes to confer a performance and/or agricultural trait of interest; editing of endogenous genes to confer a performance and/or agricultural trait of interest; modulating of endogenous genes by the CRISPR-Cas system to confer a performance and/or agricultural trait of interest. 
     In particular embodiments, the methods described herein are used to modify endogenous genes or to modify their expression without the permanent introduction into the genome of the animal, plant, algae, fungus, yeast, etc. of any foreign gene, including those encoding CRISPR components, so as to avoid the presence of foreign DNA in the genome of the plant. 
     Modified Animals 
     The organism in some embodiments of these aspects may be an animal; for example, a mammal. In certain embodiments, the organism is a non-human mammal. In an aspect, the invention provides a non-human eukaryotic organism; preferably a multicellular eukaryotic organism, comprising a eukaryotic host cell according to any of the described embodiments. In other aspects, the invention provides a eukaryotic organism; preferably a multicellular eukaryotic organism, comprising a eukaryotic host cell according to any of the described embodiments. Also, the organism may be an arthropod such as an insect. The present invention may also be extended to other agricultural applications such as, for example, farm and production animals. For example, pigs have many features that make them attractive as biomedical models, especially in regenerative medicine. In particular, pigs with severe combined immunodeficiency (SCID) may provide useful models for regenerative medicine, xenotransplantation (discussed also elsewhere herein), and tumor development and will aid in developing therapies for human SCID patients. Lee et al., (Proc Natl Acad Sci USA. 2014 May 20; 111(20):7260-5) utilized a reporter-guided transcription activator-like effector nuclease (TALEN) system to generated targeted modifications of recombination activating gene (RAG) 2 in somatic cells at high efficiency, including some that affected both alleles. 
     The methods of Lee et al., (Proc Natl Acad Sci USA. 2014 May 20; 111(20):7260-5) may be applied to the present invention analogously as follows. Mutated pigs are produced by targeted modification of RAG2 in fetal fibroblast cells followed by SCNT and embryo transfer. Constructs coding for CRISPR Cas and a reporter are electroporated into fetal-derived fibroblast cells. After 48 h, transfected cells expressing the green fluorescent protein are sorted into individual wells of a 96-well plate at an estimated dilution of a single cell per well. Targeted modification of RAG2 are screened by amplifying a genomic DNA fragment flanking any CRISPR Cas cutting sites followed by sequencing the PCR products. After screening and ensuring lack of off-site mutations, cells carrying targeted modification of RAG2 are used for SCNT. The polar body, along with a portion of the adjacent cytoplasm of oocyte, presumably containing the metaphase II plate, are removed, and a donor cell are placed in the perivitelline. The reconstructed embryos are then electrically porated to fuse the donor cell with the oocyte and then chemically activated. The activated embryos are incubated in Porcine Zygote Medium 3 (PZM3) with 0.5 μM Scriptaid (S7817; Sigma-Aldrich) for 14-16 h. Embryos are then washed to remove the Scriptaid and cultured in PZM3 until they were transferred into the oviducts of surrogate pigs. 
     The present invention is also applicable to modifying SNPs of other animals, such as cows. Tan et al. (Proc Natl Acad Sci USA. 2013 Oct. 8; 110(41): 16526-16531) expanded the livestock gene editing toolbox to include transcription activator-like (TAL) effector nuclease (TALEN)- and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-like (e.g. Cas9-like and/or Cas12-like)-stimulated homology-directed repair (HDR) using plasmid, rAAV, and oligonucleotide templates. Gene specific gRNA sequences were cloned into the Church lab gRNA vector (Addgene ID: 41824) according to their methods (Mali P, et al. (2013) RNA-Guided Human Genome Engineering via Cas9. Science 339(6121):823-826). The Cas9 nuclease was provided either by co-transfection of the hCas9 plasmid (Addgene ID: 41815) or mRNA synthesized from RCIScript-hCas9. This RCIScript-hCas9 was constructed by sub-cloning the XbaI-AgeI fragment from the hCas9 plasmid (encompassing the hCas9 cDNA) into the RCIScript plasmid. Similar approaches can be applied in the case of Cas-like (e.g. Cas9-like or Cas12-like) proteins. 
     Heo et al. (Stem Cells Dev. 2015 Feb. 1; 24(3):393-402. doi: 10.1089/scd.2014.0278. Epub 2014 Nov. 3) reported highly efficient gene targeting in the bovine genome using bovine pluripotent cells and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 nuclease. First, Heo et al. generate induced pluripotent stem cells (iPSCs) from bovine somatic fibroblasts by the ectopic expression of yamanaka factors and GSK3β and MEK inhibitor (2i) treatment. Heo et al. observed that these bovine iPSCs are highly similar to naïve pluripotent stem cells with regard to gene expression and developmental potential in teratomas. Moreover, CRISPR-Cas9 nuclease, which was specific for the bovine NANOG locus, showed highly efficient editing of the bovine genome in bovine iPSCs and embryos. A similar approach can be applied and/or adapted for use with the Cas-like (e.g. Cas9-like or Cas12-like) proteins of the CRISPR-Cas systems described herein. 
     Igenity® provides a profile analysis of animals, such as cows, to perform and transmit traits of economic traits of economic importance, such as carcass composition, carcass quality, maternal and reproductive traits and average daily gain. The analysis of a comprehensive Igenity® profile begins with the discovery of DNA markers (most often single nucleotide polymorphisms or SNPs). All the markers behind the Igenity® profile were discovered by independent scientists at research institutions, including universities, research organizations, and government entities such as USDA. Markers are then analyzed at Igenity® in validation populations. Igenity® uses multiple resource populations that represent various production environments and biological types, often working with industry partners from the seedstock, cow-calf, feedlot and/or packing segments of the beef industry to collect phenotypes that are not commonly available. Cattle genome databases are widely available, see, e.g., the NAGRP Cattle Genome Coordination Program (http://www.animalgenome.org/cattle/maps/db.html). Thus, the present invention maybe applied to target bovine SNPs. One of skill in the art may utilize the above protocols for targeting SNPs and apply them to bovine SNPs as described, for example, by Tan et al. or Heo et al. Qingjian Zou et al. (Journal of Molecular Cell Biology Advance Access published Oct. 12, 2015) demonstrated increased muscle mass in dogs by targeting the first exon of the dog Myostatin (MSTN) gene (a negative regulator of skeletal muscle mass). First, the efficiency of the sgRNA was validated, using cotransfection of the sgRNA targeting MSTN with a Cas9 vector into canine embryonic fibroblasts (CEFs). Thereafter, MSTN KO dogs were generated by micro-injecting embryos with normal morphology with a mixture of Cas9 mRNA and MSTN sgRNA and auto-transplantation of the zygotes into the oviduct of the same female dog. The knock-out puppies displayed an obvious muscular phenotype on thighs compared with its wild-type littermate sister. Similar approaches can be applied and/or adapted for the CRISPR-Cas systems incorporating one or more Cas-like (e.g. Cas9-like or Cas12-like) proteins described elsewhere herein. 
     Viral targets in livestock may include, in some embodiments, porcine CD163, for example on porcine macrophages. CD163 is associated with infection (thought to be through viral cell entry) by PRRSv (Porcine Reproductive and Respiratory Syndrome virus, an arterivirus). Infection by PRRSv, especially of porcine alveolar macrophages (found in the lung), results in a previously incurable porcine syndrome (“Mystery swine disease” or “blue ear disease”) that causes suffering, including reproductive failure, weight loss and high mortality rates in domestic pigs. Opportunistic infections, such as enzootic pneumonia, meningitis and ear oedema, are often seen due to immune deficiency through loss of macrophage activity. It also has significant economic and environmental repercussions due to increased antibiotic use and financial loss (an estimated $660m per year). 
     As reported by Kristin M Whitworth and Dr Randall Prather et al. (Nature Biotech 3434 published online 7 Dec. 2015) at the University of Missouri and in collaboration with Genus Plc, CD163 was targeted using CRISPR-Cas9 and the offspring of edited pigs were resistant when exposed to PRRSv. One founder male and one founder female, both of whom had mutations in exon 7 of CD163, were bred to produce offspring. The founder male possessed an 11-bp deletion in exon 7 on one allele, which results in a frameshift mutation and missense translation at amino acid 45 in domain 5 and a subsequent premature stop codon at amino acid 64. The other allele had a 2-bp addition in exon 7 and a 377-bp deletion in the preceding intron, which were predicted to result in the expression of the first 49 amino acids of domain 5, followed by a premature stop code at amino acid 85. The sow had a 7 bp addition in one allele that when translated was predicted to express the first 48 amino acids of domain 5, followed by a premature stop codon at amino acid 70. The sow&#39;s other allele was unamplifiable. Selected offspring were predicted to be a null animal (CD163−/−), i.e. a CD163 knock out. 
     Accordingly, in some embodiments, porcine alveolar macrophages may be targeted by the CRISPR proteins (e.g. Cas-like (e.g. Cas9-like or Cas12-like) proteins) described herein. In some embodiments, porcine CD163 may be targeted by the CRISPR protein. In some embodiments, porcine CD163 may be knocked out through induction of a DSB or through insertions or deletions, for example targeting deletion or modification of exon 7, including one or more of those described above, or in other regions of the gene, for example deletion or modification of exon 5. 
     An edited pig and its progeny are also envisaged, for example a CD163 knock out pig. This may be for livestock, breeding or modelling purposes (i.e. a porcine model). Semen comprising the gene knock out is also provided. 
     CD163 is a member of the scavenger receptor cysteine-rich (SRCR) superfamily. Based on in vitro studies SRCR domain 5 of the protein is the domain responsible for unpackaging and release of the viral genome. As such, other members of the SRCR superfamily may also be targeted in order to assess resistance to other viruses. PRRSV is also a member of the mammalian arterivirus group, which also includes murine lactate dehydrogenase-elevating virus, simian hemorrhagic fever virus and equine arteritis virus. The arteriviruses share important pathogenesis properties, including macrophage tropism and the capacity to cause both severe disease and persistent infection. Accordingly, arteriviruses, and in particular murine lactate dehydrogenase-elevating virus, simian hemorrhagic fever virus and equine arteritis virus, may be targeted, for example through porcine CD163 or homologues thereof in other species, and murine, simian and equine models and knockout also provided. 
     Indeed, this approach may be extended to viruses or bacteria that cause other livestock diseases that may be transmitted to humans, such as Swine Influenza Virus (SIV) strains which include influenza C and the subtypes of influenza A known as H1N1, H1N2, H2N1, H3N1, H3N2, and H2N3, as well as pneumonia, meningitis and oedema mentioned above. 
     Kabadi et al. (Nucleic Acids Res. 2014 Oct. 29; 42(19):e147. doi: 10.1093/nar/gku749. Epub 2014 Aug. 13) developed a single lentiviral system to express a Cas9 variant, a reporter gene and up to four sgRNAs from independent RNA polymerase III promoters that are incorporated into the vector by a convenient Golden Gate cloning method. Each sgRNA was efficiently expressed and can mediate multiplex gene editing and sustained transcriptional activation in immortalized and primary human cells. The methods of Kabadi et al. may be applied to the Cas-like (e.g. Cas9-like or Cas12-like) effector protein system of the present invention. 
     Modified Plants and Algae 
     The present invention also provides plants cells obtainable and obtained by the methods provided herein. The improved plants obtained by the methods described herein may be useful in food or feed production through expression of genes which, for instance ensure tolerance to plant pests, herbicides, drought, low or high temperatures, excessive water, etc. 
     The improved plants obtained by the methods described herein, especially crops and algae may be useful in food or feed production through expression of, for instance, higher protein, carbohydrate, nutrient or vitamin levels than would normally be seen in the wildtype. In this regard, improved plants, especially pulses and tubers are preferred. 
     Improved algae or other plants such as rape may be particularly useful in the production of vegetable oils or biofuels such as alcohols (especially methanol and ethanol), for instance. These may be engineered to express or overexpress high levels of oil or alcohols for use in the oil or biofuel industries. 
     The invention also provides for improved parts of a plant. Plant parts include, but are not limited to, leaves, stems, roots, tubers, seeds, endosperm, ovule, and pollen. Plant parts as envisaged herein may be viable, nonviable, regeneratable, and/or non-regeneratable. 
     It is also encompassed herein to provide plant cells and plants generated according to the methods of the invention. Gametes, seeds, embryos, either zygotic or somatic, progeny or hybrids of plants comprising the genetic modification, which are produced by traditional breeding methods, are also included within the scope of the present invention. Such plants may contain a heterologous or foreign DNA sequence inserted at or instead of a target sequence. Alternatively, such plants may contain only an alteration (mutation, deletion, insertion, substitution) in one or more nucleotides. As such, such plants will only be different from their progenitor plants by the presence of the particular modification. 
     In some aspects, the modified organism is a plant. In general, the term “plant” relates to any various photosynthetic, eukaryotic, unicellular or multicellular organism of the kingdom Plantae characteristically growing by cell division, containing chloroplasts, and having cell walls comprised of cellulose. The term plant encompasses monocotyledonous and dicotyledonous plants. Specifically, the plants are intended to comprise without limitation angiosperm and gymnosperm plants such as acacia, alfalfa, amaranth, apple, apricot, artichoke, ash tree, asparagus, avocado, banana, barley, beans, beet, birch, beech, blackberry, blueberry, broccoli, Brussel&#39;s sprouts, cabbage, canola, cantaloupe, carrot, cassava, cauliflower, cedar, a cereal, celery, chestnut, cherry, Chinese cabbage, citrus, clementine, clover, coffee, corn, cotton, cowpea, cucumber, cypress, eggplant, elm, endive, eucalyptus, fennel, figs, fir, geranium, grape, grapefruit, groundnuts, ground cherry, gum hemlock, hickory, kale, kiwifruit, kohlrabi, larch, lettuce, leek, lemon, lime, locust, pine, maidenhair, maize, mango, maple, melon, millet, mushroom, mustard, nuts, oak, oats, oil palm, okra, onion, orange, an ornamental plant or flower or tree,  papaya , palm, parsley, parsnip, pea, peach, peanut, pear, peat, pepper, persimmon, pigeon pea, pine, pineapple, plantain, plum, pomegranate, potato, pumpkin, radicchio, radish, rapeseed, raspberry, rice, rye, sorghum, safflower, sallow, soybean, spinach, spruce, squash, strawberry, sugar beet, sugarcane, sunflower, sweet potato, sweet corn, tangerine, tea, tobacco, tomato, trees, triticale, turf grasses, turnips, vine, walnut, watercress, watermelon, wheat, yams, yew, and zucchini. The term plant also encompasses Algae, which are mainly photoautotrophs unified primarily by their lack of roots, leaves and other organs that characterize higher plants. 
     The methods for genome editing using the CRISPR-Cas system as described herein can be used to confer desired traits on essentially any plant. A wide variety of plants and plant cell systems may be engineered for the desired physiological and agronomic characteristics described herein using the nucleic acid constructs of the present disclosure and the various transformation methods mentioned above. In preferred embodiments, target plants and plant cells for engineering include, but are not limited to, those monocotyledonous and dicotyledonous plants, such as crops including grain crops (e.g., wheat, maize, rice, millet, barley), fruit crops (e.g., tomato, apple, pear, strawberry, orange), forage crops (e.g., alfalfa), root vegetable crops (e.g., carrot, potato, sugar beets, yam), leafy vegetable crops (e.g., lettuce, spinach); flowering plants (e.g., petunia, rose, chrysanthemum), conifers and pine trees (e.g., pine fir, spruce); plants used in phytoremediation (e.g., heavy metal accumulating plants); oil crops (e.g., sunflower, rape seed) and plants used for experimental purposes (e.g.,  Arabidopsis ). Thus, the methods and CRISPR-Cas systems can be used over a broad range of plants, such as for example with dicotyledonous plants belonging to the orders Magniolales, Illiciales, Laurales, Piperales, Aristochiales, Nymphaeales, Ranunculales, Papeverales, Sarraceniaceae, Trochodendrales, Hamamelidales, Eucomiales, Leitneriales, Myricales, Fagales, Casuarinales, Caryophyllales, Batales, Polygonales, Plumbaginales, Dilleniales, Theales, Malvales, Urticales, Lecythidales, Violales, Salicales, Capparales, Ericales, Diapensales, Ebenales, Primulales, Rosales, Fabales, Podostemales, Haloragales, Myrtales, Cornales, Proteales, San tales, Rafflesiales, Celastrales, Euphorbiales, Rhamnales, Sapindales, Juglandales, Geraniales, Polygalales, Umbellales, Gentianales, Polemoniales, Lamiales, Plantaginales, Scrophulariales, Campanulales, Rubiales, Dipsacales, and Asterales; the methods and CRISPR-Cas systems can be used with monocotyledonous plants such as those belonging to the orders Alismatales, Hydrocharitales, Najadales, Triuridales, Commelinales, Eriocaulales, Restionales, Poales, Juncales, Cyperales, Typhales, Bromeliales, Zingiberales, Arecales, Cyclanthales, Pandanales, Arales, Lilliales, and Orchid ales, or with plants belonging to Gymnospermae, e.g., those belonging to the orders Pinales, Ginkgoales, Cycadales, Araucariales, Cupressales and Gnetales. 
     The CRISPR-Cas systems and methods of use described herein can be used over a broad range of plant species, included in the non-limitative list of dicot, monocot or gymnosperm genera hereunder:  Atropa, Alseodaphne, Anacardium, Arachis, Beilschmiedia, Brassica, Carthamus, Cocculus, Croton, Cucumis, Citrus, Citrullus, Capsicum, Catharanthus, Cocos, Coffea, Cucurbita, Daucus, Duguetia, Eschscholzia, Ficus, Fragaria, Glaucium, Glycine, Gossypium, Helianthus, Hevea, Hyoscyamus, Lactuca, Landolphia, Linum, Litsea, Lycopersicon, Lupinus, Manihot, Majorana, Malus, Medicago, Nicotiana, Olea, Parthenium, Papaver, Persea, Phaseolus, Pistacia, Pisum, Pyrus, Prunus, Raphanus, Ricinus, Senecio, Sinomenium, Stephania, Sinapis, Solanum, Theobroma, Trifolium, Trigonella, Vicia, Vinca, Vilis , and  Vigna ; and the genera  Allium, Andropogon, Aragrostis, Asparagus, Avena, Cynodon, Elaeis, Festuca, Festulolium, Heterocallis, Hordeum, Lemna, Lolium, Musa, Oryza, Panicum, Pannesetum, Phleum, Poa, Secale, Sorghum, Triticum, Zea, Abies, Cunninghamia, Ephedra, Picea, Pinus , and  Pseudotsuga.    
     The CRISPR-Cas systems and methods of use can also be used over a broad range of “algae” or “algae cells”; including for example algae selected from several eukaryotic phyla, including the Rhodophyta (red algae), Chlorophyta (green algae), Phaeophyta (brown algae), Bacillariophyta (diatoms), Eustigmatophyta and dinoflagellates as well as the prokaryotic phylum Cyanobacteria (blue-green algae). The term “algae” includes for example algae selected from: Amphora,  Anabaena, Anikstrodesmis, Botryococcus, Chaetoceros, Chlamydomonas, Chlorella, Chlorococcum, Cyclotella, Cylindrotheca, Dunaliella, Emiliana, Euglena, Hematococcus, Isochrysis, Monochrysis, Monoraphidium, Nannochloris, Nannnochloropsis, Navicula, Nephrochloris, Nephroselmis, Nitzschia, Nodularia, Nostoc, Oochromonas, Oocystis, Oscillartoria, Pavlova, Phaeodactylum, Playtmonas, Pleurochrysis, Porhyra, Pseudoanabaena, Pyramimonas, Stichococcus, Synechococcus, Synechocystis, Tetraselmis, Thalassiosira , and  Trichodesmium.    
     A part of a plant, i.e., a “plant tissue” may be treated according to the methods of the present invention to produce an improved plant. Plant tissue also encompasses plant cells. The term “plant cell” as used herein refers to individual units of a living plant, either in an intact whole plant or in an isolated form grown in in vitro tissue cultures, on media or agar, in suspension in a growth media or buffer or as a part of higher organized unites, such as, for example, plant tissue, a plant organ, or a whole plant. 
     A “protoplast” refers to a plant cell that has had its protective cell wall completely or partially removed using, for example, mechanical or enzymatic means resulting in an intact biochemical competent unit of living plant that can reform their cell wall, proliferate and regenerate grow into a whole plant under proper growing conditions. 
     The term “transformation” broadly refers to the process by which a plant host is genetically modified by the introduction of DNA by means of Agrobacteria or one of a variety of chemical or physical methods. As used herein, the term “plant host” refers to plants, including any cells, tissues, organs, or progeny of the plants. Many suitable plant tissues or plant cells can be transformed and include, but are not limited to, protoplasts, somatic embryos, pollen, leaves, seedlings, stems, calli, stolons, microtubers, and shoots. A plant tissue also refers to any clone of such a plant, seed, progeny, propagule whether generated sexually or asexually, and descendants of any of these, such as cuttings or seed. 
     The term “transformed” as used herein, refers to a cell, tissue, organ, or organism into which a foreign DNA molecule, such as a construct, has been introduced. The introduced DNA molecule may be integrated into the genomic DNA of the recipient cell, tissue, organ, or organism such that the introduced DNA molecule is transmitted to the subsequent progeny. In these embodiments, the “transformed” or “transgenic” cell or plant may also include progeny of the cell or plant and progeny produced from a breeding program employing such a transformed plant as a parent in a cross and exhibiting an altered phenotype resulting from the presence of the introduced DNA molecule. Preferably, the transgenic plant is fertile and capable of transmitting the introduced DNA to progeny through sexual reproduction. 
     The term “progeny”, such as the progeny of a transgenic plant, is one that is born of, begotten by, or derived from a plant or the transgenic plant. The introduced DNA molecule may also be transiently introduced into the recipient cell such that the introduced DNA molecule is not inherited by subsequent progeny and thus not considered “transgenic”. 
     The term “plant promoter” as used herein is a promoter capable of initiating transcription in plant cells, whether or not its origin is a plant cell. Exemplary suitable plant promoters include, but are not limited to, those that are obtained from plants, plant viruses, and bacteria such as  Agrobacterium  or  Rhizobium  which comprise genes expressed in plant cells. 
     One or more components of the CRISPR-Cas system described herein can be stably or transiently integrated into the genome of plants and plant cells. 
     In particular embodiments, it is envisaged that the polynucleotides encoding the components of the CRISPR-Cas system are introduced for stable integration into the genome of a plant cell. In these embodiments, the design of the transformation vector or the expression system can be adjusted depending on for when, where and under what conditions the guide RNA and/or the Cas-like (e.g. Cas9-like or Cas12-like) protein gene(s) are expressed. 
     In particular embodiments, it is envisaged to introduce the components of the CRISPR-Cas system stably into the genomic DNA of a plant cell. Additionally or alternatively, it is envisaged to introduce the components of the CRISPR-Cas system for stable integration into the DNA of a plant organelle such as, but not limited to a plastid, e mitochondrion or a chloroplast. 
     The expression system for stable integration into the genome of a plant cell may contain one or more of the following elements: a promoter element that can be used to express the RNA and/or CRISPR-Cas enzyme in a plant cell; a 5′ untranslated region to enhance expression; an intron element to further enhance expression in certain cells, such as monocot cells; a multiple-cloning site to provide convenient restriction sites for inserting the guide RNA and/or the CRISPR-Cas gene sequences and other desired elements; and a 3′ untranslated region to provide for efficient termination of the expressed transcript. 
     The elements of the expression system may be on one or more expression constructs which are either circular such as a plasmid or transformation vector, or non-circular such as linear double stranded DNA. 
     In a particular embodiment, a Cfp1 CRISPR expression system comprises at least: 
     a nucleotide sequence encoding a guide RNA (gRNA) that hybridizes with a target sequence in a plant, and wherein the guide RNA comprises a guide sequence and a direct repeat sequence, and a nucleotide sequence encoding a CRISPR-Cas protein, wherein components (a) or (b) are located on the same or on different constructs, and whereby the different nucleotide sequences can be under control of the same or a different regulatory element operable in a plant cell. 
     DNA construct(s) containing the components of the CRISPR-Cas system, and, where applicable, template sequence may be introduced into the genome of a plant, plant part, or plant cell by a variety of conventional techniques. The process generally comprises the steps of selecting a suitable host cell or host tissue, introducing the construct(s) into the host cell or host tissue, and regenerating plant cells or plants therefrom. 
     In particular embodiments, the DNA construct may be introduced into the plant cell using techniques such as but not limited to electroporation, microinjection, aerosol beam injection of plant cell protoplasts, or the DNA constructs can be introduced directly to plant tissue using biolistic methods, such as DNA particle bombardment (see also Fu et al., Transgenic Res. 2000 February; 9(1):11-9). The basis of particle bombardment is the acceleration of particles coated with gene/s of interest toward cells, resulting in the penetration of the protoplasm by the particles and typically stable integration into the genome. (see e.g. Klein et al, Nature (1987), Klein et ah, Bio/Technology (1992), Casas et ah, Proc. Natl. Acad. Sci. USA (1993)). 
     In particular embodiments, the DNA constructs containing components of the CRISPR-Cas system may be introduced into the plant by  Agrobacterium -mediated transformation. The DNA constructs may be combined with suitable T-DNA flanking regions and introduced into a conventional  Agrobacterium tumefaciens  host vector. The foreign DNA can be incorporated into the genome of plants by infecting the plants or by incubating plant protoplasts with  Agrobacterium  bacteria, containing one or more Ti (tumor-inducing) plasmids. (see e.g. Fraley et al., (1985), Rogers et al., (1987) and U.S. Pat. No. 5,563,055). 
     The CRISPR systems provided herein can be used to introduce targeted double-strand or single-strand breaks and/or to introduce into one or more plant cells or entire plants gene activator and or repressor systems and without being limitative, can be used for gene targeting, gene replacement, targeted mutagenesis, targeted deletions or insertions, targeted inversions and/or targeted translocations. By co-expression of multiple targeting polynucleotides (e.g.) RNAs directed to achieve multiple modifications in a single cell, multiplexed genome modification can be ensured. This technology can be used to high-precision engineering of plants with improved characteristics, including enhanced nutritional quality, increased resistance to diseases and resistance to biotic and abiotic stress, and increased production of commercially valuable plant products or heterologous compounds. 
     In particular embodiments, the methods described herein are used to modify endogenous genes or to modify their expression without the permanent introduction into the genome of the plant, including those encoding CRISPR components, so as to avoid the presence of foreign DNA in the genome of the plant. This can be of interest as the regulatory requirements for non-transgenic plants are less rigorous. 
     Exemplary genes conferring agronomic traits include, but are not limited to genes that confer resistance to pests or diseases; genes involved in plant diseases, such as those listed in WO 2013046247; genes that confer resistance to herbicides, fungicides, or the like; genes involved in (abiotic) stress tolerance. Other aspects of the use of the CRISPR-Cas system include, but are not limited to: create (male) sterile plants; increasing the fertility stage in plants/algae etc.; generate genetic variation in a crop of interest; affect fruit-ripening; increasing storage life of plants/algae etc.; reducing allergen in plants/algae etc.; ensure a value added trait (e.g. nutritional improvement); Screening methods for endogenous genes of interest; biofuel, fatty acid, organic acid, etc. production. 
     The CRISPR systems provided herein can be used to introduce targeted double-strand or single-strand breaks and/or to introduce gene activator and or repressor systems and without being limitative, can be used for gene targeting, gene replacement, targeted mutagenesis, targeted deletions or insertions, targeted inversions and/or targeted translocations. By co-expression of multiple targeting RNAs directed to achieve multiple modifications in a single cell, multiplexed genome modification can be ensured. This technology can be used to high-precision engineering of plants with improved characteristics, including enhanced nutritional quality, increased resistance to diseases and resistance to biotic and abiotic stress, and increased production of commercially valuable plant products or heterologous compounds. 
     Chloroplast Targeting 
     In particular embodiments, it is envisaged that the CRISPR-Cas system is used to specifically modify chloroplast genes or to ensure expression in the chloroplast. For this purpose use is made of chloroplast transformation methods or compartmentalization of the CRISPR-Cas components to the chloroplast. For instance, the introduction of genetic modifications in the plastid genome can reduce biosafety issues such as gene flow through pollen. 
     Methods of chloroplast transformation are known in the art and include Particle bombardment, PEG treatment, and microinjection. Additionally, methods involving the translocation of transformation cassettes from the nuclear genome to the plastid can be used as described in WO2010061186. 
     Alternatively, it is envisaged to target one or more of the CRISPR-Cas components to the plant chloroplast. This is achieved by incorporating in the expression construct a sequence encoding a chloroplast transit peptide (CTP) or plastid transit peptide, operably linked to the 5′ region of the sequence encoding the CRISPR-Cas protein. The CTP is removed in a processing step during translocation into the chloroplast. Chloroplast targeting of expressed proteins is well known to the skilled artisan (see for instance Protein Transport into Chloroplasts, 2010, Annual Review of Plant Biology, Vol. 61: 157-180). In such embodiments it is also desired to target the guide RNA to the plant chloroplast. Methods and constructs which can be used for translocating guide RNA into the chloroplast by means of a chloroplast localization sequence are described, for instance, in US 20040142476, incorporated herein by reference. Such variations of constructs can be incorporated into the expression systems of the invention to efficiently translocate the CRISPR-Cas-guide RNA. 
     Introduction of Polynucleotides in Algal Cells 
     Transgenic algae (or other plants such as rape) may be particularly useful in the production of vegetable oils or biofuels such as alcohols (especially methanol and ethanol) or other products. These may be engineered to express or overexpress high levels of oil or alcohols for use in the oil or biofuel industries. 
     U.S. Pat. No. 8,945,839 describes a method for engineering Micro-Algae ( Chlamydomonas reinhardtii  cells) species) using Cas9. Using similar tools, the methods of the CRISPR-Cas system described herein can be applied on  Chlamydomonas  species and other algae. In particular embodiments, Cas-like (e.g. Cas9-like or Cas12-like) protein(s) and guide RNA are introduced in algae expressed using a vector that expresses Cas-like (e.g. Cas9-like or Cas12-like) protein(s) under the control of a constitutive promoter such as Hsp70A-Rbc S2 or Beta2-tubulin. Guide RNA is optionally delivered using a vector containing T7 promoter. Alternatively, Cas-like (e.g. Cas9-like or Cas12-like) protein(s) mRNA and in vitro transcribed guide RNA can be delivered to algal cells. Electroporation protocols are available to the skilled person such as the standard recommended protocol from the GeneArt  Chlamydomonas  Engineering kit. 
     In particular embodiments, the endonuclease used herein is a Split Cas-like (e.g. Cas9-like or Cas12-like) enzyme. Split Cas-like (e.g. Cas9-like or Cas12-like) enzymes are preferentially used in Algae for targeted genome modification similar to that which has been described for Cas9 in WO 2015086795. Use of the Cas-like (e.g. Cas9-like or Cas12-like) split system is particularly suitable for an inducible method of genome targeting and avoids the potential toxic effect of the Cas9 overexpression within the algae cell. In particular embodiments, said Cas-like (e.g. Cas9-like or Cas12-like) proteins split domains (RuvC (inactive or active) and/or HNH domains and/or other catalytic domains) can be simultaneously or sequentially introduced into the cell such that said split Cas-like (e.g. Cas9-like or Cas12-like) domain(s) process the target nucleic acid sequence in the algae or other cell. The reduced size of the split Cas-like (e.g. Cas9-like or Cas12-like) protein compared to the wild type Cas-like (e.g. Cas9-like or Cas12-like) protein allows other methods of delivery of the CRISPR system to the cells, such as the use of Cell Penetrating Peptides as described elsewhere herein. This method is of particular interest for generating genetically modified algae. 
     Modifying Algae and Plants for Production of Vegetable Oils or Biofuels 
     Transgenic algae or other plants such as rape may be particularly useful in the production of vegetable oils or biofuels such as alcohols (especially methanol and ethanol), for instance. These may be engineered to express or overexpress high levels of oil or alcohols for use in the oil or biofuel industries. The term “biofuel” as used herein is an alternative fuel made from plant and plant-derived resources. Renewable biofuels can be extracted from organic matter whose energy has been obtained through a process of carbon fixation or are made through the use or conversion of biomass. This biomass can be used directly for biofuels or can be converted to convenient energy containing substances by thermal conversion, chemical conversion, and biochemical conversion. This biomass conversion can result in fuel in solid, liquid, or gas form. There are two types of biofuels: bioethanol and biodiesel. Bioethanol is mainly produced by the sugar fermentation process of cellulose (starch), which is mostly derived from maize and sugar cane. Biodiesel on the other hand is mainly produced from oil crops such as rapeseed, palm, and soybean. Biofuels are used mainly for transportation. 
     According to particular embodiments of the invention, the CRISPR-Cas system is used to generate lipid-rich diatoms which are useful in biofuel production. 
     In particular embodiments it is envisaged to specifically modify genes that are involved in the modification of the quantity of lipids and/or the quality of the lipids produced by the algal cell. Examples of genes encoding enzymes involved in the pathways of fatty acid synthesis can encode proteins having for instance acetyl-CoA carboxylase, fatty acid synthase, 3-ketoacyl_acyl-carrier protein synthase III, glycerol-3-phospate dehydrogenase (G3PDH), Enoyl-acyl carrier protein reductase (Enoyl-ACP-reductase), glycerol-3-phosphate acyltransferase, lysophosphatidic acyl transferase or diacylglycerol acyltransferase, phospholipid:diacylglycerol acyltransferase, phoshatidate phosphatase, fatty acid thioesterase such as palmitoyi protein thioesterase, or malic enzyme activities. In further embodiments it is envisaged to generate diatoms that have increased lipid accumulation. This can be achieved by targeting genes that decrease lipid catabolisation. Of particular interest for use in the methods of the present invention are genes involved in the activation of both triacylglycerol and free fatty acids, as well as genes directly involved in (β-oxidation of fatty acids, such as acyl-CoA synthetase, 3-ketoacyl-CoA thiolase, acyl-CoA oxidase activity and phosphoglucomutase. The CRISPR-Cas system and methods described herein can be used to specifically activate such genes in diatoms as to increase their lipid content. 
     Organisms such as microalgae are widely used for synthetic biology. Stovicek et al. (Metab. Eng. Comm., 2015; 2:13 describes genome editing of industrial yeast, for example,  Saccharomyces cerevisiae , to efficiently produce robust strains for industrial production. Stovicek used a CRISPR-Cas9 system codon-optimized for yeast to simultaneously disrupt both alleles of an endogenous gene and knock in a heterologous gene. Cas9 and gRNA were expressed from genomic or episomal 2μ-based vector locations. The authors also showed that gene disruption efficiency could be improved by optimization of the levels of Cas9 and gRNA expression. Hlavová et al. (Biotechnol. Adv. 2015) discusses development of species or strains of microalgae using techniques such as CRISPR to target nuclear and chloroplast genes for insertional mutagenesis and screening. The methods of Stovicek and Hlavová may be applied and/or adapted to the Cas-like (e.g. Cas9-like or Cas12-like) effector protein system of the present invention. 
     U.S. Pat. No. 8,945,839 describes a method for engineering Micro-Algae ( Chlamydomonas reinhardtii  cells) species) using Cas9. Using similar tools, the methods of the CRISPR-Cas system described herein can be applied on  Chlamydomonas  species and other algae. In particular embodiments, Cas-like (e.g. Cas9-like or Cas12-like) protein(s) and guide RNA are introduced in algae expressed using a vector that expresses the Cas-like (e.g. Cas9-like or Cas12-like) protein(s) under the control of a constitutive promoter such as Hsp70A-Rbc S2 or Beta2-tubulin. Guide RNA will be delivered using a vector containing T7 promoter. Alternatively, Cas-like (e.g. Cas9-like or Cas12-like) mRNA(s) and in vitro transcribed guide RNA can be delivered to algal cells. Electroporation protocol follows standard recommended protocol from the GeneArt  Chlamydomonas  Engineering kit 
     In particular embodiments, the methods using the CRISPR-Cas system as described herein are used to alter the properties of the cell wall in order to facilitate access by key hydrolyzing agents for a more efficient release of sugars for fermentation. In particular embodiments, the biosynthesis of cellulose and/or lignin are modified. Cellulose is the major component of the cell wall. The biosynthesis of cellulose and lignin are co-regulated. By reducing the proportion of lignin in a plant the proportion of cellulose can be increased. In particular embodiments, the methods described herein are used to downregulate lignin biosynthesis in the plant so as to increase fermentable carbohydrates. More particularly, the methods described herein are used to downregulate at least a first lignin biosynthesis gene selected from the group consisting of 4-coumarate 3-hydroxylase (C3H), phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), hydroxycinnamoyl transferase (HCT), caffeic acid O-methyltransferase (COMT), caffeoyl CoA 3-O-methyltransferase (CCoAOMT), ferulate 5-hydroxylase (F5H), cinnamyl alcohol dehydrogenase (CAD), cinnamoyl CoA-reductase (CCR), 4-coumarate-CoA ligase (4CL), monolignol-lignin-specific glycosyltransferase, and aldehyde dehydrogenase (ALDH) as disclosed in WO 2008064289 A2. 
     In particular embodiments, the methods described herein are used to produce plant mass that produces lower levels of acetic acid during fermentation (see also WO 2010096488). More particularly, the methods disclosed herein are used to generate mutations in homologs to Cas1L to reduce polysaccharide acetylation. 
     Transient Expression of CRISPR-Cas Systems and Components in Plant Cells 
     In particular embodiments, it is envisaged that the guide RNA and/or Cas-like (e.g. Cas9-like or Cas12-like) gene are transiently expressed in the plant cell. In these embodiments, the CRISPR-Cas system can ensure modification of a target gene only when both the guide RNA and the Cas-like (e.g. Cas9-like or Cas12-like) protein(s) is/are present in a cell, such that genomic modification can further be controlled. As the expression of the Cas-like (e.g. Cas9-like or Cas12-like) protein(s) is transient, plants regenerated from such plant cells typically contain no foreign DNA. In particular embodiments, the Cas-like (e.g. Cas9-like or Cas12-like) protein(s) is stably expressed by the plant cell and the guide sequence is transiently expressed. 
     In particular embodiments, the CRISPR-Cas system components can be introduced in the plant cells using a plant viral vector (Scholthof et al. 1996, Annu Rev Phytopathol. 1996; 34:299-323). In further particular embodiments, said viral vector is a vector from a DNA virus. For example, geminivirus (e.g., cabbage leaf curl virus, bean yellow dwarf virus, wheat dwarf virus, tomato leaf curl virus, maize streak virus, tobacco leaf curl virus, or tomato golden mosaic virus) or nanovirus (e.g.,  Faba  bean necrotic yellow virus). In other particular embodiments, said viral vector is a vector from an RNA virus. For example, tobravirus (e.g., tobacco rattle virus, tobacco mosaic virus), potexvirus (e.g., potato virus X), or hordeivirus (e.g., barley stripe mosaic virus). The replicating genomes of plant viruses are non-integrative vectors. 
     In particular embodiments, the vector used for transient expression of CRISPR-Cas constructs is for instance a pEAQ vector, which is tailored for  Agrobacterium -mediated transient expression (Sainsbury F. et al., Plant Biotecnol. J. 2009 September; 7(7):682-93) in the protoplast. Precise targeting of genomic locations was demonstrated using a modified Cabbage Leaf Curl virus (CaLCuV) vector to express gRNAs in stable transgenic plants expressing a CRISPR enzyme (Scientific Reports 5, Article number: 14926 (2015), doi:10.1038/srep14926). 
     In particular embodiments, double-stranded DNA fragments encoding the guide RNA and/or the Cas-like (e.g. Cas9-like or Cas12-like) gene(s) can be transiently introduced into the plant cell. In such embodiments, the introduced double-stranded DNA fragments are provided in sufficient quantity to modify the cell but do not persist after a contemplated period of time has passed or after one or more cell divisions. Methods for direct DNA transfer in plants are known by the skilled artisan (see for instance Davey et al. Plant Mol Biol. 1989 September; 13(3):273-85.) 
     In other embodiments, an RNA polynucleotide encoding the Cas-like (e.g. Cas9-like or Cas12-like) protein(s) is/are introduced into the plant cell, which is then translated and processed by the host cell generating the protein in sufficient quantity to modify the cell (in the presence of at least one guide RNA) but which does not persist after a contemplated period of time has passed or after one or more cell divisions. Methods for introducing mRNA to plant protoplasts for transient expression are known by the skilled artisan (see for instance in Gallie, Plant Cell Reports (1993), 13; 119-122). 
     Combinations of the different methods described above are also envisaged. 
     Detecting Modifications in the Plant Genome—Selectable Markers 
     In particular embodiments, where the method involves modification of an endogenous target gene of the plant genome, any suitable method can be used to determine, after the plant, plant part or plant cell is infected or transfected with the CRISPR-Cas system, whether gene targeting or targeted mutagenesis has occurred at the target site. Where the method involves introduction of a transgene, a transformed plant cell, callus, tissue or plant may be identified and isolated by selecting or screening the engineered plant material for the presence of the transgene or for traits encoded by the transgene. Physical and biochemical methods may be used to identify plant or plant cell transformants containing inserted gene constructs or an endogenous DNA modification. These methods include but are not limited to: 1) Southern analysis or PCR amplification for detecting and determining the structure of the recombinant DNA insert or modified endogenous genes; 2) Northern blot, Si RNase protection, primer-extension or reverse transcriptase-PCR amplification for detecting and examining RNA transcripts of the gene constructs; 3) enzymatic assays for detecting enzyme or ribozyme activity, where such gene products are encoded by the gene construct or expression is affected by the genetic modification; 4) protein gel electrophoresis, Western blot techniques, immunoprecipitation, or enzyme-linked immunoassays, where the gene construct or endogenous gene products are proteins. Additional techniques, such as in situ hybridization, enzyme staining, and immunostaining, also may be used to detect the presence or expression of the recombinant construct or detect a modification of endogenous gene in specific plant organs and tissues. The methods for doing all these assays are well known to those skilled in the art. 
     Additionally (or alternatively), the expression system encoding the CRISPR-Cas components is typically designed to comprise one or more selectable or detectable markers that provide a means to isolate or efficiently select cells that contain and/or have been modified by the CRISPR-Cas system at an early stage and on a large scale. 
     In the case of  Agrobacterium -mediated transformation, the marker cassette may be adjacent to or between flanking T-DNA borders and contained within a binary vector. In another embodiment, the marker cassette may be outside of the T-DNA. A selectable marker cassette may also be within or adjacent to the same T-DNA borders as the expression cassette or may be somewhere else within a second T-DNA on the binary vector (e.g., a 2 T-DNA system). 
     For particle bombardment or with protoplast transformation, the expression system can comprise one or more isolated linear fragments or may be part of a larger construct that might contain bacterial replication elements, bacterial selectable markers or other detectable elements. The expression cassette(s) comprising the polynucleotides encoding the guide and/or Cas-like (e.g. Cas9-like and/or Cas12-like) proteins may be physically linked to a marker cassette or may be mixed with a second nucleic acid molecule encoding a marker cassette. The marker cassette is comprised of necessary elements to express a detectable or selectable marker that allows for efficient selection of transformed cells. 
     The selection procedure for the cells based on the selectable marker will depend on the nature of the marker gene. In particular embodiments, use is made of a selectable marker, i.e. a marker which allows a direct selection of the cells based on the expression of the marker. A selectable marker can confer positive or negative selection and is conditional or non-conditional on the presence of external substrates (Miki et al. 2004, 107(3): 193-232). Most commonly, antibiotic or herbicide resistance genes are used as a marker, whereby selection is be performed by growing the engineered plant material on media containing an inhibitory amount of the antibiotic or herbicide to which the marker gene confers resistance. Examples of such genes are genes that confer resistance to antibiotics, such as hygromycin (hpt) and kanamycin (nptII), and genes that confer resistance to herbicides, such as phosphinothricin (bar) and chlorosulfuron (als). 
     Transformed plants and plant cells may also be identified by screening for the activities of a visible marker, typically an enzyme capable of processing a colored substrate (e.g., the β-glucuronidase, luciferase, B or C1 genes). Such selection and screening methodologies are well known to those skilled in the art. 
     Plant Cultures and Regeneration 
     In particular embodiments, plant cells which have a modified genome and that are produced or obtained by any of the methods described herein, can be cultured to regenerate a whole plant which possesses the transformed or modified genotype and thus the desired phenotype. Conventional regeneration techniques are well known to those skilled in the art. Particular examples of such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, and typically relying on a biocide and/or herbicide marker which has been introduced together with the desired nucleotide sequences. In further particular embodiments, plant regeneration is obtained from cultured protoplasts, plant callus, explants, organs, pollens, embryos or parts thereof (see e.g. Evans et al. (1983), Handbook of Plant Cell Culture, Klee et al (1987) Ann. Rev. of Plant Phys.). 
     In particular embodiments, transformed or improved plants as described herein can be self-pollinated to provide seed for homozygous improved plants of the invention (homozygous for the DNA modification) or crossed with non-transgenic plants or different improved plants to provide seed for heterozygous plants. Where a recombinant DNA was introduced into the plant cell, the resulting plant of such a crossing is a plant which is heterozygous for the recombinant DNA molecule. Both such homozygous and heterozygous plants obtained by crossing from the improved plants and comprising the genetic modification (which can be a recombinant DNA) are referred to herein as “progeny”. Progeny plants are plants descended from the original transgenic plant and containing the genome modification or recombinant DNA molecule introduced by the methods provided herein. Alternatively, genetically modified plants can be obtained by one of the methods described supra using the Cfp1 enzyme whereby no foreign DNA is incorporated into the genome. Progeny of such plants, obtained by further breeding may also contain the genetic modification. Breedings are performed by any breeding methods that are commonly used for different crops (e.g., Allard, Principles of Plant Breeding, John Wiley &amp; Sons, NY, U. of CA, Davis, Calif., 50-98 (1960). 
     Generation of Plants with Enhanced Agronomic Traits 
     The Cas-like (e.g. Cas9-like and/or Cas12-like)-based CRISPR systems provided herein can be used to introduce targeted double-strand or single-strand breaks and/or to introduce gene activator and or repressor systems and without being limitative, can be used for gene targeting, gene replacement, targeted mutagenesis, targeted deletions or insertions, targeted inversions and/or targeted translocations. By co-expression of multiple targeting RNAs directed to achieve multiple modifications in a single cell, multiplexed genome modification can be ensured. This technology can be used to high-precision engineering of plants with improved characteristics, including enhanced nutritional quality, increased resistance to diseases and resistance to biotic and abiotic stress, and increased production of commercially valuable plant products or heterologous compounds. 
     In particular embodiments, the CRISPR-Cas system as described herein is used to introduce targeted double-strand breaks (DSB) in an endogenous DNA sequence. The DSB activates cellular DNA repair pathways, which can be harnessed to achieve desired DNA sequence modifications near the break site. This is of interest where the inactivation of endogenous genes can confer or contribute to a desired trait. In particular embodiments, homologous recombination with a template sequence is promoted at the site of the DSB, in order to introduce a gene of interest. 
     In particular embodiments, the CRISPR-Cas system may be used as a generic nucleic acid binding protein with fusion to or being operably linked to a functional domain for activation and/or repression of endogenous plant genes. Exemplary functional domains may include but are not limited to translational initiator, translational activator, translational repressor, nucleases, in particular ribonucleases, a spliceosome, beads, a light inducible/controllable domain or a chemically inducible/controllable domain. Typically, in these embodiments, the Cas-like (e.g. Cas9-like and/or Cas12-like) protein(s) comprises at least one mutation, such that it has no more than 5% of the activity of the Cas-like (e.g. Cas9-like and/or Cas12-like) protein(s) not having the at least one mutation; the guide RNA comprises a guide sequence capable of hybridizing to a target sequence. 
     The methods described herein generally result in the generation of “improved plants” in that they have one or more desirable traits compared to the wildtype plant. In particular embodiments, the plants, plant cells or plant parts obtained are transgenic plants, comprising an exogenous DNA sequence incorporated into the genome of all or part of the cells of the plant. In particular embodiments, non-transgenic genetically modified plants, plant parts or cells are obtained, in that no exogenous DNA sequence is incorporated into the genome of any of the plant cells of the plant. In such embodiments, the improved plants are non-transgenic. Where only the modification of an endogenous gene is ensured and no foreign genes are introduced or maintained in the plant genome, the resulting genetically modified crops contain no foreign genes and can thus basically be considered non-transgenic. The different applications of the CRISPR-Cas system for plant genome editing are described more in detail below. 
     In further particular embodiments, crop plants can be improved by influencing specific plant traits. For example, by developing pesticide-resistant plants, improving disease resistance in plants, improving plant insect and nematode resistance, improving plant resistance against parasitic weeds, improving plant drought tolerance, improving plant nutritional value, improving plant stress tolerance, avoiding self-pollination, plant forage digestibility biomass, grain yield etc. A few specific non-limiting examples are provided hereinbelow. 
     In addition to targeted mutation of single genes, Cas-like CRISPR complexes can be designed to allow targeted mutation of multiple genes, deletion of chromosomal fragment, site-specific integration of transgene, site-directed mutagenesis in vivo, and precise gene replacement or allele swapping in plants. Therefore, the methods described herein have broad applications in gene discovery and validation, mutational and cisgenic breeding, and hybrid breeding. These applications facilitate the production of a new generation of genetically modified crops with various improved agronomic traits such as herbicide resistance, disease resistance, abiotic stress tolerance, high yield, and superior quality. 
     Introduction of One or More Foreign Genes to Confer an Agricultural Trait of Interest 
     The invention provides methods of genome editing or modifying sequences associated with or at a target locus of interest wherein the method comprises introducing a Cas-like (e.g. Cas9-like and/or Cas12-like) effector protein complex(es) into a plant cell, whereby the Cas-like (e.g. Cas9-like and/or Cas12-like) effector protein complex(es) effectively functions to integrate a DNA insert, e.g. encoding a foreign gene of interest, into the genome of the plant cell. In preferred embodiments the integration of the DNA insert is facilitated by HR with an exogenously introduced DNA template or repair template. Typically, the exogenously introduced DNA template or repair template is delivered together with the Cas-like (e.g. Cas9-like and/or Cas12-like) effector protein complex(es) or one component or a polynucleotide vector for expression of a component of the complex(es). 
     The CRISPR-Cas systems provided herein allow for targeted gene delivery. It has become increasingly clear that the efficiency of expressing a gene of interest is to a great extent determined by the location of integration into the genome. The present methods allow for targeted integration of the foreign gene into a desired location in the genome. The location can be selected based on information of previously generated events or can be selected by methods disclosed elsewhere herein. 
     In particular embodiments, the methods provided herein include (a) introducing into the cell a CRISPR-Cas complex comprising a guide RNA, comprising a direct repeat and a guide sequence, wherein the guide sequence hybridizes to a target sequence that is endogenous to the plant cell; (b) introducing into the plant cell a Cas-like (e.g. Cas9-like and/or Cas12-like) effector molecule(s), which complexes with the guide RNA when the guide sequence hybridizes to the target sequence and induces a double strand break at or near the sequence to which the guide sequence is targeted; and (c) introducing into the cell a nucleotide sequence encoding an HDR repair template which encodes the gene of interest and which is introduced into the location of the DS break as a result of HDR. In particular embodiments, the step of introducing can include delivering to the plant cell one or more polynucleotides encoding Cas-like (e.g. Cas9-like and/or Cas12-like) effector protein(s), the guide RNA and the repair template. In particular embodiments, the polynucleotides are delivered into the cell by a DNA virus (e.g., a geminivirus) or an RNA virus (e.g., a tobravirus). In particular embodiments, the introducing steps include delivering to the plant cell a T-DNA containing one or more polynucleotide sequences encoding the Cas-like (e.g. Cas9-like and/or Cas12-like) effector protein(s) the guide RNA and the repair template, where the delivering is via  Agrobacterium . The nucleic acid sequence encoding the Cas-like (e.g. Cas9-like and/or Cas12-like) effector protein(s) can be operably linked to a promoter, such as a constitutive promoter (e.g., a cauliflower mosaic virus 35S promoter), or a cell specific or inducible promoter. In particular embodiments, the polynucleotide is introduced by microprojectile bombardment. In particular embodiments, the method further includes screening the plant cell after the introducing steps to determine whether the repair template i.e. the gene of interest has been introduced. In particular embodiments, the methods include the step of regenerating a plant from the plant cell. In further embodiments, the methods include cross breeding the plant to obtain a genetically desired plant lineage. Examples of foreign genes encoding a trait of interest are listed below. 
     Editing of Endogenous Genes to Confer an Agricultural Trait of Interest 
     The invention provides methods of genome editing or modifying sequences associated with or at a target locus of interest wherein the method comprises introducing one or more Cas-like (e.g. Cas9-like and/or Cas12-like) effector protein complex(es) into a plant cell, whereby the Cas-like (e.g. Cas9-like and/or Cas12-like) complex(es) modifies the expression of an endogenous gene of the plant. This can be achieved in different ways. In particular embodiments, the elimination of expression of an endogenous gene is desirable and the CRISPR-Cas complex is used to target and cleave an endogenous gene so as to modify gene expression. In these embodiments, the methods provided herein include (a) introducing into the plant cell a CRISPR-Cas complex comprising a guide RNA, comprising a direct repeat and a guide sequence, wherein the guide sequence hybridizes to a target sequence within a gene of interest in the genome of the plant cell; and (b) introducing into the cell a Cas-like (e.g. Cas9-like and/or Cas12-like) effector protein(s), which upon binding to the guide RNA comprises a guide sequence that is hybridized to the target sequence, ensures a double strand break at or near the sequence to which the guide sequence is targeted; In particular embodiments, the step of introducing can include delivering to the plant cell one or more polynucleotides encoding Cas-like (e.g. Cas9-like and/or Cas12-like) effector protein(s) and the guide RNA. 
     In particular embodiments, the polynucleotides are delivered into the cell by a DNA virus (e.g., a geminivirus) or an RNA virus (e.g., a tobravirus). In particular embodiments, the introducing steps include delivering to the plant cell a T-DNA containing one or more polynucleotide sequences encoding the Cas-like (e.g. Cas9-like and/or Cas12-like) effector protein (s) and the guide RNA, where the delivering is via  Agrobacterium . The polynucleotide sequence encoding the components of the CRISPR-Cas system can be operably linked to a promoter, such as a constitutive promoter (e.g., a cauliflower mosaic virus 35S promoter), or a cell specific or inducible promoter. In particular embodiments, the polynucleotide is introduced by microprojectile bombardment. In particular embodiments, the method further includes screening the plant cell after the introducing steps to determine whether the expression of the gene of interest has been modified. In particular embodiments, the methods include the step of regenerating a plant from the plant cell. In further embodiments, the methods include cross breeding the plant to obtain a genetically desired plant lineage. 
     In particular embodiments of the methods described above, disease resistant crops are obtained by targeted mutation of disease susceptibility genes or genes encoding negative regulators (e.g. Mlo gene) of plant defense genes. In a particular embodiment, herbicide-tolerant crops are generated by targeted substitution of specific nucleotides in plant genes such as those encoding acetolactate synthase (ALS) and protoporphyrinogen oxidase (PPO). In particular embodiments drought and salt tolerant crops by targeted mutation of genes encoding negative regulators of abiotic stress tolerance, low amylose grains by targeted mutation of Waxy gene, rice or other grains with reduced rancidity by targeted mutation of major lipase genes in aleurone layer, etc. In particular embodiments. A more extensive list of endogenous genes encoding a traits of interest are listed below. 
     Modulating of Endogenous Genes by the CRISPR-Cas System to Confer an Agricultural Trait of Interest 
     Also provided herein are methods for modulating (i.e. activating or repressing) endogenous gene expression using the Cas-like (e.g. Cas9-like and/or Cas12-like) protein(s) provided herein. Such methods make use of distinct RNA sequence(s) which are targeted to the plant genome by the Cas-like (e.g. Cas9-like and/or Cas12-like) complex(es). More particularly the distinct RNA sequence(s) bind to two or more adaptor proteins (e.g. aptamers) whereby each adaptor protein is associated with one or more functional domains and wherein at least one of the one or more functional domains associated with the adaptor protein have one or more activities comprising methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, DNA integration activity RNA cleavage activity, DNA cleavage activity or nucleic acid binding activity; The functional domains are used to modulate expression of an endogenous plant gene so as to obtain the desired trait. Typically, in these embodiments, the Cas-like (e.g. Cas9-like and/or Cas12-like) effector protein(s) has one or more mutations such that it has no more than 5% of the nuclease activity of the Cas-like (e.g. Cas9-like and/or Cas12-like) effector protein(s) not having the at least one mutation. 
     In particular embodiments, the methods provided herein include the steps of (a) introducing into the cell a CRISPR-Cas complex comprising a guide RNA, comprising a direct repeat and a guide sequence, wherein the guide sequence hybridizes to a target sequence that is endogenous to the plant cell; (b) introducing into the plant cell a Cas-like (e.g. Cas9-like and/or Cas12-like) effector molecule(s) which complexes with the guide RNA when the guide sequence hybridizes to the target sequence; and wherein either the guide RNA is modified to comprise a distinct RNA sequence (aptamer) binding to a functional domain and/or the Cas-like (e.g. Cas9-like and/or Cas12-like) effector protein(s) is modified in that it is linked to a functional domain. In particular embodiments, the step of introducing can include delivering to the plant cell one or more polynucleotides encoding the (modified) Cas-like (e.g. Cas9-like and/or Cas12-like) effector protein(s) and the (modified) guide RNA. The details the components of the CRISPR-Cas system for use in these methods are described elsewhere herein. 
     In particular embodiments, the polynucleotides are delivered into the cell by a DNA virus (e.g., a geminivirus) or an RNA virus (e.g., a tobravirus). In particular embodiments, the introducing steps include delivering to the plant cell a T-DNA containing one or more polynucleotide sequences encoding the Cas-like (e.g. Cas9-like and/or Cas12-like) effector protein (s) and the guide RNA, where the delivering is via  Agrobacterium . The nucleic acid sequence encoding the one or more components of the RISPR-Cas system can be operably linked to a promoter, such as a constitutive promoter (e.g., a cauliflower mosaic virus 35S promoter), or a cell specific or inducible promoter. In particular embodiments, the polynucleotide is introduced by microprojectile bombardment. In particular embodiments, the method further includes screening the plant cell after the introducing steps to determine whether the expression of the gene of interest has been modified. In particular embodiments, the methods include the step of regenerating a plant from the plant cell. In further embodiments, the methods include cross breeding the plant to obtain a genetically desired plant lineage. A more extensive list of endogenous genes encoding a traits of interest are listed below. 
     The CRISPR-Cas systems described here can be used to modify polyploid plants. Many plants are polyploid, which means they carry duplicate copies of their genomes—sometimes as many as six, as in wheat. The methods according to the present invention, which make use of the CRISPR-Cas effector protein can be “multiplexed” to affect all copies of a gene, or to target dozens of genes at once. For instance, in particular embodiments, the methods of the present invention are used to simultaneously ensure a loss of function mutation in different genes responsible for suppressing defenses against a disease. In particular embodiments, the methods of the present invention are used to simultaneously suppress the expression of the TaMLO-A1, TaMLO-B1 and TaMLO-D1 nucleic acid sequence in a wheat plant cell and regenerating a wheat plant therefrom, in order to ensure that the wheat plant is resistant to powdery mildew (see also WO2015109752). 
     Described herein are exemplary genes conferring agronomic traits. As described herein above, in particular embodiments, the invention encompasses the use of the CRISPR-Cas system as described herein for the insertion of a DNA of interest, including one or more plant expressible gene(s). In further particular embodiments, the invention encompasses methods and tools using the CRISPR-Cas system as described herein for partial or complete deletion of one or more plant expressed gene(s). In other further particular embodiments, the invention encompasses methods and tools using the CRISPR-Cas system as described herein to ensure modification of one or more plant-expressed genes by mutation, substitution, insertion of one of more nucleotides. In other particular embodiments, the invention encompasses the use of CRISPR-Cas system as described herein to ensure modification of expression of one or more plant-expressed genes by specific modification of one or more of the regulatory elements directing expression of said genes. 
     In particular embodiments, the invention encompasses methods which involve the introduction of exogenous genes and/or the targeting of endogenous genes and their regulatory elements, including but not limited to any of those further described below. 
     Genes that Confer Resistance to Pests or Diseases 
     In some embodiments, the modified plant or cell thereof can be modified to contain a gene or gene variant that can confer disease resistance to the plant or cell thereof. In some embodiments, an exogenous gene is introduced. In other embodiments, an endogenous gene can be modified to a disease-resistant variant of the endogenous gene. A plant can be transformed with cloned resistance genes to engineer plants that are resistant to specific pathogen strains. See, e.g., Jones et al., Science 266:789 (1994) (cloning of the tomato Cf-9 gene for resistance to  Cladosporium fulvum ); Martin et al., Science 262:1432 (1993) (tomato Pto gene for resistance to  Pseudomonas syringae  pv. tomato encodes a protein kinase); Mindrinos et al., Cell 78:1089 (1994) (Arabidops may be RSP2 gene for resistance to  Pseudomonas syringae ). A plant gene that is upregulated or down regulated during pathogen infection can be engineered for pathogen resistance. See, e.g., Thomazella et al., bioRxiv 064824; doi: https://doi.org/10.1101/064824 Epub. Jul. 23, 2016 (tomato plants with deletions in the SIDMR6-1 which is normally upregulated during pathogen infection). In some embodiments, the modified plant can be modified to express a gene that is resistant to specific pathogens by the CRISPR-Cas systems described herein. 
     In some embodiments, the modified plant can be modified to express one or more genes conferring resistance to a pest, such as soybean cyst nematode. See e.g., PCT Application WO 96/30517; PCT Application WO 93/19181. 
     In some embodiments, the modified plant can be modified with one or more genes whose gene products can repel, deter, and/or kill a plant pest (e.g. insect, animal, or other organism that is detrimental to the plant or another plant (e.g. in the case of a trap crop)). In some embodiments, such genes can be  Bacillus thuringiensis  proteins&#39; genes, (see, e.g., Geiser et al., Gene 48:109 (1986)); lectins&#39; gene(s) (see e.g. Van Damme et al., Plant Molec. Biol. 24:25 (1994); a vitamin-binding protein gene (e.g. avidin or avidin homologue) (see e.g., PCT application US93/06487), genes encoding enzyme inhibitors (e.g. protease or proteinase inhibitors and amylase inhibitors) (see e.g., Abe et al., J. Biol. Chem. 262:16793 (1987), Huub et al., Plant Molec. Biol. 21:985 (1993)), Sumitani et al., Biosci. Biotech. Biochem. 57:1243 (1993) and U.S. Pat. No. 5,494,813); insect-specific hormones or pheromones (e.g. ecdysteroid or juvenile hormone, a variant thereof, a mimetic based thereon, or an antagonist or agonist thereof) (see e.g. Hammock et al., Nature 344:458 (1990)); genes encoding insect-specific peptides which, upon expression, disrupts the physiology of the affected pest (see e.g. Regan, J. Biol. Chem. 269:9 (1994) and Pratt et al., Biochem. Biophys. Res. Comm. 163:1243 (1989). See also U.S. Pat. No. 5,266,317); genes encoding insect-specific venom or proteins thereof produced by a snake, a wasp, or any other organism (see e.g., Pang et al., Gene 116: 165 (1992)); genes encoding enzymes responsible for a hyperaccumulation of a monoterpene, a sesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivative, or another nonprotein molecule with insecticidal activity; Enzymes involved in the modification, including the post-translational modification, of a biologically active molecule; for example, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme, a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, a phosphatase, a kinase, a phosphorylase, a polymerase, an elastase, a chitinase and a glucanase, whether natural or synthetic (see e.g., PCT application WO93/02197, Kramer et al., Insect Biochem. Molec. Biol. 23:691 (1993) and Kawalleck et al., Plant Molec. Biol. 21:673 (1993)); genes encoding molecules that can stimulate signal transduction (see e.g., Botella et al., Plant Molec. Biol. 24:757 (1994), and Griess et al., Plant Physiol. 104:1467 (1994)). gene(s) encoding viral-invasive proteins or a complex toxin derived therefrom (Beachy et al., Ann. rev. Phytopathol. 28:451 (1990)); gene(s) encoding developmental-arrestive proteins produced in nature by a pathogen or a parasite see e.g., Lamb et al., Bio/Technology 10:1436 (1992) and Toubart et al., Plant J. 2:367 (1992)); gene(s) encoding a developmental-arrestive protein produced in nature by a plant (see e.g., Logemann et al., Bio/Technology 10:305 (1992)) and combinations thereof. 
     In plants, pathogens are often host-specific. For example, some  Fusarium  species will cause tomato wilt but attacks only tomato, and other  Fusarium  species attack only wheat. Plants have existing and induced defenses to resist most pathogens. Mutations and recombination events across plant generations lead to genetic variability that gives rise to susceptibility, especially as pathogens reproduce with more frequency than plants. In plants there can be non-host resistance, e.g., the host and pathogen are incompatible or there can be partial resistance against all races of a pathogen, typically controlled by many genes and/or also complete resistance to some races of a pathogen but not to other races. Such resistance is typically controlled by a few genes. Using methods and components of the CRISPR-Cas-like system, a new tool now exists to induce specific mutations in anticipation hereon. Accordingly, one can analyze the genome of sources of resistance genes, and in plants having desired characteristics or traits, use the method and components of the CRISPR-Cas system to induce the rise of resistance genes. The present systems can do so with more precision than previous mutagenic agents and hence accelerate and improve plant breeding programs. 
     In some embodiments, the plant or cell(s) thereof can be modified to contain one or more genes involved in plant diseases, such as those that confer resistance to one or more plant diseases, such as any one or more of those listed in PCT Publication WO 2013046247. Exemplary rice diseases/disease causing organisms that the modified plant can be resistant to are, without limitation,  Magnaporthe grisea, Cochliobolus miyabeanus, Rhizoctonia solani , and  Gibberella fujikuroi.    
     Exemplary wheat diseases/disease causing organisms that the modified plant can be resistant to are, without limitation,  Erysiphe graminis, Fusarium graminearum, F. avenaceum, F. culmorum, Microdochium nivale, Puccinia striiformis, P. graminis, P. recondita, Micronectriella nivale, Typhula  sp.,  Ustilago tritici, Tilletia caries, Pseudocercosporella herpotrichoides, Mycosphaerella graminicola, Stagonospora nodorum , and  Pyrenophora tritici - repentis.    
     Exemplary barley diseases/disease causing organisms that the modified plant can be resistant to are, without limitation,  Erysiphe graminis, Fusarium graminearum, F. avenaceum, F. culmorum, Microdochium nivale, Puccinia striiformis, P. graminis, P. hordei, Ustilago nuda, Rhynchosporium secalis, Pyrenophora teres, Cochliobolus sativus, Pyrenophora graminea , and  Rhizoctonia solani.    
     Exemplary maize diseases/disease causing organisms that the modified plant can be resistant to are, without limitation,  Ustilago maydis, Cochliobolus heterostrophus, Gloeocercospora sorghi, Puccinia polysora, Cercospora zeae - maydis, Rhizoctonia solani.    
     Exemplary citrus diseases/disease causing organisms that the modified plant can be resistant to are, without limitation,  Diaporthe citri, Elsinoe fawcetti, Penicillium digitatum, P. italicum, Phytophthora parasitica , and  Phytophthora citrophthora.    
     Exemplary apple diseases/disease causing organisms that the modified plant can be resistant to are, without limitation,  Monilinia mali, Valsa ceratosperma, Podosphaera leucotricha, Alternaria alternata  apple pathotype,  Venturia inaequalis, Colletotrichum acutatum, Phytophtora cactorum.    
     Exemplary pear diseases/disease causing organisms that the modified plant can be resistant to are, without limitation,  Venturia nashicola, V. pirina, Alternaria alternata  Japanese pear pathotype,  Gymnosporangium haraeanum , and  Phytophtora cactorum.    
     Exemplary peach diseases/disease causing organisms that the modified plant can be resistant to are, without limitation,  Monilinia fructicola, Cladosporium carpophilum , and  Phomopsis  sp. 
     Exemplary grape diseases/disease causing organisms that the modified plant can be resistant to are, without limitation,  Elsinoe ampelina, Glomerella cingulata, Uninula necator, Phakopsora ampelopsidis, Guignardia bidwellii , and  Plasmopara viticola.    
     Exemplary persimmon diseases/disease causing organisms that the modified plant can be resistant to are, without limitation,  Gloesporium kaki, Cercospora kaki , and  Mycosphaerela nawae.    
     Exemplary gourd diseases/disease causing organisms that the modified plant can be resistant to are, without limitation,  Colletotrichum lagenarium, Sphaerotheca fuliginea, Mycosphaerella melonis, Fusarium oxysporum, Pseudoperonospora cubensis , and  Phytophthora  sp.,  Pythium  sp. 
     Exemplary tomato diseases/disease causing organisms that the modified plant can be resistant to are, without limitation,  Alternaria solani, Cladosporium fulvum, Phytophthora infestans; Pseudomonas syringae  pv. Tomato;  Phytophthora capsici ; and  Xanthomonas.    
     Exemplary eggplant diseases/disease causing organisms that the modified plant can be resistant to are, without limitation,  Phomopsis vexans  and  Erysiphe cichoracearum.    
     Exemplary Brassicaceous vegetable diseases/disease causing organisms that the modified plant can be resistant to are, without limitation,  Alternaria japonica, Cercosporella brassicae, Plasmodiophora brassicae , and  Peronospora parasitica.    
     Exemplary Welsh onion diseases/disease causing organisms that the modified plant can be resistant to are, without limitation,  Puccinia allii  and  Peronospora destructor.    
     Exemplary Welsh onion diseases/disease causing organisms that the modified plant can be resistant to are, without limitation  Cercospora kikuchii, Elsinoe glycines, Diaporthe phaseolorum  var.  sojae, Septoria glycines, Cercospora sojina, Phakopsora pachyrhizi, Phytophthora sojae, Rhizoctonia solani, Corynespora casiicola , and  Sclerotinia sclerotiorum.    
     Exemplary kidney bean diseases/disease causing organisms that the modified plant can be resistant to are, without limitation,  Colletrichum lindemthianum.    
     Exemplary peanut diseases/disease causing organisms that the modified plant can be resistant to are, without limitation,  Cercospora personata, Cercospora arachidicola , and  Sclerotium rolfsii.    
     Exemplary pea diseases/disease causing organisms that the modified plant can be resistant to are, without limitation,  Erysiphe pisi.    
     Exemplary potato diseases/disease causing organisms that the modified plant can be resistant to are, without limitation,  Alternaria solani, Phytophthora infestans, Phytophthora erythroseptica, Spongospora subterranean , and f. sp.  Subterranean.    
     Exemplary strawberry diseases/disease causing organisms that the modified plant can be resistant to are, without limitation,  Sphaerotheca humuli  and  Glomerella cingulate.    
     Exemplary tea diseases/disease causing organisms that the modified plant can be resistant to are, without limitation,  Exobasidium reticulatum, Elsinoe leucospila, Pestalotiopsis  sp., and  Colletotrichum theae - sinensis.    
     Exemplary tobacco diseases/disease causing organisms that the modified plant can be resistant to are, without limitation,  Alternaria longipes, Erysiphe cichoracearum, Colletotrichum tabacum, Peronospora tabacina , and  Phytophthora nicotianae.    
     Exemplary rapeseed diseases/disease causing organisms that the modified plant can be resistant to are, without limitation,  Sclerotinia sclerotiorum , and  Rhizoctonia solani.    
     Exemplary cotton diseases/disease causing organisms that the modified plant can be resistant to are, without limitation,  Rhizoctonia solani.    
     Exemplary beet diseases/disease causing organisms that the modified plant can be resistant to are, without limitation,  Cercospora beticola, Thanatephorus cucumeris, Thanatephorus cucumeris , and  Aphanomyces cochlioides.    
     Exemplary rose diseases/disease causing organisms that the modified plant can be resistant to are, without limitation,  Diplocarpon rosae, Sphaerotheca pannosa , and  Peronospora sparsa.    
     Exemplary chrysanthemum and asteraceae diseases/disease causing organisms that the modified plant can be resistant to are, without limitation,  Bremia lactuca, Septoria chrysanthemi - indici , and  Puccinia horiana.    
     Exemplary radish diseases/disease causing organisms that the modified plant can be resistant to are, without limitation,  Alternaria brassicicola.    
     Exemplary zoysia diseases/disease causing organisms that the modified plant can be resistant to are, without limitation,  Sclerotinia homeocarpa , and  Rhizoctonia solani.    
     Exemplary banana diseases/disease causing organisms that the modified plant can be resistant to are, without limitation,  Mycosphaerella fijiensis  and  Mycosphaerella musicola.    
     Exemplary sunflower diseases/disease causing organisms that the modified plant can be resistant to are, without limitation, and  Plasmopara halstedii.    
     Exemplary seed or initial stage of plant growth diseases/disease causing organisms that the modified plant can be resistant to are, without limitation,  Aspergillus  spp.,  Penicillium  spp.,  Fusarium  spp.,  Gibberella  spp.,  Tricoderma  spp.,  Thielaviopsis  spp.,  Rhizopus  spp.,  Mucor  spp.,  Corticium  spp.,  Rhoma  spp.,  Rhizoctonia  spp.,  Diplodia  spp., and the like. 
     Other exemplary diseases/disease causing organisms that the modified plant can be resistant to are, without limitation,  Pythium aphanidermatum, Pythium debarianum, Pythium graminicola, Pythium irregulare, Pythium ultimum, Botrytis cinerea, Sclerotinia sclerotiorum, Polymixa  spp.,  Olpidium  spp. 
     Genes that Confer Resistance to Herbicides 
     In some embodiments, the CRISPR-Cas systems described herein can be used to modify a plant or cell thereof such that the modified plant or cell thereof contains one or more genes that confer herbicide resistance to the plant. 
     In some embodiments, the modified plant or cell thereof can contain one or more genes that confer resistance to herbicides that inhibit the growing point or meristem, such as an imidazolinone or a sulfonylurea, for example, by Lee et al., EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet. 80:449 (1990), respectively. 
     In some embodiments, the modified plant or cell thereof can contain one or more genes that confer glyphosate tolerance (e.g., mutant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPs) genes, aroA genes and glyphosate acetyl transferase (GAT) genes, respectively), or resistance to other phosphono compounds such as by glufosinate (e.g., phosphinothricin acetyl transferase (PAT) genes from  Streptomyces  species, including  Streptomyces hygroscopicus  and  Streptomyces viridichromogenes ), and to pyridinoxy or phenoxy proprionic acids and cyclohexones (e.g., ACCase inhibitor-encoding genes. See, for example, U.S. Pat. Nos. 4,940,835 and 6,248,876, 4,769,061, EP No. 0 333 033 and U.S. Pat. No. 4,975,374. See also EP No. 0242246, DeGreef et al., Bio/Technology 7:61 (1989), Marshall et al., Theor. Appl. Genet. 83:435 (1992), and WO 2005012515 to Castle et. al. and WO 2005107437). 
     In some embodiments, the modified plant or cell thereof can contain one or more genes that confer resistance to herbicides that inhibit photosynthesis, such as a triazine (e.g., psbA and gs+ genes) or a benzonitrile (e.g., nitrilase gene), and glutathione S-transferase in Przibila et al., Plant Cell 3:169 (1991), U.S. Pat. No. 4,810,648, and Hayes et al., Biochem. J. 285: 173 (1992). 
     In some embodiments, the modified plant or cell thereof can contain one or more genes encoding enzymes that can detoxify a herbicide or a mutant glutamine synthase enzyme that is resistant to inhibition, e.g. n U.S. patent application Ser. No. 11/760,602. Or a detoxifying enzyme is an enzyme encoding a phosphinothricin acetyltransferase (such as the bar or pat protein from  Streptomyces  species). Phosphinothricin acetyltransferases are for example described in U.S. Pat. Nos. 5,561,236; 5,648,477; 5,646,024; 5,273,894; 5,637,489; 5,276,268; 5,739,082; 5,908,810 and 7,112,665. 
     In some embodiments, the modified plant or cell thereof can contain one or more genes encoding hydroxyphenylpyruvatedioxygenases (HPPD) inhibitors, i.e., naturally occurring HPPD resistant enzymes, or genes encoding a mutated or chimeric HPPD enzyme as described in WO 96/38567, WO 99/24585, and WO 99/24586, WO 2009/144079, WO 2002/046387, or U.S. Pat. No. 6,768,044. 
     Genes Involved in Abiotic Stress Tolerance 
     In some embodiments, the CRISPR-Cas systems described herein can be used to modify a plant or a cell thereof such that the modified or cell thereof plant contains one or more genes that confer abiotic stress tolerance to the plant. 
     In some embodiments, the modified plant or cell thereof can contain one or more transgenes capable of reducing the expression and/or the activity of poly(ADP-ribose) polymerase (PARP) gene in the plant cells or plants as described in WO 00/04173 or WO/2006/045633. 
     In some embodiments, the modified plant or cell thereof can contain one or more transgenes capable of reducing the expression and/or the activity of the PARG encoding genes of the plants or plants cells, as described e.g. in WO 2004/090140. 
     In some embodiments, the modified plant or cell thereof can contain one or more transgenes coding for a plant-functional enzyme of the nicotineamide adenine dinucleotide salvage synthesis pathway including nicotinamidase, nicotinate phosphoribosyltransferase, nicotinic acid mononucleotide adenyl transferase, nicotinamide adenine dinucleotide synthetase or nicotine amide phosphorybosyltransferase as described e.g. in EP 04077624.7, WO 2006/133827, PCT/EP07/002,433, EP 1999263, or WO 2007/107326. 
     In some embodiments, the modified plant or cell thereof can be modified to contain one or more genes encoding enzyme(s) involved in carbohydrate biosynthesis. Such enzymes include those described in e.g. EP 0571427, WO 95/04826, EP 0719338, WO 96/15248, WO 96/19581, WO 96/27674, WO 97/11188, WO 97/26362, WO 97/32985, WO 97/42328, WO 97/44472, WO 97/45545, WO 98/27212, WO 98/40503, WO99/58688, WO 99/58690, WO 99/58654, WO 00/08184, WO 00/08185, WO 00/08175, WO 00/28052, WO 00/77229, WO 01/12782, WO 01/12826, WO 02/101059, WO 03/071860, WO 2004/056999, WO 2005/030942, WO 2005/030941, WO 2005/095632, WO 2005/095617, WO 2005/095619, WO 2005/095618, WO 2005/123927, WO 2006/018319, WO 2006/103107, WO 2006/108702, WO 2007/009823, WO 00/22140, WO 2006/063862, WO 2006/072603, WO 02/034923, EP 06090134.5, EP 06090228.5, EP 06090227.7, EP 07090007.1, EP 07090009.7, WO 01/14569, WO 02/79410, WO 03/33540, WO 2004/078983, WO 01/19975, WO 95/26407, WO 96/34968, WO 98/20145, WO 99/12950, WO 99/66050, WO 99/53072, U.S. Pat. No. 6,734,341, WO 00/11192, WO 98/22604, WO 98/32326, WO 01/98509, WO 01/98509, WO 2005/002359, U.S. Pat. Nos. 5,824,790, 6,013,861, WO 94/04693, WO 94/09144, WO 94/11520, WO 95/35026 or WO 97/20936. 
     In some embodiments, the modified plant or cell thereof can be modified to contain one or more genes encoding enzyme(s) involved in the production of polyfructose, especially of the inulin and levan-type, as disclosed in EP 0663956, WO 96/01904, WO 96/21023, WO 98/39460, and WO 99/24593. In some embodiments, the modified plant or cell thereof can be modified to contain one or more genes encoding enzyme(s) involved in the production of alpha-1,4-glucans as disclosed in WO 95/31553, US 2002031826, U.S. Pat. Nos. 6,284,479, 5,712,107, WO 97/47806, WO 97/47807, WO 97/47808 and WO 00/14249. In some embodiments, the modified plant or cell thereof can be modified to contain one or more genes encoding enzyme(s) involved in the production of alpha-1,6 branched alpha-1,4-glucans, as disclosed in WO 00/73422, the production of alternan, as disclosed in e.g. WO 00/47727, WO 00/73422, EP 06077301.7, U.S. Pat. No. 5,908,975 and EP 0728213. In some embodiments, the modified plant or cell thereof can be modified to contain one or more genes encoding enzyme(s) involved in the production of hyaluronan, as for example disclosed in WO 2006/032538, WO 2007/039314, WO 2007/039315, WO 2007/039316, JP 2006304779, and WO 2005/012529. 
     In some embodiments, the modified plant or cell thereof can be modified to contain one or more genes that improve drought resistance. For example, WO 2013122472 discloses that the absence or reduced level of functional Ubiquitin Protein Ligase protein (UPL) protein, more specifically, UPL3, leads to a decreased need for water or improved resistance to drought of said plant. In some embodiments, the modified plant or cell thereof can be modified to contain one or more genes that cause the absence or reduced level of functional Ubiquitin Protein Ligase protein (UPL) protein, more specifically, UPL3. In some embodiments, this can include knocking out a UPL gene, such as UPL3. 
     Other examples of transgenic plants with increased drought tolerance are disclosed in, for example, US 2009/0144850, US 2007/0266453, and WO 2002/083911. US2009/0144850 describes a plant displaying a drought tolerance phenotype due to altered expression of a DR02 nucleic acid. US 2007/0266453 describes a plant displaying a drought tolerance phenotype due to altered expression of a DR03 nucleic acid and WO 2002/08391 1 describes a plant having an increased tolerance to drought stress due to a reduced activity of an ABC transporter which is expressed in guard cells. Another example is the work by Kasuga and co-authors (1999), who describe that overexpression of cDNA encoding DREB1 A in transgenic plants activated the expression of many stress tolerance genes under normal growing conditions and resulted in improved tolerance to drought, salt loading, and freezing. However, the expression of DREB1A also resulted in severe growth retardation under normal growing conditions (Kasuga (1999) Nat Biotechnol 17(3) 287-291). In some embodiments, the CRISPR-Cas systems described herein can be used to modify a plant to contain any of these genes associated with drought tolerance. 
     Increasing the Fertility Stage in Plants 
     The non-Class I CRISPR-Cas systems described herein can be used to generate male sterile plants. Hybrid plants typically have advantageous agronomic traits compared to inbred plants. However, for self-pollinating plants, the generation of hybrids can be challenging. In different plant types, genes have been identified which are important for plant fertility, more particularly male fertility. For instance, in maize, at least two genes have been identified which are important in fertility (Amitabh Mohanty International Conference on New Plant Breeding Molecular Technologies Technology Development and Regulation, Oct. 9-10, 2014, Jaipur, India; Svitashev et al. Plant Physiol. 2015 October; 169(2):931-45; Djukanovic et al. Plant J. 2013 December; 76(5):888-99). The methods provided herein can be used to target genes required for male fertility so as to generate male sterile plants which can easily be crossed to generate hybrids. In particular embodiments, the CRISPR-Cas system provided herein is used for targeted mutagenesis of the cytochrome P450-like gene (MS26) or the meganuclease gene (MS45) thereby conferring male sterility to the maize plant. Maize plants which are as such genetically altered can be used in hybrid breeding programs. 
     In particular embodiments, the methods provided herein are used to prolong the fertility stage of a plant such as of a rice plant. For instance, a rice fertility stage gene such as Ehd3 can be targeted in order to generate a mutation in the gene and plantlets can be selected for a prolonged regeneration plant fertility stage (as described in CN 104004782). 
     Generating Genetic Variation 
     The non-class I CRISPR-Cas system described herein can be used to generate genetic variation in a crop of interest. The availability of wild germplasm and genetic variations in crop plants is the key to crop improvement programs, but the available diversity in germplasms from crop plants is limited. The present invention envisages methods for generating a diversity of genetic variations in a germplasm of interest. In this application of the CRISPR-Cas system a library of guide RNAs targeting different locations in the plant genome is provided and is introduced into plant cells together with the Cas-like (e.g. Cas9-like and/or Cas12-like) effector protein(s). In this way a collection of genome-scale point mutations and gene knock-outs can be generated. In particular embodiments, the methods comprise generating a plant part or plant from the cells so obtained and screening the cells for a trait of interest. The target genes can include both coding and non-coding regions. In particular embodiments, the trait is stress tolerance and the method is a method for the generation of stress-tolerant crop varieties 
     Modulating Fruit Ripening 
     The non-Class I CRISPR Cas systems described herein can be used to affect fruit-ripening. Ripening is a normal phase in the maturation process of fruits and vegetables. Only a few days after it starts it renders a fruit or vegetable inedible. This process brings significant losses to both farmers and consumers. In some embodiments the CRISPR-Cas systems described herein can be used to introduce one or more genes or modify one or more endogenous genes such that ethylene production is altered, such as decreased. In some embodiments, CRISPR-Cas systems described herein can be used to introduce one or more genes or modify one or more endogenous genes such that ACC (1-aminocyclopropane-1-carboxylic acid) synthase gene expression or ACC synthase levels are reduced and/or its function is altered, e.g., reduced. ACC synthase is the enzyme responsible for the conversion of S-adenosylmethionine (SAM) to ACC; the second to the last step in ethylene biosynthesis. In some embodiments, the CRISPR-Cas systems described herein can be used to introduce an antisense (“mirror-image”) or truncated copy of the ACC synthase gene into the plant&#39;s genome. 
     In some embodiments reduction of ethylene production can be achieved by introducing an ACC deaminase. In some embodiments, the CRISPR-Cas systems described herein can be used to introduce an ACC deaminase gene into the plant&#39;s genome. An exemplary ACC deaminase gene can be that from  Pseudomonas chlororaphis , a common nonpathogenic soil bacterium. It converts ACC to a different compound thereby reducing the amount of ACC available for ethylene production. 
     In some embodiments reduction of ethylene production can be achieved by introducing a SAM hydrolase. In some embodiments, the CRISPR-Cas systems described herein can introduce a SAM hydrolase gene into the plant&#39;s genome. This approach is similar to ACC deaminase wherein ethylene production is hindered when the amount of its precursor metabolite is reduced; in this case SAM is converted to homoserine. In some embodiments the gene encoding the SAM hydrolase is from  E. coli  T3 bacteriophage. 
     In some embodiments reduction of ethylene production can be achieved by suppression of ACC oxidase. In some embodiments, the CRISPR-Cas systems described herein can be used to introduce one or more genes that result in and suppression of ACC oxidase gene expression. ACC oxidase is the enzyme which catalyzes the oxidation of ACC to ethylene, the last step in the ethylene biosynthetic pathway. Using the methods described herein, down regulation of the ACC oxidase gene results in the suppression of ethylene production, thereby delaying fruit ripening. 
     In particular embodiments, additionally or alternatively to the modifications described above, the methods and CRISPR-Cas systems described herein are used to modify ethylene receptors, so as to interfere with ethylene signals obtained by the fruit. In particular embodiments, the CRISPR-Cas systems described herein are used to introduce and/or modify one or more genes that result in altered, and more specifically decreased or suppressed, expression of the ETR1 gene, encoding an ethylene binding protein is modified. In particular embodiments, additionally or alternatively to the modifications described above, the methods and CRISPR-Cas systems described herein are used to modify expression of the gene encoding Polygalacturonase (PG), which is the enzyme responsible for the breakdown of pectin, the substance that maintains the integrity of plant cell walls. Pectin breakdown occurs at the start of the ripening process resulting in the softening of the fruit. Accordingly, in particular embodiments, the methods and CRISPR-Cas systems described herein are used to introduce a mutation in the PG gene or to suppress activation of the PG gene in order to reduce the amount of PG enzyme produced thereby delaying pectin degradation. 
     Increasing Storage Life of Plants and Plant Products 
     In particular embodiments, the methods and CRISPR-Cas systems described herein are used to modify one or more genes involved in the production of compounds which affect storage life of the plant or plant part. In some embodiments, the modification is in a gene that prevents the accumulation of reducing sugars in potato tubers. Upon high-temperature processing, these reducing sugars react with free amino acids, resulting in brown, bitter-tasting products and elevated levels of acrylamide, which is a potential carcinogen. In particular embodiments, the methods and CRISPR-Cas systems provided herein are used to reduce or inhibit expression of the vacuolar invertase gene (VInv), which encodes a protein that breaks down sucrose to glucose and fructose (Clasen et al. DOI: 10.1111/pbi.12370). 
     Nutritionally Improved Plants 
     In particular embodiments the CRISPR-Cas system described herein is used to produce nutritionally improved agricultural crops. In particular embodiments, the methods provided herein are adapted to generate “functional foods”, i.e. a modified food or food ingredient that may provide a health benefit beyond the traditional nutrients it contains and or “nutraceutical”, i.e. substances that may be considered a food or part of a food and provides health benefits, including the prevention and treatment of disease. In particular embodiments, the nutraceutical is useful in the prevention and/or treatment of one or more of cancer, diabetes, cardiovascular disease, and hypertension. 
     Examples of nutritionally improved crops include, but are not limited to, those discussed in Newell-McGloughlin, Plant Physiology, July 2008, Vol. 147, pp. 939-953). In some embodiments, the CRISPR-Cas systems described herein can be used to modify a plant&#39;s protein quality, content and/or amino acid composition, such as have been described for Bahiagrass (Luciani et al. 2005, Florida Genetics Conference Poster), Canola (Roesler et al., 1997, Plant Physiol 113 75-81), Maize (Cromwell et al, 1967, 1969 J Anim Sci 26 1325-1331, O&#39;Quin et al. 2000 J Anim Sci 78 2144-2149, Yang et al. 2002, Transgenic Res 11 11-20, Young et al. 2004, Plant J 38 910-922), Potato (Yu J and Ao, 1997 Acta Bot Sin 39 329-334; Chakraborty et al. 2000, Proc Natl Acad Sci USA 97 3724-3729; Li et al. 2001) Chin Sci Bull 46 482-484, Rice (Katsube et al. 1999, Plant Physiol 120 1063-1074), Soybean (Dinkins et al. 2001, Rapp 2002, In Vitro Cell Dev Biol Plant 37 742-747), Sweet Potato (Egnin and Prakash 1997, In Vitro Cell Dev Biol 33 52A). 
     In some embodiments, the CRISPR-Cas systems described herein can be used to modify a plant&#39;s essential amino acid content, such as has been described for Canola (Falco et al. 1995, Bio/Technology 13 577-582), Lupin (White et al. 2001, J Sci Food Agric 81 147-154), Maize (Lai and Messing, 2002, Agbios 2008 GM crop database (Mar. 11, 2008)), Potato (Zeh et al. 2001, Plant Physiol 127 792-802), Sorghum (Zhao et al. 2003, Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 413-416), Soybean (Falco et al. 1995 Bio/Technology 13 577-582; Galili et al. 2002 Crit Rev Plant Sci 21 167-204). 
     In some embodiments, the CRISPR-Cas systems described herein can be used to modify a plant&#39;s oils and fatty acids, such as for Canola (Dehesh et al. (1996) Plant J 9 167-172 [PubMed]; Del Vecchio (1996) INFORM International News on Fats, Oils and Related Materials 7 230-243; Roesler et al. (1997) Plant Physiol 113 75-81 [PMC free article] [PubMed]; Froman and Ursin (2002, 2003) Abstracts of Papers of the American Chemical Society 223 U35; James et al. (2003) Am J Clin Nutr 77 1140-1145 [PubMed]; Agbios (2008, above); coton (Chapman et al. (2001). J Am Oil Chem Soc 78 941-947; Liu et al. (2002) J Am Coll Nutr 21 205S-211S [PubMed]; O&#39;Neill (2007) Australian Life Scientist. http://www.biotechnews.com.au/index.php/id;866694817;fp;4;fpid;2 (Jun. 17, 2008), Linseed (Abbadi et al., 2004, Plant Cell 16: 2734-2748), Maize (Young et al., 2004, Plant J 38 910-922), oil palm (Jalani et al. 1997, J Am Oil Chem Soc 74 1451-1455; Parveez, 2003, AgBiotechNet 113 1-8), Rice (Anai et al., 2003, Plant Cell Rep 21 988-992), Soybean (Reddy and Thomas, 1996, Nat Biotechnol 14 639-642; Kinney and Kwolton, 1998, Blackie Academic and Professional, London, pp 193-213), Sunflower (Arcadia, Biosciences 2008). 
     In some embodiments, the CRISPR-Cas systems described herein can be used to modify a plant&#39;s carbohydrate content, such as Fructans described for Chicory (Smeekens (1997) Trends Plant Sci 2 286-287, Sprenger et al. (1997) FEBS Lett 400 355-358, Sévenier et al. (1998) Nat Biotechnol 16 843-846), Maize (Caimi et al. (1996) Plant Physiol 110 355-363), Potato (Hellwege et al., 1997 Plant J 12 1057-1065), Sugar Beet (Smeekens et al. 1997, above), Inulin, such as described for Potato (Hellewege et al. 2000, Proc Natl Acad Sci USA 97 8699-8704), Starch, such as described for Rice (Schwall et al. (2000) Nat Biotechnol 18 551-554, Chiang et al. (2005) Mol Breed 15 125-143), 
     In some embodiments, the CRISPR-Cas systems described herein can be used to modify a plant&#39;s vitamins and carotenoid content, such as described for Canola (Shintani and DellaPenna (1998) Science 282 2098-2100), Maize (Rocheford et al. (2002). J Am Coll Nutr 21 191S-198S, Cahoon et al. (2003) Nat Biotechnol 21 1082-1087, Chen et al. (2003) Proc Natl Acad Sci USA 100 3525-3530), Mustardseed (Shewmaker et al. (1999) Plant J 20 401-412, Potato (Ducreux et al., 2005, J Exp Bot 56 81-89), Rice (Ye et al. (2000) Science 287 303-305, Strawberry (Agius et al. (2003), Nat Biotechnol 21 177-181), Tomato (Rosati et al. (2000) Plant J 24 413-419, Fraser et al. (2001) J Sci Food Agric 81 822-827, Mehta et al. (2002) Nat Biotechnol 20 613-618, Diaz de la Garza et al. (2004) Proc Natl Acad Sci USA 101 13720-13725, Enfissi et al. (2005) Plant Biotechnol J 3 17-27, DellaPenna (2007) Proc Natl Acad Sci USA 104 3675-3676. 
     In some embodiments, the CRISPR-Cas systems described herein can be used to modify a plant&#39;s functional secondary metabolites, such as described for Apple (stilbenes, Szankowski et al. (2003) Plant Cell Rep 22: 141-149), Alfalfa (resveratrol, Hipskind and Paiva (2000) Mol Plant Microbe Interact 13 551-562), Kiwi (resveratrol, Kobayashi et al. (2000) Plant Cell Rep 19 904-910), Maize and Soybean (flavonoids, Yu et al. (2000) Plant Physiol 124 781-794), Potato (anthocyanin and alkaloid glycoside, Lukaszewicz et al. (2004) J Agric Food Chem 52 1526-1533), Rice (flavonoids &amp; resveratrol, Stark-Lorenzen et al. (1997) Plant Cell Rep 16 668-673, Shin et al. (2006) Plant Biotechnol J 4 303-315), Tomato (+resveratrol, chlorogenic acid, flavonoids, stilbene; Rosati et al. (2000) above, Muir et al. (2001) Nature 19 470-474, Niggeweg et al. (2004) Nat Biotechnol 22 746-754, Giovinazzo et al. (2005) Plant Biotechnol J 3 57-69), wheat (caffeic and ferulic acids, resveratrol; United Press International (2002)). 
     In some embodiments, the CRISPR-Cas systems described herein can be used to modify a plant&#39;s mineral availabilities and/or content such as described for Alfalfa (phytase, Austin-Phillips et al. (1999) http://www.molecularfarming.com/nonmedical.html), Lettuce (iron, Goto et al. (2000) Theor Appl Genet 100 658-664), Rice (iron, Lucca et al. (2002) J Am Coll Nutr 21 184S-190S), Maize, Soybean and wheat (phytase, Drakakaki et al. (2005) Plant Mol Biol 59 869-880, Denbow et al. (1998) Poult Sci 77 878-881, Brinch-Pedersen et al. (2000) Mol Breed 6 195-206). 
     In particular embodiments, the value-added trait is related to the envisaged health benefits of the compounds present in the plant. For instance, in particular embodiments, the value-added crop is obtained by applying the methods and CRISPR-Cas systems described herein to modify and/or induce/increase the synthesis of one or more of the following compounds: 
     a) Carotenoids, such as α-Carotene present in carrots which Neutralizes free radicals that may cause damage to cells or β-Carotene present in various fruits and vegetables which neutralizes free radicals;
 
b) Lutein, such as that present in green vegetables which contributes to maintenance of healthy vision;
 
c) Lycopene present in tomato and tomato products, which is believed to reduce the risk of prostate cancer;
 
d) Zeaxanthin, present in citrus and maize, which contributes to maintenance of healthy vision;
 
e) dietary fiber, such as insoluble fiber present in wheat bran which may reduce the risk of breast and/or colon cancer and β-Glucan present in oat, soluble fiber present in  Psyllium  and whole cereal grains which may reduce the risk of cardiovascular disease (CVD)
 
f) Fatty acids, such as ω-β fatty acids which may reduce the risk of CVD and improve mental and visual functions, conjugated linoleic acid, which may improve body composition, may decrease risk of certain cancers and GLA which may reduce inflammation risk of cancer and CVD, may improve body composition;
 
g) Flavonoids, such as Hydroxycinnamates, present in wheat which have Antioxidant-like activities, may reduce risk of degenerative diseases, flavonols, catechins and tannins present in fruits and vegetables which neutralize free radicals and may reduce risk of cancer
 
h) Glucosinolates, indoles, and isothiocyanates, such as Sulforaphane, present in Cruciferous vegetables (broccoli, kale, and horseradish), which neutralize free radicals, may reduce risk of cancer;
 
i) phenolics, such as stilbenes present in grape (may reduce risk of degenerative diseases, heart disease, and cancer, may have longevity effect), caffeic acid and ferulic acid present in vegetables and citrus (have antioxidant-like activities and may reduce risk of degenerative diseases, heart disease, and eye disease), and epicatechin present in cacao (has antioxidant-like activities and may reduce risk of degenerative diseases and heart disease);
 
j) Plant stanols/sterols present in maize, soy, wheat and wooden oils, which may reduce risk of coronary heart disease by lowering blood cholesterol levels;
 
k) Fructans, inulins, fructo-oligosaccharides present in Jerusalem artichoke, shallot, onion powder, which may improve gastrointestinal health;
 
l) saponins present in soybean, which may lower LDL cholesterol;
 
m) soybean protein present in soybean, which may reduce risk of heart disease;
 
n) phytoestrogens such as isoflavones present in soybean, which may reduce menopause symptoms, such as hot flashes, may reduce osteoporosis and CVD and lignans present in flax, rye and vegetables, which may protect against heart disease and some cancers, may lower LDL cholesterol, total cholesterol;
 
o) sulfides and thiols such as diallyl sulphide present in onion, garlic, olive, leek and scallions and Allyl methyl trisulfide, dithiolthiones present in cruciferous vegetables, which may lower LDL cholesterol and helps to maintain healthy immune system; and
 
p) tannins, such as proanthocyanidins, present in cranberry, cocoa, which may improve urinary tract health and may reduce risk of CVD and high blood pressure.
 
     In addition, the methods and CRISPR-Cas systems described herein can be used to modify the protein/starch functionality, shelf life, taste/aesthetics, fiber quality, and allergen, antinutrient, and toxin reduction traits of a plant or a cell thereof. 
     In some embodiments, a method of using the CRISPR-Cas systems described herein to produce plants with nutritional added value can include introducing into a plant cell a gene encoding an enzyme involved in the production of a component of added nutritional value using the CRISPR-Cas system as described herein and regenerating a plant from said plant cell, said plant characterized in an increase expression of said component of added nutritional value. In particular embodiments, the CRISPR-Cas system is used to modify the endogenous synthesis of these compounds indirectly, e.g. by modifying one or more transcription factors that controls the metabolism of this compound. Methods for introducing a gene of interest into a plant cell and/or modifying an endogenous gene using the CRISPR-Cas system are described elsewhere herein. 
     Some specific examples of modifications in plants that have been modified to confer value-added traits are: plants with modified fatty acid metabolism, for example, by transforming a plant with an antisense gene of stearyl-ACP desaturase to increase stearic acid content of the plant. See Knultzon et al., Proc. Natl. Acad. Sci. U.S.A. 89:2624 (1992). Another example involves decreasing phytate content, for example by cloning and then reintroducing DNA associated with the single allele which may be responsible for maize mutants characterized by low levels of phytic acid. See Raboy et al, Maydica 35:383 (1990). 
     Similarly, expression of the maize ( Zea mays ) Tfs C1 and R, which regulate the production of flavonoids in maize aleurone layers under the control of a strong promoter, resulted in a high accumulation rate of anthocyanins in  Arabidopsis  ( Arabidopsis thaliana ), presumably by activating the entire pathway (Bruce et al., 2000, Plant Cell 12:65-80). DellaPenna (Welsch et al., 2007 Annu Rev Plant Biol 57: 711-73 8) found that Tf RAP2.2 and its interacting partner SINAT2 increased carotenogenesis in  Arabidopsis  leaves. Expressing the Tf Dof1 induced the up-regulation of genes encoding enzymes for carbon skeleton production, a marked increase of amino acid content, and a reduction of the Glc level in transgenic  Arabidopsis  (Yanagisawa, 2004 Plant Cell Physiol 45: 386-391), and the DOF Tf AtDof1.1 (OBP2) up-regulated all steps in the glucosinolate biosynthetic pathway in  Arabidopsis  (Skirycz et al., 2006 Plant J 47: 10-24). 
     Reducing Allergen in Plants 
     In particular embodiments, the methods and CRISPR-Cas systems described herein can be used to generate plants with a reduced level of allergens, making them safer for the consumer. In particular embodiments, the methods can include modifying expression of one or more genes responsible for the production of plant allergens. For instance, in particular embodiments, the methods comprise down-regulating expression of a Lol p5 gene in a plant cell, such as a ryegrass plant cell and regenerating a plant therefrom so as to reduce allergenicity of the pollen of said plant (Bhalla et al. 1999, Proc. Natl. Acad. Sci. USA Vol. 96: 11676-11680). 
     Peanut allergies and allergies to legumes generally are a real and serious health concern. The Cas-like (e.g. Cas9-like and/or Cas12-like) effector protein system of the present invention can be used to identify and then edit or silence genes encoding allergenic proteins of such legumes. Without limitation as to such genes and proteins, Nicolaou et al. identifies allergenic proteins in peanuts, soybeans, lentils, peas, lupin, green beans, and mung beans. See, Nicolaou et al., Current Opinion in Allergy and Clinical Immunology 2011; 11(3):222). 
     Further Applications of the CRISPR-Cas Systems in Plants 
     In particular embodiments, the CRISPR system, and preferably the CRISPR-Cas system described herein, can be used for visualization of genetic element dynamics. For example, CRISPR-Cas imaging can visualize either repetitive or non-repetitive genomic sequences, report telomere length change and telomere movements and monitor the dynamics of gene loci throughout the cell cycle (see e.g., Chen et al., Cell, 2013). These methods may also be applied to plants using the CRISPR-Cas systems described herein. 
     In some embodiments, the CRISPR-Cas systems described herein can be used for targeted gene disruption positive-selection screening in vitro and in vivo (see e.g., Malina et al., Genes and Development, 2013). These methods may also be applied to plants. 
     In particular embodiments, fusion of inactive Cas9, Cas12, Cas-like (e.g. Cas9-like and/or Cas12-like) endonucleases with histone-modifying enzymes can introduce custom changes in the complex epigenome (see e.g., Rusk et al., Nature Methods, 2014). These methods may also be applied to plants. 
     In particular embodiments, the CRISPR-Cas systems described herein, can be used to purify a specific portion of the chromatin and identify the associated proteins, thus elucidating their regulatory roles in transcription (e.g., Waldrip et al., Epigenetics, 2014). These methods may also be applied to plants. 
     In particular embodiments, present invention can be used as a therapy for virus removal in plant systems as it is able to cleave both viral DNA and RNA. Previous studies in human systems have demonstrated the success of utilizing CRISPR in targeting the single strand RNA virus, hepatitis C (see e.g., A. Price, et al., Proc. Natl. Acad. Sci, 2015) as well as the double stranded DNA virus, hepatitis B (see e.g., V. Ramanan, et al., Sci. Rep, 2015). These methods may also be adapted for using the CRISPR-Cas system described herein in plants. 
     In particular embodiments, the CRISPR-Cas systems described can be used to alter genome complexity. In further particular embodiment, the CRISPR system, and preferably the CRISPR-Cas system described herein, can be used to disrupt or alter chromosome number and generate haploid plants, which only contain chromosomes from one parent. Such plants can be induced to undergo chromosome duplication and converted into diploid plants containing only homozygous alleles (see e.g., Karimi-Ashtiyani et al., PNAS, 2015; Anton et al., Nucleus, 2014). These methods may also be applied to plants. 
     In particular embodiments, the CRISPR-Cas system described herein, can be used for self-cleavage. In these embodiments, the promotor of the Cas-like (e.g. Cas9-like and/or Cas12-like) enzyme(s) and gRNA can be a constitutive promotor and a second gRNA is introduced in the same transformation cassette, but controlled by an inducible promoter. This second gRNA can be designated to induce site-specific cleavage in the Cas-like (e.g. Cas9-like and/or Cas12-like) gene in order to create a non-functional Cas-like (e.g. Cas9-like and/or Cas12-like) protein(s). In a further particular embodiment, the second gRNA induces cleavage on both ends of the transformation cassette, resulting in the removal of the cassette from the host genome. This system offers a controlled duration of cellular exposure to the Cas enzyme and further minimizes off-target editing. Furthermore, cleavage of both ends of a CRISPR/Cas cassette can be used to generate transgene-free T0 plants with bi-allelic mutations (as described for Cas9 e.g. Moore et al., Nucleic Acids Research, 2014; Schaeffer et al., Plant Science, 2015). The methods of Moore et al. may be applied to the CRISPR-Cas systems described herein. 
     Sugano et al. (Plant Cell Physiol. 2014 March; 55(3):475-81. doi: 10.1093/pcp/pcu014. Epub 2014 Jan. 18) reports the application of CRISPR-Cas9 to targeted mutagenesis in the liverwort  Marchantia polymorpha  L., which has emerged as a model species for studying land plant evolution. The U6 promoter of  M. polymorpha  was identified and cloned to express the gRNA. The target sequence of the gRNA was designed to disrupt the gene encoding auxin response factor 1 (ARF1) in  M. polymorpha . Using  Agrobacterium -mediated transformation, Sugano et al. isolated stable mutants in the gametophyte generation of  M. polymorpha . CRISPR-Cas9-based site-directed mutagenesis in vivo was achieved using either the Cauliflower mosaic virus 35S or  M. polymorpha  EF1α promoter to express Cas9. Isolated mutant individuals showing an auxin-resistant phenotype were not chimeric. Moreover, stable mutants were produced by asexual reproduction of T1 plants. Multiple arf1 alleles were easily established using CRIPSR-Cas9-based targeted mutagenesis. The methods of Sugano et al. may be applied to the CRISPR-Cas systems described herein. 
     Ling et al. (BMC Plant Biology 2014, 14:327) developed a CRISPR-Cas9 binary vector set based on the pGreen or pCAMBIA backbone, as well as a gRNA This toolkit requires no restriction enzymes besides BsaI to generate final constructs harboring maize-codon optimized Cas9 and one or more gRNAs with high efficiency in as little as one cloning step. The toolkit was validated using maize protoplasts, transgenic maize lines, and transgenic  Arabidopsis  lines and was shown to exhibit high efficiency and specificity. Using this toolkit, targeted mutations of three  Arabidopsis  genes were detected in transgenic seedlings of the T1 generation. The multiple-gene mutations could be inherited by the next generation. (guide RNA) module vector set, as a toolkit for multiplex genome editing in plants. The toolbox of Lin et al. may be applied to the CRISPR-Cas systems described herein. 
     Protocols for targeted plant genome editing via CRISPR-Cas systems described herein are also available based on those disclosed for the CRISPR-Cas9 system in volume 1284 of the series Methods in Molecular Biology pp 239-255 10 Feb. 2015. A detailed procedure to design, construct, and evaluate dual gRNAs for plant codon optimized Cas9 (pcoCas9) mediated genome editing using  Arabidopsis thaliana  and  Nicotiana benthamiana  protoplasts s model cellular systems are described. Strategies to apply the CRISPR-Cas9 system to generating targeted genome modifications in whole plants are also discussed. The protocols described in the chapter may be applied to the CRISPR-Cas systems described herein. 
     Ma et al. (Mol Plant. 2015 Aug. 3; 8(8):1274-84. doi: 10.1016/j.molp.2015.04.007) reports robust CRISPR-Cas9 vector system, utilizing a plant codon optimized Cas9 gene, for convenient and high-efficiency multiplex genome editing in monocot and dicot plants. Ma et al. designed PCR-based procedures to rapidly generate multiple sgRNA expression cassettes, which can be assembled into the binary CRISPR-Cas9 vectors in one round of cloning by Golden Gate ligation or Gibson Assembly. With this system, Ma et al. edited 46 target sites in rice with an average 85.4% rate of mutation, mostly in biallelic and homozygous status. Ma et al. provide examples of loss-of-function gene mutations in T0 rice and T1 Arabidopsis plants by simultaneous targeting of multiple (up to eight) members of a gene family, multiple genes in a biosynthetic pathway, or multiple sites in a single gene. The methods of Ma et al. may be applied to the CRISPR-Cas systems described herein. 
     Lowder et al. (Plant Physiol. 2015 Aug. 21. pii: pp. 00636.2015) developed a CRISPR-Cas9 toolbox that allows for multiplex genome editing and transcriptional regulation of expressed, silenced or non-coding genes in plants. This toolbox provides a protocol and reagents to quickly and efficiently assemble functional CRISPR-Cas9 T-DNA constructs for monocots and dicots using Golden Gate and Gateway cloning methods. It comes with a full suite of capabilities, including multiplexed gene editing and transcriptional activation or repression of plant endogenous genes. T-DNA based transformation technology is fundamental to modern plant biotechnology, genetics, molecular biology and physiology. As such, a method for the assembly of Cas9 (WT, nickase or dCas9) and gRNA(s) into a T-DNA destination-vector of interest can be used with the CRISPR-Cas systems described herein. This assembly method is based on both Golden Gate assembly and MultiSite Gateway recombination. Three modules are used for this assembly. The first module is a Cas9 entry vector, which contains promoterless Cas9 or its derivative genes flanked by attL1 and attR5 sites. The second module is a gRNA entry vector which contains entry gRNA expression cassettes flanked by attL5 and attL2 sites. The third module includes attR1-attR2-containing destination T-DNA vectors that provide promoters of choice for Cas9 expression. The toolbox of Lowder et al. may be applied to the CRISPR-Cas systems described herein. 
     Wang et al. (bioRxiv 051342; doi: https://doi.org/10.1101/051342; Epub. May 12, 2016) demonstrate editing of homologous copies of four genes affecting important agronomic traits in hexaploid wheat using a multiplexed gene editing construct with several gRNA-tRNA units under the control of a single promoter. The methods of Wang et al., can be applied to the CRISPR-Cas systems described herein. 
     The CRISPR-Cas systems described herein can be used to modify one or more genes in a tree. The CRISPR-Cas systems described herein can be used for modification of herbaceous systems (see, e.g., Belhaj et al., Plant Methods 9: 39 and Harrison et al., Genes &amp; Development 28: 1859-1872). In some embodiments, the CRISPR Cas systems described herein can be used to target single nucleotide polymorphisms (SNPs) in trees (see, e.g., Zhou et al., New Phytologist, Volume 208, Issue 2, pages 298-301, October 2015). Zhou et al., applied a CRISPR-Cas system in the woody perennial  Populus  using the 4-coumarate:CoA ligase (4CL) gene family as a case study and achieved 100% mutational efficiency for two 4CL genes targeted, with every transformant examined carrying biallelic modifications. The CRISPR-Cas system of Zhou et al., was highly sensitive to single nucleotide polymorphisms (SNPs), as cleavage for a third 4CL gene was abolished due to SNPs in the target sequence. These methods may be applied to the CRISPR-Cas systems described herein. In some embodiments, two 4CL genes, 4CL1 and 4CL2, associated with lignin and flavonoid biosynthesis, respectively can be targeted and modified by the CRISPR-Cas systems described herein. The  Populus tremula  x alba clone 717-1B4 routinely used for transformation is divergent from the genome-sequenced  Populus trichocarpa . Therefore, in some embodiments, the 4CL1 and 4CL2 gRNAs can be designed from the reference genome are interrogated with in-house 717 RNA-Seq data to ensure the absence of SNPs which could limit Cas efficiency. A third gRNA can be designed for 4CL5, a genome duplicate of 4CL1, is also included. The corresponding 717 sequence can harbor one SNP in each allele near/within the PAM, both of which are expected to abolish targeting by the 4CL5-gRNA. All three gRNA target sites are located within the first exon. For 717 transformation, the gRNA can be expressed from the  Medicago  U6.6 promoter, along with a human codon-optimized Cas under control of the CaMV 35S promoter in a binary vector. Transformation with the Cas-only vector can serve as a control. Randomly selected 4CL1 and 4CL2 lines are subjected to amplicon-sequencing. The data can then be processed and biallelic mutations are confirmed in all cases. 
     Modified Insects 
     In some embodiments, the CRISPR-Cas systems described herein can be used to modify one or more polynucleotides in an arthropod such as an insect. In some embodiments, the modification can improve or reduce the insect&#39;s resistance to a pesticide or other environmental chemical, improve an insect&#39;s resistance to a disease or disease causing organism, and/or can reduce an insect&#39;s ability to be a host or vector for a disease causing organism or pathogen. Other beneficial modifications that can be introduced by the CRISPR-Cas systems described herein into an insect will be appreciated in view of this disclosure. 
     Exemplary insects for modification can include, but are not limited to, any of those in the following orders: Apocrita (includes ants, bees, and wasps), Coleoptera (includes beetles and weevils), Lepidoptera (includes butterflies and moths), Trichoptera (includes caddisflies), Blattodea (includes cockroaches), Orthoptera (includes crickets, grasshoppers, and katydids), Diplura (includes diplurans), Odonata (includes dragonflies and damselflies), Dermaptera (includes earwigs), Siphonaptera (includes fleas), Diptera (includes flies), Mantophasmotodea (includes gladiator bugs), Hemiptera (includes hemipterans), Homoptera (includes momopterans), Grylloblatodea (includes icebugs), Neuroptera (includes lacewings), Phthiraptera (includes lice), Manotodea (includes mantids), Ephemoptera (includes mayflies), Meglaoptera (includes megalopterans), Psoceoptera (includes Psocids), Mecoptera (includes scorpionflies), Plecoptera (includes stoneflies), Strepsiptera (includes strepsipterans), Isoptera (includes termites), Thysanoptera (includes thrips), Herteroptera (includes true bugs, e.g. assassin bugs, bat bugs, bedbugs, lace bugs, stink bugs, etc.) Embioptera (includes webspinners), Phasmida (includes walkingsticks), and Apterygota (includes apterygote). 
     Modified Fungi 
     In some embodiments, the CRISPR-Cas systems described herein can be used to modify one or more polynucleotides in a fungus. In particular embodiments, the CRISPR-Cas system described herein can be used for genome editing of yeast cells. Methods for transforming yeast cells which can be used to introduce polynucleotides encoding the CRISPR-Cas system components are well known to the artisan and are reviewed by Kawai et al., 2010, Bioeng Bugs. 2010 November-December; 1(6): 395-403). Non-limiting examples include transformation of yeast cells by lithium acetate treatment (which may further include carrier DNA and PEG treatment), bombardment or by electroporation. Other methods of delivering the CRISPR-Cas systems are described elsewhere herein. 
     As used herein, a “fungal cell” refers to any type of eukaryotic cell within the kingdom of fungi. Phyla within the kingdom of fungi include Ascomycota, Basidiomycota, Blastocladiomycota, Chytridiomycota, Glomeromycota, Microsporidia, and Neocallimastigomycota. Fungal cells may include yeasts, molds, and filamentous fungi. In some embodiments, the fungal cell is a yeast cell. 
     As used herein, the term “yeast cell” refers to any fungal cell within the phyla Ascomycota and Basidiomycota. Yeast cells may include budding yeast cells, fission yeast cells, and mold cells. Without being limited to these organisms, many types of yeast used in laboratory and industrial settings are part of the phylum Ascomycota. In some embodiments, the yeast cell is an  S. cerevisiae, Kluyveromyces marxianus , or  Issatchenkia orientalis  cell. Other yeast cells may include without limitation  Candida  spp. (e.g.,  Candida albicans ),  Yarrowia  spp. (e.g.,  Yarrowia lipolytica ),  Pichia  spp. (e.g.,  Pichia pastoris ),  Kluyveromyces  spp. (e.g.,  Kluyveromyces lactis  and  Kluyveromyces marxianus ),  Neurospora  spp. (e.g.,  Neurospora crassa ),  Fusarium  spp. (e.g.,  Fusarium oxysporum ), and Issatchenkia spp. (e.g., Issatchenkia  orientalis , a.k.a.  Pichia  kudriavzevii and  Candida acidothermophilum ). In some embodiments, the fungal cell is a filamentous fungal cell. As used herein, the term “filamentous fungal cell” refers to any type of fungal cell that grows in filaments, i.e., hyphae or mycelia. Examples of filamentous fungal cells may include without limitation  Aspergillus  spp. (e.g.,  Aspergillus niger ),  Trichoderma  spp. (e.g.,  Trichoderma reesei ),  Rhizopus  spp. (e.g.,  Rhizopus oryzae ), and  Mortierella  spp. (e.g.,  Mortierella isabellina ). 
     In some embodiments, the fungal cell modified is an industrial strain. As used herein, “industrial strain” refers to any strain of fungal cell used in or isolated from an industrial process, e.g., production of a product on a commercial or industrial scale. Industrial strain may refer to a fungal species that is typically used in an industrial process, or it may refer to an isolate of a fungal species that may be also used for non-industrial purposes (e.g., laboratory research). Examples of industrial processes may include fermentation (e.g., in production of food or beverage products), distillation, biofuel production, production of a compound, and production of a polypeptide. Examples of industrial strains may include, without limitation, JAY270 and ATCC4124. 
     In some embodiments, the fungal cell modified is a polyploid cell. As used herein, a “polyploid” cell may refer to any cell whose genome is present in more than one copy. A polyploid cell may refer to a type of cell that is naturally found in a polyploid state, or it may refer to a cell that has been induced to exist in a polyploid state (e.g., through specific regulation, alteration, inactivation, activation, or modification of meiosis, cytokinesis, or DNA replication). A polyploid cell may refer to a cell whose entire genome is polyploid, or it may refer to a cell that is polyploid in a particular genomic locus of interest. Without wishing to be bound to theory, it is thought that the abundance of gRNA may more often be a rate-limiting component in genome engineering of polyploid cells than in haploid cells, and thus the methods using the CRISPR-Cas CRISPRS system described herein may take advantage of using a certain fungal cell type. 
     In some embodiments, the fungal cell modified is a diploid cell. As used herein, a “diploid” cell may refer to any cell whose genome is present in two copies. A diploid cell may refer to a type of cell that is naturally found in a diploid state, or it may refer to a cell that has been induced to exist in a diploid state (e.g., through specific regulation, alteration, inactivation, activation, or modification of meiosis, cytokinesis, or DNA replication). For example, the  S. cerevisiae  strain S228C may be maintained in a haploid or diploid state. A diploid cell may refer to a cell whose entire genome is diploid, or it may refer to a cell that is diploid in a particular genomic locus of interest. In some embodiments, the fungal cell is a haploid cell. As used herein, a “haploid” cell may refer to any cell whose genome is present in one copy. A haploid cell may refer to a type of cell that is naturally found in a haploid state, or it may refer to a cell that has been induced to exist in a haploid state (e.g., through specific regulation, alteration, inactivation, activation, or modification of meiosis, cytokinesis, or DNA replication). For example, the  S. cerevisiae  strain S228C may be maintained in a haploid or diploid state. A haploid cell may refer to a cell whose entire genome is haploid, or it may refer to a cell that is haploid in a particular genomic locus of interest. 
     Modifying Yeast for Biofuel Production 
     The CRISPR-Cas systems described herein can be used bioethanol production by recombinant micro-organisms, such as yeast. to generate biofuel or biopolymers from fermentable sugars and optionally to be able to degrade plant-derived lignocellulose derived from agricultural waste as a source of fermentable sugars. In some embodiments, a CRISPR-Cas system, such as a CRISPR-Cas complex, can be used to introduce foreign genes required for biofuel production into micro-organisms and/or to modify endogenous genes why may interfere with the biofuel synthesis. In some embodiments, a method can include introducing into a micro-organism, such as a yeast, one or more nucleotide sequence encoding enzymes involved in the conversion of pyruvate to ethanol or another product of interest, where the one or more nucleotide sequences can be introduced using a CRISPR-Cas system described herein. In some embodiments, the methods ensure the introduction of one or more polynucleotides that encode enzyme(s) which allows the micro-organism to degrade cellulose, such as a cellulase, where the introduction of the one or more polynucleotides is facilitated by a CRISPR-Cas system described herein. In yet further embodiments, the CRISPR-Cas system described herein is used to modify endogenous metabolic pathways which compete with the biofuel production pathway. 
     In some embodiments, the method can include introducing at least one heterologous nucleic acid or increase expression of at least one endogenous nucleic acid encoding a plant cell wall degrading enzyme, such that said micro-organism is capable of expressing said nucleic acid and of producing and secreting said plant cell wall degrading enzyme; 
     introducing at least one heterologous nucleic acid or increase expression of at least one endogenous nucleic acid encoding an enzyme that converts pyruvate to acetaldehyde optionally combined with at least one heterologous nucleic acid encoding an enzyme that converts acetaldehyde to ethanol such that said host cell is capable of expressing said nucleic acid; and/or 
     modifying at least one nucleic acid encoding for an enzyme in a metabolic pathway in said host cell, wherein said pathway produces a metabolite other than acetaldehyde from pyruvate or ethanol from acetaldehyde, and wherein said modification results in a reduced production of said metabolite, or to introduce at least one nucleic acid encoding for an inhibitor of said enzyme. 
     The CRISPR-Cas system described herein can be used to generate modified yeast having improved xylose or cellobiose utilization. Thus, described herein are modified yeast having improved xylose or cellobiose utilization. 
     In particular embodiments, the CRISPR-Cas system described herein may be applied to select for improved xylose or cellobiose utilizing yeast strains. Error-prone PCR can be used to amplify one (or more) genes involved in the xylose utilization or cellobiose utilization pathways. Examples of genes involved in xylose utilization pathways and cellobiose utilization pathways may include, without limitation, those described in Ha, S. J., et al. (2011) Proc. Natl. Acad. Sci. USA 108(2):504-9 and Galazka, J. M., et al. (2010) Science 330(6000):84-6. Resulting libraries of double-stranded DNA molecules, each comprising a random mutation in such a selected gene could be co-transformed with the components of the CRISPR-Cas system into a yeast strain (for instance S288C) and strains can be selected with enhanced xylose or cellobiose utilization capacity, as described in WO2015138855. 
     The CRISPR-Cas systems described herein can be used to generate improved yeasts strains for use in isoprenoid biosynthesis. 
     Tadas Jakočiūnas et al. described the successful application of a multiplex CRISPR/Cas9 system for genome engineering of up to 5 different genomic loci in one transformation step in baker&#39;s yeast  Saccharomyces cerevisiae  (Metabolic Engineering Volume 28, March 2015, Pages 213-222) resulting in strains with high mevalonate production, a key intermediate for the industrially important isoprenoid biosynthesis pathway. In particular embodiments, the CRISPR-Cas systems described herein may be applied in a multiplex genome engineering method as described herein for identifying additional high producing yeast strains for use in isoprenoid synthesis. 
     The CRISPR-Cas systems described herein can be used to generate lactic acid producing yeasts strains. 
     In another embodiment, successful application of a multiplex CRISPR-Cas system is encompassed. In analogy with Vratislav Stovicek et al. (Metabolic Engineering Communications, Volume 2, December 2015, Pages 13-22), improved lactic acid-producing strains can be designed and obtained in a single transformation event. In a particular embodiment, the CRISPR-Cas system described herein is used for simultaneously inserting the heterologous lactate dehydrogenase gene and disruption of two endogenous genes PDC1 and PDC5 genes. 
     Modified Microorganisms 
     The non-Class I CRISPR-Cas systems described herein can be expressed in and can be used to generate modified micro-organisms. 
     In certain embodiments, the modified micro-organisms can be capable of fatty acid production. In particular embodiments, the CRISPR-Cas systems described herein can be used to generate genetically engineered micro-organisms capable of the production of fatty esters, such as fatty acid methyl esters (“FAME”) and fatty acid ethyl esters (“FAEE”), In some embodiments, host cells can be engineered to produce fatty esters from a carbon source, such as an alcohol, present in the medium, by expression or overexpression of a gene encoding a thioesterase, a gene encoding an acyl-CoA synthase, and a gene encoding an ester synthase. Accordingly, the methods provided herein are used to modify a micro-organisms so as to overexpress or introduce a thioesterase gene, a gene encoding an acyl-CoA synthase, and a gene encoding an ester synthase. In particular embodiments, the thioesterase gene is selected from tesA, ‘tesA, tesB, fatB, fatB2, fatB3, fatA1, or fatA. In particular embodiments, the gene encoding an acyl-CoA synthase is selected from fadDJadK, BH3103, pfl-4354, EAV15023, fadD1, fadD2, RPC_4074, fadDD35, fadDD22, faa39, or an identified gene encoding an enzyme having the same properties. In particular embodiments, the gene encoding an ester synthase is a gene encoding a synthase/acyl-CoA:diacylglycerl acyltransferase from  Simmondsia chinensis, Acinetobacter  sp. ADP,  Alcanivorax borkumensis, Pseudomonas aeruginosa, Fundibacter jadensis, Arabidopsis thaliana , or  Alkaligenes eutrophus , or a variant thereof. 
     In some embodiments, the CRISPR-Cas systems described herein are used to modify a microorganism such that the modified microorganism has decreased expression of at least one of a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis. In particular embodiments one or more of these genes is inactivated, such as by introduction of a mutation. In particular embodiments, the gene encoding an acyl-CoA dehydrogenase is fadE. In particular embodiments, the gene encoding a transcriptional regulator of fatty acid biosynthesis encodes a DNA transcription repressor, for example, fabR. 
     In some embodiments, the CRISPR-Cas systems described herein are used to modify a microorganism such that the modified microorganism has reduced expression of at least one of a gene encoding a pyruvate formate lyase, a gene encoding a lactate dehydrogenase, or both. In particular embodiments, the gene encoding a pyruvate formate lyase is pflB. In particular embodiments, the gene encoding a lactate dehydrogenase is IdhA. In particular embodiments, one or more of these genes is inactivated, such as by introduction of a mutation therein. 
     In particular embodiments, the micro-organism modified is selected from the genus  Escherichia, Bacillus, Lactobacillus, Rhodococcus, Synechococcus, Synechoystis, Pseudomonas, Aspergillus, Trichoderma, Neurospora, Fusarium, Humicola, Rhizomucor, Kluyveromyces, Pichia, Mucor, Myceliophtora, Penicillium, Phanerochaete, Pleurotus, Trametes, Chrysosporium, Saccharomyces, Stenotrophamonas, Schizosaccharomyces, Yarrowia , or  Streptomyces.    
     The CRISPR-Cas system described herein can be used to generate modified micro-organisms capable of organic acid production. Thus, described herein are modified micro-organisms capable of producing organic acids. 
     The CRISPR-Cas systems provided herein are further used to engineer micro-organisms capable of organic acid production, more particularly from pentose or hexose sugars. In particular embodiments, the methods comprise introducing into a micro-organism an exogenous LDH gene. In particular embodiments, the organic acid production in said micro-organisms is additionally or alternatively increased by using the CRISPR-Cas systems described herein to inactivate endogenous genes encoding proteins involved in an endogenous metabolic pathway which produces a metabolite other than the organic acid of interest and/or wherein the endogenous metabolic pathway consumes the organic acid. In particular embodiments, the modification ensures that the production of the metabolite other than the organic acid of interest is reduced. In some embodiments, the CRISPR-Cas systems described herein can introduce at least one engineered gene deletion and/or inactivation of an endogenous pathway in which the organic acid is consumed or a gene encoding a product involved in an endogenous pathway which produces a metabolite other than the organic acid of interest. In particular embodiments, the CRISPR-Cas systems described herein introduce at least one engineered gene deletion or inactivation is in one or more gene encoding an enzyme selected from the group consisting of pyruvate decarboxylase (pdc), fumarate reductase, alcohol dehydrogenase (adh), acetaldehyde dehydrogenase, phosphoenolpyruvate carboxylase (ppc), D-lactate dehydrogenase (d-ldh), L-lactate dehydrogenase (l-ldh), lactate 2-monooxygenase. In further embodiments the at least one engineered gene deletion and/or inactivation is in an endogenous gene encoding pyruvate decarboxylase (pdc). 
     In further embodiments, the CRISPR-Cas system is used to modify a micro-organism to produce lactic acid by introducing at least one engineered gene deletion and/or inactivation, which can be an endogenous gene encoding lactate dehydrogenase. In some embodiments, the micro-organism comprises at least one engineered gene deletion or inactivation of an endogenous gene encoding a cytochrome-dependent lactate dehydrogenase, such as a cytochrome B2-dependent L-lactate dehydrogenase. 
     The following additional references can be adapted and applied though the CRISPR-Cas systems described herein to produce various modified micro-organisms: PCT Publications WO2016/099887; WO2016/025131; WO2016/073433; WO2017/066175; WO2017/100158; WO 2017/105991; WO2017/106414; WO2016/100272; WO2016/100571; WO 2016/100568; WO 2016/100562; and WO 2017/019867. 
     Delivery of the CRISPR-Cas Systems 
     Formulations 
     General Discussion 
     One or more of the polypeptides, polynucleotides, CRISPR-Cas complexes, vectors, cells, virus particles, nanoparticles, other delivery particles, and combinations thereof described herein can be included in a formulation, such as a pharmaceutical formulation. Thus, also within the scope of this disclosure are formulations, such as pharmaceutical formulations, containing one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein. One or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein can be provided to a subject in need thereof alone or as an active ingredient, such as in a pharmaceutical formulation. As such, also described herein are pharmaceutical formulations containing an amount of one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein. In some embodiments, the pharmaceutical formulation can contain an effective amount of the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein. The pharmaceutical formulations described herein can be administered to a subject in need thereof. 
     Also described herein are pharmaceutical formulations that can contain an amount, effective amount, and/or least effective amount, and/or therapeutically effective amount of one or more compounds, molecules, compositions, vectors, vector systems, cells, or a combination thereof (which are also referred to as the primary active agent or ingredient elsewhere herein) described in greater detail elsewhere herein a pharmaceutically acceptable carrier. When present, the compound can optionally be present in the pharmaceutical formulation as a pharmaceutically acceptable salt. In some embodiments, the pharmaceutical formulation can include, such as an active ingredient, a CRISPR-Cas system or component thereof described herein and/or a modified cell, and optionally one or more auxiliary active ingredients or agents. 
     The pharmaceutical formulations described herein can be administered via any suitable method or route to a subject in need thereof. Suitable administration routes can include, but are not limited to auricular (otic), buccal, conjunctival, cutaneous, dental, electro-osmosis, endocervical, endosinusial, endotracheal, enteral, epidural, extra-amniotic, extracorporeal, hemodialysis, infiltration, interstitial, intra abdominal, intra-amniotic, intra-arterial, intra-articular, intrabiliary, intrabronchial, intrabursal, intracardiac, intracartilaginous, intracaudal, intracavernous, intracavitary, intracerebral, intracisternal, intracorneal, intracoronal (dental), intracoronary, intracorporus cavernosum, intradermal, intradiscal, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intragingival, intraileal, intralesional, intraluminal, intralymphatic, intramedullary, intrameningeal, intramuscular, intraocular, intraovarian, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrasinal, intraspinal, intrasynovial, intratendinous, intratesticular, intrathecal, intrathoracic, intratubular, intratumor, intratym panic, intrauterine, intravascular, intravenous, intravenous bolus, intravenous drip, intraventricular, intravesical, intravitreal, iontophoresis, irrigation, laryngeal, nasal, nasogastric, occlusive dressing technique, ophthalmic, oral, oropharyngeal, other, parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, respiratory (inhalation), retrobulbar, soft tissue, subarachnoid, subconjunctival, subcutaneous, sublingual, submucosal, topical, transdermal, transmucosal, transplacental, transtracheal, transtympanic, ureteral, urethral, and/or vaginal administration, and/or any combination of the above administration routes, which typically depends on the disease to be treated and/or the active ingredient(s). 
     Where appropriate, compounds, molecules, compositions, vectors, vector systems, cells, or a combination thereof described in greater detail elsewhere herein can be provided to a subject in need thereof as an ingredient, such as an active ingredient or agent, in a pharmaceutical formulation. As such, also described are pharmaceutical formulations containing one or more of the compounds and salts thereof, or pharmaceutically acceptable salts thereof described herein. Suitable salts include, hydrobromide, iodide, nitrate, bisulfate, phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate, napthalenesulfonate, propionate, malonate, mandelate, malate, phthalate, and pamoate. 
     In some embodiments, the subject to which the CRISPR-Cas systems, components thereof, modified cells, vectors, etc. described herein can have or be suspected of having a disease, such as a genetic or epigenetic disease. Exemplary diseases are described in greater detail elsewhere herein. As used herein, “agent” refers to any substance, compound, molecule, and the like, which can be biologically active or otherwise can induce a biological and/or physiological effect on a subject to which it is administered to. An agent can be a primary active agent, or in other words, the component(s) of a composition to which the whole or part of the effect of the composition is attributed. An agent can be a secondary agent, or in other words, the component(s) of a composition to which an additional part and/or other effect of the composition is attributed. 
     In certain embodiments, the nucleic acid component (e.g. gRNA, modified gRNA, sgRNA, modified sgRNA, the inactivated AAV-CRISPR enzyme (with or without functional domains), and the binding protein with one or more functional domains, may each individually be comprised in a composition or pharmaceutical formulation as described herein and administered to a host individually or collectively. Alternatively, these components may be provided in a single composition for administration to a host, e.g., the AAV-CRISPR enzyme can deliver the RNA or guide or sgRNA or modified sgRNA and/or other components of the CRISPR system. Administration to a host may be performed via viral vectors, advantageously using the AAV-CRISPR enzyme as the delivery vehicle, although other vehicles can be used to deliver components other than the enzyme of the CRISPR system, and such viral vectors can be, for example, lentiviral vector, adenoviral vector, AAV vector. Several variations are appropriate to elicit a genomic locus event, including DNA cleavage, gene activation, or gene deactivation. Using the provided compositions, the person skilled in the art can advantageously and specifically target single or multiple loci with the same or different functional domains to elicit one or more genomic locus events. The compositions may be applied in a wide variety of methods for screening in libraries in cells and functional modeling in vivo (e.g., gene activation of lincRNA and identification of function; gain-of-function modeling; loss-of-function modeling; the use the compositions of the invention to establish cell lines and transgenic animals for optimization and screening purposes as described elsewhere herein). 
     Pharmaceutically Acceptable Carriers and Auxiliary Ingredients and Agents 
     In certain embodiments, the pharmaceutical formulation containing an amount of one or more of the polypeptides, polynucleotides, CRISPR-Cas complexes, vectors, cells, virus particles, nanoparticles, other delivery particles, and combinations thereof described herein can further include a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxy methylcellulose, and polyvinyl pyrrolidone, which do not deleteriously react with the active composition. 
     The pharmaceutical formulations can be sterilized, and if desired, mixed with auxiliary agents, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances, and the like which do not deleteriously react with the active composition. 
     In addition to an amount of one or more of the polypeptides, polynucleotides, CRISPR-Cas complexes, vectors, cells, virus particles, nanoparticles, other delivery particles, and combinations thereof described herein, the pharmaceutical formulation can also include an effective amount of an auxiliary active agent, including but not limited to, polynucleotides, amino acids, peptides, polypeptides, antibodies, aptamers, ribozymes, hormones, immunomodulators, antipyretics, anxiolytics, antipsychotics, analgesics, antispasmodics, anti-inflammatories, anti-histamines, anti-infectives, chemotherapeutics, and combinations thereof. 
     Where co-therapies or multiple pharmaceutical formulations are to be delivered to a subject and/or a cell, the different therapies or formulations can be administered sequentially or simultaneously. Sequential administration is administration where an appreciable amount of time occurs between administrations, such as more than about 15, 20, 30, 45, 60 minutes or more. The time between administrations in sequential administration can be on the order of hours, days, months, or even years, depending on the active agent present in each administration. Simultaneous administration refers to administration of two or more formulations at the same time or substantially at the same time (e.g. within seconds or just a few minutes apart), where the intent is that the formulations be administered together at the same time. 
     Suitable hormones include, but are not limited to, amino-acid derived hormones (e.g. melatonin and thyroxine), small peptide hormones and protein hormones (e.g. thyrotropin-releasing hormone, vasopressin, insulin, growth hormone, luteinizing hormone, follicle-stimulating hormone, and thyroid-stimulating hormone), eicosanoids (e.g. arachidonic acid, lipoxins, and prostaglandins), and steroid hormones (e.g. estradiol, testosterone, tetrahydro testosteron Cortisol). Suitable immunomodulators include, but are not limited to, prednisone, azathioprine, 6-MP, cyclosporine, tacrolimus, methotrexate, interleukins (e.g. IL-2, IL-7, and IL-12), cytokines (e.g. interferons (e.g. IFN-α, IFN-β, IFN-ε, IFN-K, IFN-ω, and IFN-γ), granulocyte colony-stimulating factor, and imiquimod), chemokines (e.g. CCL3, CCL26 and CXCL7), cytosine phosphate-guanosine, oligodeoxynucleotides, glucans, antibodies, and aptamers). 
     Suitable antipyretics include, but are not limited to, non-steroidal anti-inflammants (e.g. ibuprofen, naproxen, ketoprofen, and nimesulide), aspirin and related salicylates (e.g. choline salicylate, magnesium salicylae, and sodium salicaylate), paracetamol/acetaminophen, metamizole, nabumetone, phenazone, and quinine. 
     Suitable anxiolytics include, but are not limited to, benzodiazepines (e.g. alprazolam, bromazepam, chlordiazepoxide, clonazepam, clorazepate, diazepam, flurazepam, lorazepam, oxazepam, temazepam, triazolam, and tofisopam), serotenergic antidepressants (e.g. selective serotonin reuptake inhibitors, tricyclic antidepressants, and monoamine oxidase inhibitors), mebicar, afobazole, selank, bromantane, emoxypine, azapirones, barbiturates, hydroxyzine, pregabalin, validol, and beta blockers. 
     Suitable antipsychotics include, but are not limited to, benperidol, bromoperidol, droperidol, haloperidol, moperone, pipaperone, timiperone, fluspirilene, penfluridol, pimozide, acepromazine, chlorpromazine, cyamemazine, dizyrazine, fluphenazine, levomepromazine, mesoridazine, perazine, pericyazine, perphenazine, pipotiazine, prochlorperazine, promazine, promethazine, prothipendyl, thioproperazine, thioridazine, trifluoperazine, triflupromazine, chlorprothixene, clopenthixol, flupentixol, tiotixene, zuclopenthixol, clotiapine, loxapine, prothipendyl, carpipramine, clocapramine, molindone, mosapramine, sulpiride, veralipride, amisulpride, amoxapine, aripiprazole, asenapine, clozapine, blonanserin, iloperidone, lurasidone, melperone, nemonapride, olanzapine, paliperidone, perospirone, quetiapine, remoxipride, risperidone, sertindole, trimipramine, ziprasidone, zotepine, alstonie, befeprunox, bitopertin, brexpiprazole, cannabidiol, cariprazine, pimavanserin, pomaglumetad methionil, vabicaserin, xanomeline, and zicronapine. 
     Suitable analgesics include, but are not limited to, paracetamol/acetaminophen, nonsteroidal anti-inflammants (e.g. ibuprofen, naproxen, ketoprofen, and nimesulide), COX-2 inhibitors (e.g. rofecoxib, celecoxib, and etoricoxib), opioids (e.g. morphine, codeine, oxycodone, hydrocodone, dihydromorphine, pethidine, buprenorphine), tramadol, norepinephrine, flupiretine, nefopam, orphenadrine, pregabalin, gabapentin, cyclobenzaprine, scopolamine, methadone, ketobemidone, piritramide, and aspirin and related salicylates (e.g. choline salicylate, magnesium salicylate, and sodium salicylate). 
     Suitable antispasmodics include, but are not limited to, mebeverine, papverine, cyclobenzaprine, carisoprodol, orphenadrine, tizanidine, metaxalone, methodcarbamol, chlorzoxazone, baclofen, dantrolene, baclofen, tizanidine, and dantrolene. Suitable anti-inflammatories include, but are not limited to, prednisone, non-steroidal anti-inflammants (e.g. ibuprofen, naproxen, ketoprofen, and nimesulide), COX-2 inhibitors (e.g. rofecoxib, celecoxib, and etoricoxib), and immune selective anti-inflammatory derivatives (e.g. submandibular gland peptide-T and its derivatives). 
     Suitable anti-histamines include, but are not limited to, H1-receptor antagonists (e.g. acrivastine, azelastine, bilastine, brompheniramine, buclizine, bromodiphenhydramine, carbinoxamine, cetirizine, chlorpromazine, cyclizine, chlorpheniramine, clemastine, cyproheptadine, desloratadine, dexbromapheniramine, dexchlorpheniramine, dimenhydrinate, dimetindene, diphenhydramine, doxylamine, ebasine, embramine, fexofenadine, hydroxyzine, levocetirzine, loratadine, meclozine, mirtazapine, olopatadine, orphenadrine, phenindamine, pheniramine, phenyltoloxamine, promethazine, pyrilamine, quetiapine, rupatadine, tripelennamine, and triprolidine), H2-receptor antagonists (e.g. cimetidine, famotidine, lafutidine, nizatidine, rafitidine, and roxatidine), tritoqualine, catechin, cromoglicate, nedocromil, and p2-adrenergic agonists. 
     Suitable anti-infectives include, but are not limited to, amebicides (e.g. nitazoxanide, paromomycin, metronidazole, tinidazole, chloroquine, miltefosine, amphotericin b, and iodoquinol), aminoglycosides (e.g. paromomycin, tobramycin, gentamicin, amikacin, kanamycin, and neomycin), anthelmintics (e.g. pyrantel, mebendazole, ivermectin, praziquantel, abendazole, thiabendazole, oxamniquine), antifungals (e.g. azole antifungals (e.g. itraconazole, fluconazole, posaconazole, ketoconazole, clotrimazole, miconazole, and voriconazole), echinocandins (e.g. caspofungin, anidulafungin, and micafungin), griseofulvin, terbinafine, flucytosine, and polyenes (e.g. nystatin, and amphotericin b), antimalarial agents (e.g. pyrimethamine/sulfadoxine, artemether/lumefantrine, atovaquone/proquanil, quinine, hydroxychloroquine, mefloquine, chloroquine, doxycycline, pyrimethamine, and halofantrine), antituberculosis agents (e.g. aminosalicylates (e.g. aminosalicylic acid), isoniazid/rifampin, isoniazid/pyrazinamide/rifampin, bedaquiline, isoniazid, ethambutol, rifampin, rifabutin, rifapentine, capreomycin, and cycloserine), antivirals (e.g. amantadine, rimantadine, abacavir/lamivudine, emtricitabine/tenofovir, cobicistat/elvitegravir/emtricitabine/tenofovir, efavirenz/emtricitabine/tenofovir, avacavir/lamivudine/zidovudine, lamivudine/zidovudine, emtricitabine/tenofovir, emtricitabine/opinavir/ritonavir/tenofovir, interferon alfa-2v/ribavirin, peginterferon alfa-2b, maraviroc, raltegravir, dolutegravir, enfuvirtide, foscarnet, fomivirsen, oseltamivir, zanamivir, nevirapine, efavirenz, etravirine, rilpivirine, delaviridine, nevirapine, entecavir, lamivudine, adefovir, sofosbuvir, didanosine, tenofovir, avacivr, zidovudine, stavudine, emtricitabine, xalcitabine, telbivudine, simeprevir, boceprevir, telaprevir, lopinavir/ritonavir, fosamprenvir, dranuavir, ritonavir, tipranavir, atazanavir, nelfinavir, amprenavir, indinavir, sawuinavir, ribavirin, valcyclovir, acyclovir, famciclovir, ganciclovir, and valganciclovir), carbapenems (e.g. doripenem, meropenem, ertapenem, and cilastatin/imipenem), cephalosporins (e.g. cefadroxil, cephradine, cefazolin, cephalexin, cefepime, ceflaroline, loracarbef, cefotetan, cefuroxime, cefprozil, loracarbef, cefoxitin, cefaclor, ceftibuten, ceftriaxone, cefotaxime, cefpodoxime, cefdinir, cefixime, cefditoren, cefizoxime, and ceftazidime), glycopeptide antibiotics (e.g. vancomycin, dalbavancin, oritavancin, and telvancin), glycylcyclines (e.g. tigecycline), leprostatics (e.g. clofazimine and thalidomide), lincomycin and derivatives thereof (e.g. clindamycin and lincomycin), macrolides and derivatives thereof (e.g. telithromycin, fidaxomicin, erythromycin, azithromycin, clarithromycin, dirithromycin, and troleandomycin), linezolid, sulfamethoxazole/trimethoprim, rifaximin, chloramphenicol, fosfomycin, metronidazole, aztreonam, bacitracin, penicillins (amoxicillin, ampicillin, bacampicillin, carbenicillin, piperacillin, ticarcillin, amoxicillin/clavulanate, ampicillin/sulbactam, piperacillin/tazobactam, clavulanate/ticarcillin, penicillin, procaine penicillin, oxaxillin, dicloxacillin, and nafcillin), quinolones (e.g. lomefloxacin, norfloxacin, ofloxacin, qatifloxacin, moxifloxacin, ciprofloxacin, levofloxacin, gemifloxacin, moxifloxacin, cinoxacin, nalidixic acid, enoxacin, grepafloxacin, gatifloxacin, trovafloxacin, and sparfloxacin), sulfonamides (e.g. sulfamethoxazole/trimethoprim, sulfasalazine, and sulfasoxazole), tetracyclines (e.g. doxycycline, demeclocycline, minocycline, doxycycline/salicyclic acid, doxycycline/omega-3 polyunsaturated fatty acids, and tetracycline), and urinary anti-infectives (e.g. nitrofurantoin, methenamine, fosfomycin, cinoxacin, nalidixic acid, trimethoprim, and methylene blue). 
     Suitable chemotherapeutics include, but are not limited to, paclitaxel, brentuximab vedotin, doxorubicin, 5-FU (fluorouracil), everolimus, pemetrexed, melphalan, pamidronate, anastrozole, exemestane, nelarabine, ofatumumab, bevacizumab, belinostat, tositumomab, carmustine, bleomycin, bosutinib, busulfan, alemtuzumab, irinotecan, vandetanib, bicalutamide, lomustine, daunorubicin, clofarabine, cabozantinib, dactinomycin, ramucirumab, cytarabine, Cytoxan, cyclophosphamide, decitabine, dexamethasone, docetaxel, hydroxyurea, decarbazine, leuprolide, epirubicin, oxaliplatin, asparaginase, estramustine, cetuximab, vismodegib, asparginase  Erwinia chrysanthemi , amifostine, etoposide, flutamide, toremifene, fulvestrant, letrozole, degarelix, pralatrexate, methotrexate, floxuridine, obinutuzumab, gemcitabine, afatinib, imatinib mesylatem, carmustine, eribulin, trastuzumab, altretamine, topotecan, ponatinib, idarubicin, ifosfamide, ibrutinib, axitinib, interferon alfa-2a, gefitinib, romidepsin, ixabepilone, ruxolitinib, cabazitaxel, ado-trastuzumab emtansine, carfilzomib, chlorambucil, sargramostim, cladribine, mitotane, vincristine, procarbazine, megestrol, trametinib, mesna, strontium-89 chloride, mechlorethamine, mitomycin, busulfan, gemtuzumab ozogamicin, vinorelbine, filgrastim, pegfilgrastim, sorafenib, nilutamide, pentostatin, tamoxifen, mitoxantrone, pegaspargase, denileukin diftitox, alitretinoin, carboplatin, pertuzumab, cisplatin, pomalidomide, prednisone, aldesleukin, mercaptopurine, zoledronic acid, lenalidomide, rituximab, octretide, dasatinib, regorafenib, histrelin, sunitinib, siltuximab, omacetaxine, thioguanine (tioguanine), dabrafenib, erlotinib, bexarotene, temozolomide, thiotepa, thalidomide, BCG, temsirolimus, bendamustine hydrochloride, triptorelin, aresnic trioxide, lapatinib, valrubicin, panitumumab, vinblastine, bortezomib, tretinoin, azacitidine, pazopanib, teniposide, leucovorin, crizotinib, capecitabine, enzalutamide, ipilimumab, goserelin, vorinostat, idelalisib, ceritinib, abiraterone, epothilone, tafluposide, azathioprine, doxifluridine, vindesine, and all-trans retinoic acid. 
     Effective Amounts 
     In some embodiments, the amount of the primary active agent and/or optional auxiliary active agent can be an effective amount, least effective amount, and/or therapeutically effective amount. The effective amount, least effective amount, and/or therapeutically effective amount of the primary and optional auxiliary active agent described elsewhere herein contained in the pharmaceutical formulation can range from about 0 to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 pg, ng, μg, mg, g, pL, nL, μL, mL, or L or be any numerical value with any of these ranges. In some embodiments, the effective amount, least effective amount, and/or therapeutically effective amount can be an effective concentration, least effective concentration, and/or therapeutically effective concentration, which can each range from about 0 to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 pM, nM, μM, mM, or M or be any numerical value with any of these ranges. 
     In other embodiments, the effective amount, least effective amount, and/or therapeutically effective amount of the auxiliary active agent can range from about 0 to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 IU or be any numerical value with any of these ranges. 
     In some embodiments, a primary active agent can be present in the pharmaceutical formulation can range from about 0 to 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.9, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9% w/w, v/v, or w/v of the pharmaceutical formulation. 
     In some embodiments, the auxiliary active agent, when optionally present, can range from about 0 to 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.9, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9% w/w, v/v, or w/v of the pharmaceutical formulation. 
     In some embodiments where a cell population is delivered, the effective amount of cells can range from about 1×10 1 /mL to 1×10 20 /mL or more, such as about 1×10′/mL, 1×10 2 /mL, 1×10 3 /mL, 1×10 4 /mL, 1×10 5 /mL, 1×10 6 /mL, 1×10 7 /mL, 1×10 8 /mL, 1×10 9 /mL, 1×10 19 /mL, 1×10″/mL, 1×10 12 /mL, 1×10 13 /mL, 1×10 14 /mL, 1×10 15 /mL, 1×10 16 /mL, 1×10 17 /mL, 1×10 18 /mL, 1×10 19 /mL, to/or about 1×10 20 /mL. 
     In some embodiments, the amount or effective amount, particularly where an infective particle is being delivered (e.g. a virus particle having a CRISPR-Cas system or component thereof as a cargo), the effective amount of virus particles can be expressed as a titer (plaque forming units per unit of volume) or as a MOI (multiplicity of infection). In some embodiments, the effective amount can be 1×10 1  particles per pL, nL, μL, mL, or L to 1×10 20 /particles per pL, nL, μL, mL, or L or more, such as about 1×10 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 , 1×10 18 , 1×10 19 , to/or about 1×10 20  particles per pL, nL, μL, mL, or L. In some embodiments, the effective titer can be about 1×10 1  transforming units per pL, nL, μL, mL, or L to 1×10 20 /transforming units per pL, nL, μL, mL, or L or more, such as about 1×10 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 , 1×10 18 , 1×10 19 , to/or about 1×10 20  transforming units per pL, nL, μL, mL, or L. In some embodiments, the MOI of the pharmaceutical formulation can range from about 0.1 to 10 or more, such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10 or more. 
     In some embodiments, the amount or effective amount of the one or more of the polypeptides, polynucleotides, CRISPR-Cas complexes, vectors, cells, virus particles, nanoparticles, other delivery particles, and combinations thereof described herein contained in the pharmaceutical formulation can range from about 1 μg/kg to about 10 mg/kg based upon the bodyweight of the subject in need thereof or average bodyweight of the specific patient population to which the pharmaceutical formulation can be administered. The amount of the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein in the pharmaceutical formulation can range from about 1 μg to about 10 g, from about 10 nL to about 10 ml. In certain embodiments where the pharmaceutical formulation contains one or more cells, the amount can range from about 1 cell to 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 1 , 1×10 8 , 1×10 9 , 1×10 10  or more cells. In certain embodiments where the pharmaceutical formulation contains one or more cells, the amount can range from about 1 cell to 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10  or more cells per nL, μL, mL, or L. 
     In embodiments where there is an auxiliary active agent contained in the pharmaceutical formulation, the effective amount of the auxiliary active agent will vary depending on the auxiliary active agent. 
     When optionally present in the pharmaceutical formulation, the auxiliary active agent can be included in the pharmaceutical formulation or can exist as a stand-alone compound or pharmaceutical formulation that can be administered contemporaneously or sequentially with the compound, derivative thereof, or pharmaceutical formulation thereof. In yet other embodiments, the effective amount of the auxiliary active agent can range from about 0 to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9% w/w, v/v, or w/v of the total auxiliary active agent pharmaceutical formulation. In additional embodiments, the effective amount of the auxiliary active agent can range from about 0 to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9% w/w, v/v, or w/v of the total pharmaceutical formulation. 
     Dosage Forms 
     In some embodiments, the pharmaceutical formulations described herein may be in a dosage form. The dosage forms can be adapted for administration by any appropriate route. Appropriate routes include, but are not limited to, oral (including buccal or sublingual), rectal, epidural, intracranial, intraocular, inhaled, intranasal, topical (including buccal, sublingual, or transdermal), vaginal, intraurethral, parenteral, intracranial, subcutaneous, intramuscular, intravenous, intraperitoneal, intradermal, intraosseous, intracardiac, intraarticular, intracavernous, intrathecal, intravitreal, intracerebral, gingival, subgingival, intracerebroventricular, and intradermal. Such formulations may be prepared by any method known in the art. 
     Dosage forms adapted for oral administration can be discrete dosage units such as capsules, pellets or tablets, powders or granules, solutions, or suspensions in aqueous or nonaqueous liquids; edible foams or whips, or in oil-in-water liquid emulsions or water-in-oil liquid emulsions. In some embodiments, the pharmaceutical formulations adapted for oral administration also include one or more agents which flavor, preserve, color, or help disperse the pharmaceutical formulation. Dosage forms prepared for oral administration can also be in the form of a liquid solution that can be delivered as foam, spray, or liquid solution. In some embodiments, the oral dosage form can contain about 1 ng to 1000 g of a pharmaceutical formulation containing a therapeutically effective amount or an appropriate fraction thereof of the targeted effector fusion protein and/or complex thereof or composition containing the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein. The oral dosage form can be administered to a subject in need thereof. 
     Where appropriate, the dosage forms described herein can be microencapsulated. 
     The dosage form can also be prepared to prolong or sustain the release of any ingredient. In some embodiments, the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein can be the ingredient whose release is delayed. In other embodiments, the release of an optionally included auxiliary ingredient is delayed. Suitable methods for delaying the release of an ingredient include, but are not limited to, coating or embedding the ingredients in material in polymers, wax, gels, and the like. Delayed release dosage formulations can be prepared as described in standard references such as “Pharmaceutical dosage form tablets,” eds. Liberman et. al. (New York, Marcel Dekker, Inc., 1989), “Remington—The science and practice of pharmacy”, 20th ed., Lippincott Williams &amp; Wilkins, Baltimore, Md., 2000, and “Pharmaceutical dosage forms and drug delivery systems”, 6th Edition, Ansel et al., (Media, Pa.: Williams and Wilkins, 1995). These references provide information on excipients, materials, equipment, and processes for preparing tablets and capsules and delayed release dosage forms of tablets and pellets, capsules, and granules. The delayed release can be anywhere from about an hour to about 3 months or more. 
     Examples of suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic acid polymers and copolymers, and methacrylic resins that are commercially available under the trade name EUDRAGIT® (Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides. 
     Coatings may be formed with a different ratio of water-soluble polymer, water insoluble polymers, and/or pH dependent polymers, with or without water insoluble/water soluble non polymeric excipient, to produce the desired release profile. The coating is either performed on the dosage form (matrix or simple) which includes, but is not limited to, tablets (compressed with or without coated beads), capsules (with or without coated beads), beads, particle compositions, “ingredient as is” formulated as, but not limited to, suspension form or as a sprinkle dosage form. 
     Dosage forms adapted for topical administration can be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, or oils. In some embodiments for treatments of the eye or other external tissues, for example the mouth or the skin, the pharmaceutical formulations are applied as a topical ointment or cream. When formulated in an ointment, the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein can be formulated with a paraffinic or water-miscible ointment base. In some embodiments, the active ingredient can be formulated in a cream with an oil-in-water cream base or a water-in-oil base. Dosage forms adapted for topical administration in the mouth include lozenges, pastilles, and mouth washes. 
     Dosage forms adapted for nasal or inhalation administration include aerosols, solutions, suspension drops, gels, or dry powders. In some embodiments, the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein is contained in a dosage form adapted for inhalation is in a particle-size-reduced form that is obtained or obtainable by micronization. In some embodiments, the particle size of the size reduced (e.g. micronized) compound or salt or solvate thereof, is defined by a D50 value of about 0.5 to about 10 microns as measured by an appropriate method known in the art. Dosage forms adapted for administration by inhalation also include particle dusts or mists. Suitable dosage forms wherein the carrier or excipient is a liquid for administration as a nasal spray or drops include aqueous or oil solutions/suspensions of an active ingredient (e.g. the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein and/or auxiliary active agent), which may be generated by various types of metered dose pressurized aerosols, nebulizers, or insufflators. 
     In some embodiments, the dosage forms can be aerosol formulations suitable for administration by inhalation. In some of these embodiments, the aerosol formulation can contain a solution or fine suspension of the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein and a pharmaceutically acceptable aqueous or nonaqueous solvent. Aerosol formulations can be presented in single or multi-dose quantities in sterile form in a sealed container. For some of these embodiments, the sealed container is a single dose or multi-dose nasal or an aerosol dispenser fitted with a metering valve (e.g. metered dose inhaler), which is intended for disposal once the contents of the container have been exhausted. 
     Where the aerosol dosage form is contained in an aerosol dispenser, the dispenser contains a suitable propellant under pressure, such as compressed air, carbon dioxide, or an organic propellant, including but not limited to a hydrofluorocarbon. The aerosol formulation dosage forms in other embodiments are contained in a pump-atomizer. The pressurized aerosol formulation can also contain a solution or a suspension of one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein. In further embodiments, the aerosol formulation can also contain co-solvents and/or modifiers incorporated to improve, for example, the stability and/or taste and/or fine particle mass characteristics (amount and/or profile) of the formulation. Administration of the aerosol formulation can be once daily or several times daily, for example 2, 3, 4, or 8 times daily, in which 1, 2, or 3 doses are delivered each time. 
     For some dosage forms suitable and/or adapted for inhaled administration, the pharmaceutical formulation is a dry powder inhalable formulation. In addition to the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein, an auxiliary active ingredient, and/or pharmaceutically acceptable salt thereof, such a dosage form can contain a powder base such as lactose, glucose, trehalose, manitol, and/or starch. In some of these embodiments, the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein is in a particle-size reduced form. In further embodiments, a performance modifier, such as L-leucine or another amino acid, cellobiose octaacetate, and/or metals salts of stearic acid, such as magnesium or calcium stearate. 
     In some embodiments, the aerosol dosage forms can be arranged so that each metered dose of aerosol contains a predetermined amount of an active ingredient, such as the one or more of the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein. 
     Dosage forms adapted for vaginal administration can be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulations. Dosage forms adapted for rectal administration include suppositories or enemas. 
     Dosage forms adapted for parenteral administration and/or adapted for any type of injection (e.g. intravenous, intraperitoneal, subcutaneous, intramuscular, intradermal, intraosseous, epidural, intracardiac, intraarticular, intracavernous, gingival, subginigival, intrathecal, intravireal, intracerebral, and intracerebroventricular) can include aqueous and/or nonaqueous sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, solutes that render the composition isotonic with the blood of the subject, and aqueous and nonaqueous sterile suspensions, which can include suspending agents and thickening agents. The dosage forms adapted for parenteral administration can be presented in a single-unit dose or multi-unit dose containers, including but not limited to sealed ampoules or vials. The doses can be lyophilized and resuspended in a sterile carrier to reconstitute the dose prior to administration. Extemporaneous injection solutions and suspensions can be prepared in some embodiments, from sterile powders, granules, and tablets. 
     Dosage forms adapted for ocular administration can include aqueous and/or nonaqueous sterile solutions that can optionally be adapted for injection, and which can optionally contain anti-oxidants, buffers, bacteriostats, solutes that render the composition isotonic with the eye or fluid contained therein or around the eye of the subject, and aqueous and nonaqueous sterile suspensions, which can include suspending agents and thickening agents. 
     For some embodiments, the dosage form contains a predetermined amount of the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein per unit dose. In some embodiments, the predetermined amount of the Such unit doses may therefore be administered once or more than once a day. Such pharmaceutical formulations may be prepared by any of the methods well known in the art. 
     Kits 
     Also described herein are kits that contain one or more of the one or more of the polypeptides, polynucleotides, vectors, cells, or other components described herein and combinations thereof and pharmaceutical formulations described herein. In certain embodiments, one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein can be presented as a combination kit. As used herein, the terms “combination kit” or “kit of parts” refers to the compounds, or formulations and additional components that are used to package, screen, test, sell, market, deliver, and/or administer the combination of elements or a single element, such as the active ingredient, contained therein. Such additional components include but are not limited to, packaging, syringes, blister packages, bottles, and the like. The combination kit can contain one or more of the components (e.g. one or more of the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof) or formulation thereof can be provided in a single formulation (e.g. a liquid, lyophilized powder, etc.), or in separate formulations. The separate components or formulations can be contained in a single package or in separate packages within the kit. The kit can also include instructions in a tangible medium of expression that can contain information and/or directions regarding the content of the components and/or formulations contained therein, safety information regarding the content of the components(s) and/or formulation(s) contained therein, information regarding the amounts, dosages, indications for use, screening methods, component design recommendations and/or information, recommended treatment regimen(s) for the components(s) and/or formulations contained therein. As used herein, “tangible medium of expression” refers to a medium that is physically tangible or accessible and is not a mere abstract thought or an unrecorded spoken word. “Tangible medium of expression” includes, but is not limited to, words on a cellulosic or plastic material, or data stored in a suitable computer readable memory form. The data can be stored on a unit device, such as a flash memory drive or CD-ROM or on a server that can be accessed by a user via, e.g. a web interface. 
     In one aspect, the invention provides a kit comprising one or more of the components described herein. In some embodiments, the kit comprises a vector system and instructions for using the kit. In some embodiments, the vector system comprises (a) a first regulatory element operably linked to a tracr mate sequence and one or more insertion sites for inserting one or more guide sequences upstream of the tracr mate sequence, wherein when expressed, the guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence in a eukaryotic cell, wherein the CRISPR complex comprises a CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence; and/or (b) said AAV-CRISPR enzyme optionally comprising a nuclear localization sequence. In some embodiments, the kit comprises components (a) and (b) located on or in the same or different vectors of the system, e.g., (a) can be contained in (b). In some embodiments, component (a) further comprises the tracr sequence downstream of the tracr mate sequence under the control of the first regulatory element. In some embodiments, component (a) further comprises two or more guide sequences operably linked to the first regulatory element, wherein when expressed, each of the two or more guide sequences direct sequence specific binding of a CRISPR complex to a different target sequence in a eukaryotic cell. In some embodiments, the system further comprises a third regulatory element, such as a polymerase III promoter, operably linked to said tracr sequence. In some embodiments, the tracr sequence exhibits at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of sequence complementarity along the length of the tracr mate sequence when optimally aligned. In some embodiments, the CRISPR enzyme comprises one or more nuclear localization sequences of sufficient strength to drive accumulation of said CRISPR enzyme in a detectable amount in the nucleus of a eukaryotic cell. In some embodiments, the CRISPR enzyme is a type II CRISPR system enzyme. In some embodiments, the CRISPR enzyme is a Cas-like (e.g. Cas9-like and/or Cas12-like) enzyme. In some embodiments, the Cas-like (e.g. Cas9-like and/or Cas12-like) enzyme is derived from  S. pneumoniae, S. pyogenes, S. thermophilus, F. novicida  or  S. aureus  Cas9-like (e.g., modified to have or be associated with at least one AAV), and may include further alteration or mutation of the Cas9-like, and can be a chimeric Cas9-like. In some embodiments, the coding for the AAV-CRISPR enzyme is codon-optimized for expression in a eukaryotic cell. In some embodiments, the AAV-CRISPR enzyme directs cleavage of one or two strands at the location of the target sequence. In some embodiments, the AAV-CRISPR enzyme lacks or substantially DNA strand cleavage activity (e.g., no more than 5% nuclease activity as compared with a wild type enzyme or enzyme not having the mutation or alteration that decreases nuclease activity). In some embodiments, the first regulatory element is a polymerase III promoter. In some embodiments, the second regulatory element is a polymerase II promoter. In some embodiments, the guide sequence is at least 15, 16, 17, 18, 19, 20, 25 nucleotides, or between 10-30, or between 15-25, or between 15-20 nucleotides in length. 
     In one aspect, the invention provides a kit comprising one or more of the components described herein above. In some embodiments, the kit comprises a vector system and instructions for using the kit. In some embodiments, the vector system comprises (a) a first regulatory element operably linked to a direct repeat sequence and one or more insertion sites for inserting one or more guide sequences downstream of the direct repeat sequence, wherein when expressed, the guide sequence directs sequence-specific binding of a Cas CRISPR complex to a target sequence in a eukaryotic cell, wherein the CRISPR complex comprises a Cas enzyme complexed with the protected guide RNA comprising the guide sequence that is hybridized to the target sequence and/or (b) a second regulatory element operably linked to an enzyme-coding sequence encoding said Cas enzyme comprising a nuclear localization sequence. In some embodiments, the kit comprises components (a) and (b) located on the same or different vectors of the system. In some embodiments, component (a) further comprises two or more guide sequences operably linked to the first regulatory element, wherein when expressed, each of the two or more guide sequences direct sequence specific binding of a CRISPR complex to a different target sequence in a eukaryotic cell. In some embodiments, the Cas enzyme comprises one or more nuclear localization sequences of sufficient strength to drive accumulation of said Cas enzyme in a detectable amount in the nucleus of a eukaryotic cell. In some embodiments, the Cas enzyme is  Acidaminococcus  sp. BV3L6, Lachnospiraceae bacterium MA2020 or  Francisella tularensis  1 Novicida Cas9-like, and may include mutated Cas9 derived from these organisms. The enzyme may be a Cas9 homolog or ortholog. In some embodiments, the CRISPR enzyme is codon-optimized for expression in a eukaryotic cell. In some embodiments, the CRISPR enzyme directs cleavage of one or two strands at the location of the target sequence. In some embodiments, the CRISPR enzyme lacks DNA strand cleavage activity. In some embodiments, the first regulatory element is a polymerase III promoter. In some embodiments, the second regulatory element is a polymerase II promoter. 
     In one aspect, the invention provides a kit comprising one or more of the components described herein. In some embodiments, the kit comprises a vector system and instructions for using the kit. In some embodiments, the vector system comprises (a) a first regulatory element operably linked to a direct repeat sequence and one or more insertion sites for inserting one or more guide sequences up- or downstream (whichever applicable) of the direct repeat sequence, wherein when expressed, the guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence in a eukaryotic cell, wherein the CRISPR complex comprises a Cas enzyme complexed with the guide sequence that is hybridized to the target sequence; and/or (b) a second regulatory element operably linked to an enzyme-coding sequence encoding said Cas enzyme comprising a nuclear localization sequence. Where applicable, a tracr sequence may also be provided. In some embodiments, the kit comprises components (a) and (b) located on the same or different vectors of the system. In some embodiments, component (a) further comprises two or more guide sequences operably linked to the first regulatory element, wherein when expressed, each of the two or more guide sequences direct sequence specific binding of a CRISPR complex to a different target sequence in a eukaryotic cell. In some embodiments, the Cas enzyme comprises one or more nuclear localization sequences of sufficient strength to drive accumulation of said CRISPR enzyme in a detectable amount in the nucleus of a eukaryotic cell. In some embodiments, the CRISPR enzyme is Cas-like protein described elsewhere herein. In some embodiments, the CRISPR enzyme is a Cas-like enzyme described elsewhere herein. In some aspects, the Cas-like enzyme can be modified to have or be associated with at least one DD), and may include further alteration or mutation of the Cas-like protein, and can be a chimeric Cas-like protein. In some embodiments, the DD-CRISPR enzyme is codon-optimized for expression in a eukaryotic cell. In some embodiments, the DD-CRISPR enzyme directs cleavage of one or two strands at the location of the target sequence. In some embodiments, the DD-CRISPR enzyme lacks or substantially DNA strand cleavage activity (e.g., no more than 5% nuclease activity as compared with a wild type enzyme or enzyme not having the mutation or alteration that decreases nuclease activity). In some embodiments, the first regulatory element is a polymerase III promoter. In some embodiments, the second regulatory element is a polymerase II promoter. In some embodiments, the guide sequence is at least 16, 17, 18, 19, 20, 25 nucleotides, or between 16-30, or between 16-25, or between 16-20 nucleotides in length. 
     Methods of Use 
     General Discussion 
     Generally, the CRISPR-Cas systems and components thereof described herein can be used to modify one or more polynucleotides. Such systems therefore can have various application where it is useful to modify a polynucleotide, whether it be inside or outside of a cell. Exemplary, non-limiting, methods of using the CRISPR-Cas systems and components thereof described herein as well as the modified polynucleotides, cells, and/or organisms generated by the use of the CRISPR-Cas systems described herein. 
     Methods of Modifying a Polynucleotide Using the CRISPR-Cas Systems 
     Also described herein are methods of inducing one or more mutations in a eukaryotic cell (in vitro, i.e. in an isolated eukaryotic cell) as herein discussed comprising delivering to cell a vector as herein discussed. The mutation(s) can include the introduction, deletion, or substitution of one or more nucleotides at each target sequence of cell(s) via the nucleic acid components (e.g. guide(s) RNA(s) or sgRNA(s)). The mutations can include the introduction, deletion, or substitution of 1-75 nucleotides at each target sequence of said cell(s) via the guide(s) RNA(s) or sgRNA(s). The mutations can include the introduction, deletion, or substitution of 1, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides at each target sequence of said cell(s) via the guide(s) RNA(s) or sgRNA(s). The mutations can include the introduction, deletion, or substitution of 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides at each target sequence of said cell(s) via the guide(s) RNA(s) or sgRNA(s). The mutations include the introduction, deletion, or substitution of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides at each target sequence of said cell(s) via the guide(s) RNA(s) or sgRNA(s). The mutations can include the introduction, deletion, or substitution of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides at each target sequence of said cell(s) via the guide(s) RNA(s) or sgRNA(s). The mutations can include the introduction, deletion, or substitution of 40, 45, 50, 75, 100, 200, 300, 400 or 500 nucleotides at each target sequence of said cell(s) via the guide(s) RNA(s) or sgRNA(s). 
     In some embodiments, the concentration or dosage of the CRISPR-Cas system and/or components thereof delivered to achieve polynucleotide modification can be controlled, which in some embodiments can be effective to reduce off-target effects. Optimal concentrations of Cas mRNA and guide RNA can be determined by testing different concentrations in a cellular or non-human eukaryote animal model and using deep sequencing the analyze the extent of modification at potential off-target genomic loci. Alternatively, to minimize the level of toxicity and off-target effect, Cas nickase mRNA (for example  S. pyogenes  Cas9-like with the D10A mutation) can be delivered with a pair of guide RNAs targeting a site of interest. Guide sequences and strategies to minimize toxicity and off-target effects can be as in WO 2014/093622; or, via mutation, or other strategy as described elsewhere herein. 
     In some embodiments, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence. Without wishing to be bound by theory, the tracr sequence, which may comprise or consist of all or a portion of a wild-type tracr sequence (e.g. about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of a wild-type tracr sequence), may also form part of a CRISPR complex, such as by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence that is operably linked to the guide sequence. 
     In one aspect, the invention provides a method of modifying a target polynucleotide in a eukaryotic cell. In some embodiments, the method comprises allowing a AAV-CRISPR complex to bind to the target polynucleotide, e.g., to effect cleavage of said target polynucleotide, thereby modifying the target polynucleotide, wherein the AAV-CRISPR complex comprises a AAV-CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within said target polynucleotide, wherein said guide sequence is linked to a tracr mate sequence which in turn hybridizes to a tracr sequence. In some embodiments, said cleavage comprises cleaving one or two strands at the location of the target sequence by said AAV-CRISPR enzyme. In some embodiments, said cleavage results in decreased transcription of a target gene. In some embodiments, the method further comprises repairing said cleaved target polynucleotide by homologous recombination with an exogenous template polynucleotide, wherein said repair results in a mutation comprising an insertion, deletion, or substitution of one or more nucleotides of said target polynucleotide. In some embodiments, said mutation results in one or more amino acid changes in a protein expressed from a gene comprising the target sequence. In some embodiments, the method further comprises delivering one or more vectors to said eukaryotic cell, wherein one or more vectors comprise the AAV-CRISPR enzyme and one or more vectors drive expression of one or more of: the guide sequence linked to the tracr mate sequence, and the tracr sequence. In some embodiments, said AAV-CRISPR enzyme drive expression of one or more of: the guide sequence linked to the tracr mate sequence, and the tracr sequence. In some embodiments such AAV-CRISPR enzyme are delivered to the eukaryotic cell in a subject. In some embodiments, said modifying takes place in said eukaryotic cell in a cell culture. In some embodiments, the method further comprises isolating said eukaryotic cell from a subject prior to said modifying. In some embodiments, the method further comprises returning said eukaryotic cell and/or cells derived therefrom to said subject. In some embodiments, the method comprises allowing a AAV-CRISPR complex to bind to the polynucleotide such that said binding results in increased or decreased expression of said polynucleotide; wherein the AAV-CRISPR complex comprises a AAV-CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within said polynucleotide, wherein said guide sequence is linked to a tracr mate sequence which in turn hybridizes to a tracr sequence. In some embodiments, the method further comprises delivering one or more vectors to said eukaryotic cells, wherein the one or more vectors are the AAV-CRISPR enzyme and/or drive expression of one or more of: the guide sequence linked to the tracr mate sequence, and the tracr sequence. 
     In one aspect, the invention provides a method of modifying multiple target polynucleotides in a host cell such as a eukaryotic cell. In some embodiments, the method comprises allowing a CRISPR complex to bind to multiple target polynucleotides, e.g., to effect cleavage of said multiple target polynucleotides, thereby modifying multiple target polynucleotides, wherein the CRISPR complex comprises one or more Cas enzymes complexed with multiple guide sequences each of the being hybridized to a specific target sequence within said target polynucleotide, wherein said multiple guide sequences are linked to a direct repeat sequence. Where applicable, a tracr sequence may also be provided (e.g. to provide a single guide RNA, sgRNA). In some embodiments, said cleavage comprises cleaving one or two strands at the location of each of the target sequence by said Cas enzyme. In some embodiments, said cleavage results in decreased transcription of the multiple target genes. In some embodiments, the method further comprises repairing one or more of said cleaved target polynucleotide by homologous recombination with an exogenous template polynucleotide, wherein said repair results in a mutation comprising an insertion, deletion, or substitution of one or more nucleotides of one or more of said target polynucleotides. In some embodiments, said mutation results in one or more amino acid changes in a protein expressed from a gene comprising one or more of the target sequence(s). In some embodiments, the method further comprises delivering one or more vectors to said eukaryotic cell, wherein the one or more vectors drive expression of one or more of: the Cas enzyme and the multiple guide RNA sequence linked to a direct repeat sequence. Where applicable, a tracr sequence may also be provided. In some embodiments, said vectors are delivered to the eukaryotic cell in a subject. In some embodiments, said modifying takes place in said eukaryotic cell in a cell culture. In some embodiments, the method further comprises isolating said eukaryotic cell from a subject prior to said modifying. In some embodiments, the method further comprises returning said eukaryotic cell and/or cells derived therefrom to said subject. 
     In one aspect, the invention provides a method of modifying expression of multiple polynucleotides in a eukaryotic cell. In some embodiments, the method comprises allowing a Cas CRISPR complex to bind to multiple polynucleotides such that said binding results in increased or decreased expression of said polynucleotides; wherein the Cas CRISPR complex comprises one or more Cas enzyme complexed with multiple guide sequences each specifically hybridized to its own target sequence within said polynucleotide, wherein said guide sequences are linked to a direct repeat sequence. Where applicable, a tracr sequence may also be provided. In some embodiments, the method further comprises delivering one or more vectors to said eukaryotic cells, wherein the one or more vectors drive expression of one or more of: the Cas enzyme and the multiple guide sequences linked to the direct repeat sequences. Where applicable, a tracr sequence may also be provided. The multiple guide RNAs target the multiple DNA molecules encoding the multiple gene products in a cell and the CRISPR protein may cleave the multiple DNA molecules encoding the gene products (it may cleave one or both strands or have substantially no nuclease activity), whereby expression of the multiple gene products is altered; and, wherein the CRISPR protein and the multiple guide RNAs do not naturally occur together. 
     In some embodiments, a polynucleotide in a eukaryotic cell can be modified via a CRISPR-Cas system described herein. In some embodiments, a polynucleotide in a prokaryotic cell can be modified via a CRISPR-Cas system described herein. In some embodiments, a polynucleotide in a non-human animal cell can be modified via a CRISPR-Cas system described herein. In some embodiments, a polynucleotide in a human cell can be modified via a CRISPR-Cas system described herein. In some embodiments, a polynucleotide in plant cell can be modified via a CRISPR-Cas system described herein. In some embodiments, a polynucleotide in yeast cell can be modified via a CRISPR-Cas system described herein. In some embodiments, a polynucleotide in microorganism can be modified via a CRISPR-Cas system described herein. Modification can occur in vitro, ex vivo, or in vivo. 
     CRISPR-Cas System Therapeutic Uses and Methods of Treatment 
     Also provided herein are methods of diagnosing, prognosing, treating, and/or preventing a disease, state, or condition in or of a subject. Generally, the methods of diagnosing, prognosing, treating, and/or preventing a disease, state, or condition in or of a subject can include modifying a polynucleotide in a subject or cell thereof using a CRISPR-Cas system or component thereof described herein and/or include detecting a diseased or healthy polynucleotide in a subject or cell thereof using a CRISPR-Cas system or component thereof described herein. In some embodiments, the method of treatment or prevention can include using a CRISPR-Cas system or component thereof to modify a polynucleotide of an infectious organism (e.g. bacterial or virus) within a subject or cell thereof. In some embodiments, the method of treatment or prevention can include using a CRISPR-Cas system or component thereof to modify a polynucleotide of an infectious organism or symbiotic organism within a subject. The CRISPR-Cas systems and components thereof can be used to develop models of diseases, states, or conditions. The CRISPR-Cas systems and components thereof can be used to detect a disease state or correction thereof, such as by a method of treatment or prevention described herein. The CRISPR-Cas systems and components thereof can be used to screen and select cells that can be used, for example, as treatments or preventions described herein. The CRISPR-Cas systems and components thereof can be used to develop biologically active agents that can be used to modify one or more biologic functions or activities in a subject or a cell thereof. 
     In general, the method can include delivering a CRISPR-Cas System and/or component thereof to a subject or cell thereof, or to an infectious or symbiotic organism by a suitable delivery technique and/or composition. Once administered the components can operate as described elsewhere herein to elicit a nucleic acid modification event. In some aspects, the nucleic acid modification event can occur at the genomic, epigenomic, and/or transcriptomic level. In some embodiments, DNA and/or RNA cleavage, gene activation, and/or gene deactivation can occur. Additional features, uses, and advantages are described in greater detail below. On the basis of this concept, several variations are appropriate to elicit a genomic locus event, including DNA cleavage, gene activation, or gene deactivation. Using the provided compositions, the person skilled in the art can advantageously and specifically target single or multiple loci with the same or different functional domains to elicit one or more genomic locus events. In addition to treating and/or preventing a disease in a subject, the compositions may be applied in a wide variety of methods for screening in libraries in cells and functional modeling in vivo (e.g. gene activation of lincRNA and identification of function; gain-of-function modeling; loss-of-function modeling; the use the compositions of the invention to establish cell lines and transgenic animals for optimization and screening purposes). 
     The CRISPR-Cas systems and components thereof described elsewhere herein can be used to treat and/or prevent a disease, such as a genetic and/or epigenetic disease, in a subject. The CRISPR-Cas systems and components thereof described elsewhere herein can be used to treat and/or prevent genetic infectious diseases in a subject, such as bacterial infections, viral infections, fungal infections, parasite infections, and combinations thereof. The CRISPR-Cas systems and components thereof described elsewhere herein can be used to modify the composition or profile of a microbiome in a subject, which can in turn modify the health status of the subject. The CRISPR-Cas systems described herein can be used to modify cells ex vivo, which can then be administered to the subject whereby the modified cells can treat or prevent a disease or symptom thereof. This is also referred to in some contexts as adoptive therapy. The CRISPR-Cas systems described herein can be used to treat mitochondrial diseases, where the mitochondrial disease etiology involves a mutation in the mitochondrial DNA. 
     Also provided is a method of treating a subject, e.g., a subject in need thereof, comprising inducing gene editing by transforming the subject with the polynucleotide encoding one or more components of the CRISPR-Cas system or complex or any of polynucleotides or vectors described herein and administering them to the subject. A suitable repair template may also be provided, for example delivered by a vector comprising said repair template. Also provided is a method of treating a subject, e.g., a subject in need thereof, comprising inducing transcriptional activation or repression of multiple target gene loci by transforming the subject with the polynucleotides or vectors described herein, wherein said polynucleotide or vector encodes or comprises one or more components of CRISPR-Cas system, complex or component thereof comprising multiple Cas effectors. Where any treatment is occurring ex vivo, for example in a cell culture, then it will be appreciated that the term ‘subject’ may be replaced by the phrase “cell or cell culture.” 
     Also provided is a method of treating a subject, e.g., a subject in need thereof, comprising inducing gene editing by transforming the subject with the Cas effector(s), advantageously encoding and expressing in vivo the remaining portions of the CRISPR-Cas system (e.g., RNA, guides). A suitable repair template may also be provided, for example delivered by a vector comprising said repair template. Also provided is a method of treating a subject, e.g., a subject in need thereof, comprising inducing transcriptional activation or repression by transforming the subject with the Cas effector(s) advantageously encoding and expressing in vivo the remaining portions of the CRISPR-Cas system (e.g., RNA, guides); advantageously in some embodiments the CRISPR enzyme is a catalytically inactive Cas effector and includes one or more associated functional domains. Where any treatment is occurring ex vivo, for example in a cell culture, then it will be appreciated that the term ‘subject’ may be replaced by the phrase “cell or cell culture.” 
     One or more components of the non-Class I nucleic acid targeting system described herein can be included in a composition, such as a pharmaceutical composition, and administered to a host individually or collectively. Alternatively, these components may be provided in a single composition for administration to a host. Administration to a host may be performed via viral vectors known to the skilled person or described herein for delivery to a host (e.g. lentiviral vector, adenoviral vector, AAV vector). As explained herein, use of different selection markers (e.g. for lentiviral gRNA selection) and concentration of gRNA (e.g. dependent on whether multiple gRNAs are used) may be advantageous for eliciting an improved effect. 
     Thus, also described herein are methods of inducing one or more polynucleotide modifications in a eukaryotic or prokaryotic cell or component thereof (e.g. a mitochondria) of a subject, infectious organism, and/or organism of the microbiome of the subject. The modification can include the introduction, deletion, or substitution of one or more nucleotides at a target sequence of a polynucleotide of one or more cell(s). The modification can occur in vitro, ex vivo, in situ, or in vivo. 
     In some embodiments, the method of treating or inhibiting a condition or a disease caused by one or more mutations in a genomic locus in a eukaryotic organism or a non-human organism can include manipulation of a target sequence within a coding, non-coding or regulatory element of said genomic locus in a target sequence in a subject or a non-human subject in need thereof comprising modifying the subject or a non-human subject by manipulation of the target sequence and wherein the condition or disease is susceptible to treatment or inhibition by manipulation of the target sequence including providing treatment comprising delivering a composition comprising the particle delivery system or the delivery system or the virus particle of any one of the above embodiment or the cell of any one of the above embodiment. 
     Also provided herein is the use of the particle delivery system or the delivery system or the virus particle of any one of the above embodiment or the cell of any one of the above embodiment in ex vivo or in vivo gene or genome editing; or for use in in vitro, ex vivo or in vivo gene therapy. Also provided herein are particle delivery systems, non-viral delivery systems, and/or the virus particle of any one of the above embodiments or the cell of any one of the above embodiments used in the manufacture of a medicament for in vitro, ex vivo or in vivo gene or genome editing or for use in in vitro, ex vivo or in vivo gene therapy or for use in a method of modifying an organism or a non-human organism by manipulation of a target sequence in a genomic locus associated with a disease or in a method of treating or inhibiting a condition or disease caused by one or more mutations in a genomic locus in a eukaryotic organism or a non-human organism. 
     In some embodiments, polynucleotide modification can include the introduction, deletion, or substitution of 1-75 nucleotides at each target sequence of said polynucleotide of said cell(s). The modification can include the introduction, deletion, or substitution of 1, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides at each target sequence. The modification can include the introduction, deletion, or substitution of 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides at each target sequence of said cell(s). The modification can include the introduction, deletion, or substitution of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides at each target sequence of said cell(s). The modification can include the introduction, deletion, or substitution of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides at each target sequence of said cell(s). The modification can include the introduction, deletion, or substitution of 40, 45, 50, 75, 100, 200, 300, 400 or 500 nucleotides at each target sequence of said cell(s). The modification can include the introduction, deletion, or substitution of 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100, 8200, 8300, 8400, 8500, 8600, 8700, 8800, 8900, 9000, 9100, 9200, 9300, 9400, 9500, 9600, 9700, 9800, or 9900 to 10000 nucleotides at each target sequence of said cell(s). 
     In some embodiments, the modifications can include the introduction, deletion, or substitution of nucleotides at each target sequence of said cell(s) via nucleic acid components (e.g. guide(s) RNA(s) or sgRNA(s)), such as those mediated by a CRISPR-Cas system or a component thereof described elsewhere herein. In some embodiments, the modifications can include the introduction, deletion, or substitution of nucleotides at a target or random sequence of said cell(s) via a non CRISPR-Cas system or technique. 
     The target sequences of polynucleotides to be modified to treat or prevent disease are described in greater detail below. 
     As is also discussed elsewhere herein, the CRISPR-Cas system can include a template polynucleotide (also referred to herein as template nucleic acids or template sequence). In an embodiment, the template nucleic acid alters the structure of the target position by participating in homologous recombination. In an embodiment, the template nucleic acid alters the sequence of the target position. In an embodiment, the template nucleic acid results in the incorporation of a modified, or non-naturally occurring base into the target nucleic acid. 
     The template sequence may undergo a breakage mediated or catalyzed recombination with the target sequence. In an embodiment, the template nucleic acid can include sequence that corresponds to a site on the target sequence that is cleaved, nicked, or otherwise modified by one or more Cas effector mediated cleavage event(s). In an embodiment, the template nucleic acid can include sequence that corresponds to both, a first site on the target sequence that is cleaved, nicked, or otherwise modified in a first Cas effector mediated event, and a second site on the target sequence that is cleaved in a second Cas effector mediated event. 
     In certain embodiments, the template nucleic acid can include a sequence which results in an alteration in the coding sequence of a translated sequence, e.g., one which results in the substitution of one amino acid for another in a protein product, e.g., transforming a mutant allele into a wild type allele, transforming a wild type allele into a mutant allele, and/or introducing a stop codon, insertion of an amino acid residue, deletion of an amino acid residue, or a nonsense mutation. In certain embodiments, the template nucleic acid can include sequence which results in an alteration in a non-coding sequence, e.g., an alteration in an exon or in a 5′ or 3′ non-translated or non-transcribed region. Such alterations include an alteration in a control element, e.g., a promoter, enhancer, and an alteration in a cis-acting or trans-acting control element. 
     A template nucleic acid having homology with a target position in a target gene may be used to alter the structure of a target sequence. The template sequence may be used to alter an unwanted structure, e.g., an unwanted or mutant nucleotide. The template nucleic acid may include sequence which, when integrated, results in: decreasing the activity of a positive control element; increasing the activity of a positive control element; decreasing the activity of a negative control element; increasing the activity of a negative control element; decreasing the expression of a gene; increasing the expression of a gene; increasing resistance to a disorder or disease; increasing resistance to viral entry; correcting a mutation or altering an unwanted amino acid residue conferring, increasing, abolishing or decreasing a biological property of a gene product, e.g., increasing the enzymatic activity of an enzyme, or increasing the ability of a gene product to interact with another molecule. 
     The template nucleic acid may include sequence which results in: a change in sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more nucleotides of the target sequence. In an embodiment, the template nucleic acid may be 20+/−10, 30+/−10, 40+/−10, 50+/−10, 60+/−10, 70+/−10, 80+/−10, 9 0+/−10, 100+/−10, 110+/−10, 120+/−10, 130+/−10, 140+/−10, 150+/−10, 160+/−10, 170+/−10, 1 80+/−10, 190+/−10, 200+/−10, 210+/−10, of 220+/−10 nucleotides in length. In an embodiment, the template nucleic acid may be 30+/−20, 40+/−20, 50+/−20, 60+/−20, 70+/−20, 80+/−20, 90+/−20, 100+/−20, 110+/−20, 120+/−20, 130+/−20, 140+/−20, 150+/−20, 160+/−20, 170+/−20, 180+/−20, 190+/−20, 200+/−20, 210+/−20, of 220+/−20 nucleotides in length. In an embodiment, the template nucleic acid is 10 to 1,000, 20 to 900, 30 to 800, 40 to 700, 50 to 600, 50 to 500, 50 to 400, 50 to 300, 50 to 200, or 50 to 100 nucleotides in length. 
     A template nucleic acid comprises the following components: [5′ homology arm]-[replacement sequence]-[3′ homology arm]. The homology arms provide for recombination into the chromosome, thus replacing the undesired element, e.g., a mutation or signature, with the replacement sequence. In an embodiment, the homology arms flank the most distal cleavage sites. In an embodiment, the 3′ end of the 5′ homology arm is the position next to the 5′ end of the replacement sequence. In an embodiment, the 5′ homology arm can extend at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides 5′ from the 5′ end of the replacement sequence. In an embodiment, the 5′ end of the 3′ homology arm is the position next to the 3′ end of the replacement sequence. In an embodiment, the 3′ homology arm can extend at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides 3′ from the 3′ end of the replacement sequence. 
     In certain embodiments, one or both homology arms may be shortened to avoid including certain sequence repeat elements. For example, a 5′ homology arm may be shortened to avoid a sequence repeat element. In other embodiments, a 3′ homology arm may be shortened to avoid a sequence repeat element. In some embodiments, both the 5′ and the 3′ homology arms may be shortened to avoid including certain sequence repeat elements. 
     In certain embodiments, a template nucleic acids for correcting a mutation may designed for use as a single-stranded oligonucleotide. When using a single-stranded oligonucleotide, 5′ and 3′ homology arms may range up to about 200 base pairs (bp) in length, e.g., at least 25, 50, 75, 100, 125, 150, 175, or 200 bp in length. 
     In some embodiments, the CRISPR-Cas system or component thereof can promote Non-Homologous End-Joining (NHEJ). In some embodiments, modification of a polynucleotide by a CRISPR-Cas system or a component thereof, such as a diseased polynucleotide, can include NHEJ. In some embodiments, promotion of this repair pathway by the CRISPR-Cas system or a component thereof can be used to target gene or polynucleotide specific knock-outs and/or knock-ins. In some embodiments, promotion of this repair pathway by the CRISPR-Cas system or a component thereof can be used to generate NHEJ-mediated indels. Nuclease-induced NHEJ can also be used to remove (e.g., delete) sequence in a gene of interest. Generally, NHEJ repairs a double-strand break in the DNA by joining together the two ends; however, generally, the original sequence is restored only if two compatible ends, exactly as they were formed by the double-strand break, are perfectly ligated. The DNA ends of the double-strand break are frequently the subject of enzymatic processing, resulting in the addition or removal of nucleotides, at one or both strands, prior to rejoining of the ends. This results in the presence of insertion and/or deletion (indel) mutations in the DNA sequence at the site of the NHEJ repair. In some embodiments, the indel mutation(s) generated by the CRISPR-Cas systems described herein can alter the reading frame of a polypeptide. In some embodiments, alteration of the reading frame can result in a non-functional protein. Indel mutations that insert or delete a significant amount of sequence can also alter the functionality of a protein. In some embodiments, the indel can completely destroy the functionality of a protein. 
     The indel can range in size from 1-50 or more base pairs. In some embodiments the indel can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, or 500 base pairs or more. If a double-strand break is targeted near to a short target sequence, the deletion mutations caused by the NHEJ repair often span, and therefore remove, the unwanted nucleotides. For the deletion of larger DNA segments, introducing two double-strand breaks, one on each side of the sequence, can result in NHEJ between the ends with removal of the entire intervening sequence. Both of these approaches can be used to delete specific DNA sequences. 
     In some embodiments, CRISPR-Cas system mediated NHEJ can be used in the method to delete small sequence motifs. In some embodiments, CRISPR-Cas system mediated NHEJ can be used in the method to generate NHEJ-mediate indels that can be targeted to the gene, e.g., a coding region, e.g., an early coding region of a gene of interest can be used to knockout (i.e., eliminate expression of) a gene of interest. For example, early coding region of a gene of interest includes sequence immediately following a transcription start site, within a first exon of the coding sequence, or within 500 bp of the transcription start site (e.g., less than 500, 450, 400, 350, 300, 250, 200, 150, 100 or 50 bp). In an embodiment, in which a guide RNA and Cas effector generate a double strand break for the purpose of inducing NHEJ-mediated indels, a guide RNA may be configured to position one double-strand break in close proximity to a nucleotide of the target position. In an embodiment, the cleavage site may be between 0-500 bp away from the target position (e.g., less than 500, 400, 300, 200, 100, 50, 40, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 bp from the target position). In an embodiment, in which two guide RNAs complexing with one or more Cas nickases induce two single strand breaks for the purpose of inducing NHEJ-mediated indels, two guide RNAs may be configured to position two single-strand breaks to provide for NHEJ repair a nucleotide of the target position. 
     For minimization of toxicity and off-target effect, it may be important to control the concentration of Cas mRNA and guide RNA delivered. Optimal concentrations of Cas mRNA and guide RNA can be determined by testing different concentrations in a cellular or non-human eukaryote animal model and using deep sequencing the analyze the extent of modification at potential off-target genomic loci. Alternatively, to minimize the level of toxicity and off-target effect, Cas nickase mRNA (for example  S. pyogenes  Cas9 with the D10A mutation) can be delivered with a pair of guide RNAs targeting a site of interest. Guide sequences and strategies to minimize toxicity and off-target effects can be as in WO 2014/093622 (PCT/US2013/074667); or, via mutation. Others are as described elsewhere herein. 
     Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage, nicking, and/or another modification of one or both strands in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence. In some embodiments, the tracr sequence, which may comprise or consist of all or a portion of a wild-type tracr sequence (e.g. about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of a wild-type tracr sequence), can also form part of a CRISPR complex, such as by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence that is operably linked to the guide sequence. 
     In some embodiments, a method of modifying a target polynucleotide in a cell to treat or prevent a disease can include allowing a CRISPR-Cas system or component thereof to bind to the target polynucleotide, e.g., to effect cleavage, nicking, or other modification as the CRISPR-Cas system is capable of said target polynucleotide, thereby modifying the target polynucleotide, wherein the CRISPR-Cas system or component thereof, complex with a guide sequence, and hybridize said guide sequence to a target sequence within the target polynucleotide, wherein said guide sequence is optionally linked to a tracr mate sequence, which in turn can hybridize to a tracr sequence. In some of these embodiments, the CRISPR-Cas system or component thereof can be or include a CRISPR-Cas effector complexed with a guide sequence. In some embodiments, modification can include cleaving or nicking one or two strands at the location of the target sequence by one or more components of the CRISPR-Cas system or component thereof. 
     The cleavage, nicking, or other modification capable of being performed by the CRISPR-Cas system can modify transcription of a target polynucleotide. In some embodiments, modification of transcription can include decreasing transcription of a target polynucleotide. In some embodiments, modification can include increasing transcription of a target polynucleotide. In some embodiments, the method includes repairing said cleaved target polynucleotide by homologous recombination with an exogenous template polynucleotide, wherein said repair results in a modification such as, but not limited to, an insertion, deletion, or substitution of one or more nucleotides of said target polynucleotide. In some embodiments, said modification results in one or more amino acid changes in a protein expressed from a gene comprising the target sequence. In some embodiments, the modification imparted by the CRISPR-Cas system or component thereof provides a transcript and/or protein that can correct a disease or a symptom thereof, including but not limited to, any of those described in greater detail elsewhere herein. 
     In some embodiments, the method of treating or preventing a disease can include delivering one or more vectors or vector systems to a cell, such as a eukaryotic or prokaryotic cell, wherein one or more vectors or vector systems include the CRISPR-Cas system or component thereof. In some embodiments, the vector(s) or vector system(s) can be a viral vector or vector system, such as an AAV or lentiviral vector system, which are described in greater detail elsewhere herein. In some embodiments, the method of treating or preventing a disease can include delivering one or more viral particles, such as an AAV or lentiviral particle, containing the CRISPR-Cas system or component thereof. In some embodiments, the viral particle has a tissue specific tropism. In some embodiments, the viral particle has a liver, muscle, eye, heart, pancreas, kidney, neuron, epithelial cell, endothelial cell, astrocyte, glial cell, immune cell, or red blood cell specific tropism. 
     It will be understood that the CRISPR-Cas systems according to the invention as described herein, such as the CRISPR-Cas systems for use in the methods according to the invention as described herein, may be suitably used for any type of application known for CRISPR-Cas systems, preferably in eukaryotes. In certain embodiments, the application is therapeutic, preferably therapeutic in a eukaryote organism, such as including but not limited to animals (including human), plants, algae, fungi (including yeasts), etc. In certain embodiments, the application may involve accomplishing or inducing one or more particular traits or characteristics, such as genotypic and/or phenotypic traits or characteristics, as also described elsewhere herein. 
     Treating Diseases of the Circulatory System 
     In some embodiments, the CRISPR-Cas system and/or component thereof described herein can be used to treat and/or prevent a circulatory system disease. Exemplary disease is provided, for example, in Tables 12 and 13. In some embodiments the plasma exosomes of Wahlgren et al. (Nucleic Acids Research, 2012, Vol. 40, No. 17 e130) can be used to deliver the CRISPR-Cas system and/or component thereof described herein to the blood. In some embodiments, the circulatory system disease can be treated by using a lentivirus to deliver the CRISPR-Cas system described herein to modify hematopoietic stem cells (HSCs) in vivo or ex vivo (see e.g. Drakopoulou, “Review Article, The Ongoing Challenge of Hematopoietic Stem Cell-Based Gene Therapy for β-Thalassemia,” Stem Cells International, Volume 2011, Article ID 987980, 10 pages, doi:10.4061/2011/987980, which can be adapted for use with the CRISPR-Cas systems herein in view of the description herein). In some embodiments, the circulatory system disorder can be treated by correcting HSCs as to the disease using a CRISPR-Cas system herein or a component thereof, wherein the CRISPR-Cas system optionally includes a suitable HDR repair template (see e.g. Cavazzana, “Outcomes of Gene Therapy for β-Thalassemia Major via Transplantation of Autologous Hematopoietic Stem Cells Transduced Ex Vivo with a Lentiviral βA-T87Q-Globin Vector.”; Cavazzana-Calvo, “Transfusion independence and HMGA2 activation after gene therapy of human β-thalassaemia”, Nature 467, 318-322 (16 Sep. 2010) doi:10.1038/nature09328; Nienhuis, “Development of Gene Therapy for Thalassemia, Cold Spring Harbor Perspectives in Medicine, doi: 10.1101/cshperspect.a011833 (2012), LentiGlobin BB305, a lentiviral vector containing an engineered β-globin gene (βA-T87Q); and Xie et al., “Seamless gene correction of β-thalassaemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyback” Genome Research gr.173427.114 (2014) http://www.genome.org/cgi/doi/10.1101/gr.173427.114 (Cold Spring Harbor Laboratory Press; [1599] Watts, “Hematopoietic Stem Cell Expansion and Gene Therapy” Cytotherapy 13(10):1164-1171. doi:10.3109/14653249.2011.620748 (2011), which can be adapted for use with the CRISPR-Cas systems herein in view of the description herein). In some embodiments, iPSCs can be modified using a CRISPR-Cas system described herein to correct a disease polynucleotide associated with a circulatory disease. In this regard, the teachings of Xu et al. (Sci Rep. 2015 Jul. 9; 5:12065. doi: 10.1038/srep12065) and Song et al. (Stem Cells Dev. 2015 May 1; 24(9):1053-65. doi: 10.1089/scd.2014.0347. Epub 2015 Feb. 5) with respect to modifying iPSCs can be adapted for use in view of the description herein with the CRISPR-Cas systems described herein. 
     The term “Hematopoietic Stem Cell” or “HSC” refers broadly those cells considered to be an HSC, e.g., blood cells that give rise to all the other blood cells and are derived from mesoderm; located in the red bone marrow, which is contained in the core of most bones. HSCs of the invention include cells having a phenotype of hematopoietic stem cells, identified by small size, lack of lineage (lin) markers, and markers that belong to the cluster of differentiation series, like: CD34, CD38, CD90, CD133, CD105, CD45, and also c-kit,—the receptor for stem cell factor. Hematopoietic stem cells are negative for the markers that are used for detection of lineage commitment, and are, thus, called Lin-; and, during their purification by FACS, a number of up to 14 different mature blood-lineage markers, e.g., CD13 &amp; CD33 for myeloid, CD71 for erythroid, CD19 for B cells, CD61 for megakaryocytic, etc. for humans; and, B220 (murine CD45) for B cells, Mac-1 (CD11b/CD18) for monocytes, Gr-1 for Granulocytes, Ter119 for erythroid cells, Il7Ra, CD3, CD4, CDS, CD8 for T cells, etc. Mouse HSC markers: CD34lo/−, SCA-1+, Thy1.1+/lo, CD38+, C-kit+, lin−, and Human HSC markers: CD34+, CD59+, Thy1/CD90+, CD38lo/−, C-kit/CD117+, and lin−. HSCs are identified by markers. Hence in embodiments discussed herein, the HSCs can be CD34+ cells. HSCs can also be hematopoietic stem cells that are CD34−/CD38−. Stem cells that may lack c-kit on the cell surface that are considered in the art as HSCs are within the ambit of the invention, as well as CD133+ cells likewise considered HSCs in the art. 
     In some embodiments, the treatment or prevention for treating a circulatory system or blood disease can include modifying a human cord blood cell with any modification described herein. In some embodiments, the treatment or prevention for treating a circulatory system or blood disease can include modifying a granulocyte colony-stimulating factor-mobilized peripheral blood cell (mPB) with any modification described herein. In some embodiments, the human cord blood cell or mPB can be CD34+. In some embodiments, the cord blood cell(s) or mPB cell(s) modified can be autologous. In some embodiments, the cord blood cell(s) or mPB cell(s) can be allogenic. In addition to the modification of the disease gene(s), allogenic cells can be further modified using the composition, system, described herein to reduce the immunogenicity of the cells when delivered to the recipient. Such techniques are described elsewhere herein and e.g. Cartier, “MINI-SYMPOSIUM: X-Linked Adrenoleukodystrophypa, Hematopoietic Stem Cell Transplantation and Hematopoietic Stem Cell Gene Therapy in X-Linked Adrenoleukodystrophy,” Brain Pathology 20 (2010) 857-862, which can be adapted for use with the composition, system, herein. The modified cord blood cell(s) or mPB cell(s) can be optionally expanded in vitro. The modified cord blood cell(s) or mPB cell(s) can be derived to a subject in need thereof using any suitable delivery technique. 
     The CRISPR-Cas (system may be engineered to target genetic locus or loci in HSCs. In some embodiments, the Cas effector(s) can be codon-optimized for a eukaryotic cell and especially a mammalian cell, e.g., a human cell, for instance, HSC, or iPSC and sgRNA targeting a locus or loci in HSC, such as circulatory disease, can be prepared. These may be delivered via particles. The particles may be formed by the Cas effector (e.g., Cas9) protein and the gRNA being admixed. The gRNA and Cas effector (e.g., Cas9) protein mixture can be, for example, admixed with a mixture comprising or consisting essentially of or consisting of surfactant, phospholipid, biodegradable polymer, lipoprotein and alcohol, whereby particles containing the gRNA and Cas effector (e.g. Cas9) protein may be formed. The invention comprehends so making particles and particles from such a method as well as uses thereof. Particles suitable delivery of the CRISRP-Cas systems in the context of blood or circulatory system or HSC delivery to the blood or circulatory system are described in greater detail elsewhere herein. 
     In some embodiments, after ex vivo modification the HSCs or iPCS can be expanded prior to administration to the subject. Expansion of HSCs can be via any suitable method such as that described by, Lee, “Improved ex vivo expansion of adult hematopoietic stem cells by overcoming CUL4-mediated degradation of HOXB4.” Blood. 2013 May 16; 121(20):4082-9. doi: 10.1182/blood-2012-09-455204. Epub 2013 Mar. 21. 
     In some embodiments, the HSCs or iPSCs modified can be autologous. In some embodiments, the HSCs or iPSCs can be allogenic. In addition to the modification of the disease gene(s), allogenic cells can be further modified using the CRISPR-Cas system described herein to reduce the immunogenicity of the cells when delivered to the recipient. Such techniques are described elsewhere herein and e.g. Cartier, “MINI-SYMPOSIUM: X-Linked Adrenoleukodystrophypa, Hematopoietic Stem Cell Transplantation and Hematopoietic Stem Cell Gene Therapy in X-Linked Adrenoleukodystrophy,” Brain Pathology 20 (2010) 857-862, which can be adapted for use with the CRISPR-Cas system herein. 
     Treating Diseases of the Brain 
     In some embodiments, the CRISPR-Cas systems described herein can be used to treat diseases of the brain and CNS. Delivery options for the brain include encapsulation of CRISPR enzyme and guide RNA in the form of either DNA or RNA into liposomes and conjugating to molecular Trojan horses for trans-blood brain barrier (BBB) delivery. Molecular Trojan horses have been shown to be effective for delivery of B-gal expression vectors into the brain of non-human primates. The same approach can be used to delivery vectors containing CRISPR enzyme and guide RNA. For instance, Xia C F and Boado R J, Pardridge W M (“Antibody-mediated targeting of siRNA via the human insulin receptor using avidin-biotin technology.” Mol Pharm. 2009 May-June; 6(3):747-51. doi: 10.1021/mp800194) describes how delivery of short interfering RNA (siRNA) to cells in culture, and in vivo, is possible with combined use of a receptor-specific monoclonal antibody (mAb) and avidin-biotin technology. The authors also report that because the bond between the targeting mAb and the siRNA is stable with avidin-biotin technology, and RNAi effects at distant sites such as brain are observed in vivo following an intravenous administration of the targeted siRNA, the teachings of which can be adapted for use with the CRISPR-Cas systems herein. In other embodiments, an artificial virus can be generated for CNS and/or brain delivery. See e.g. Zhang et al. (Mol Ther. 2003 January; 7(1):11-8)), the teachings of which can be adapted for use with the CRISPR-Cas systems herein. 
     Treating Hearing Diseases 
     In some embodiments the CRISPR-Cas system described herein can be used to treat a hearing disease or hearing loss in one or both ears. Deafness is often caused by lost or damaged hair cells that cannot relay signals to auditory neurons. In such cases, cochlear implants may be used to respond to sound and transmit electrical signals to the nerve cells. But these neurons often degenerate and retract from the cochlea as fewer growth factors are released by impaired hair cells. 
     In some embodiments, the CRISPR-Cas system or modified cells can be delivered to one or both ears for treating or preventing hearing disease or loss by any suitable method or technique. Suitable methods and techniques include, but are not limited to those set forth in US patent application 20120328580 describes injection of a pharmaceutical composition into the ear (e.g., auricular administration), such as into the luminae of the cochlea (e.g., the Scala media, Sc vestibulae, and Sc tympani), e.g., using a syringe, e.g., a single-dose syringe. For example, one or more of the compounds described herein can be administered by intratympanic injection (e.g., into the middle ear), and/or injections into the outer, middle, and/or inner ear; administration in situ, via a catheter or pump (see e.g. McKenna et al., (U.S. Publication No. 2006/0030837) and Jacobsen et al., (U.S. Pat. No. 7,206,639); administration in combination with a mechanical device such as a cochlear implant or a hearing aid, which is worn in the outer ear (see e.g. U.S. Publication No. 2007/0093878, which provides an exemplary cochlear implant suitable for delivery of the CRISPR-Cas systems described herein to the ear). Such methods are routinely used in the art, for example, for the administration of steroids and antibiotics into human ears. Injection can be, for example, through the round window of the ear or through the cochlear capsule. Other inner ear administration methods are known in the art (see, e.g., Salt and Plontke, Drug Discovery Today, 10:1299-1306, 2005). In some embodiments, a catheter or pump can be positioned, e.g., in the ear (e.g., the outer, middle, and/or inner ear) of a patient during a surgical procedure. In some embodiments, a catheter or pump can be positioned, e.g., in the ear (e.g., the outer, middle, and/or inner ear) of a patient without the need for a surgical procedure. 
     In general, the cell therapy methods described in US patent application 20120328580 can be used to promote complete or partial differentiation of a cell to or towards a mature cell type of the inner ear (e.g., a hair cell) in vitro. Cells resulting from such methods can then be transplanted or implanted into a patient in need of such treatment. The cell culture methods required to practice these methods, including methods for identifying and selecting suitable cell types, methods for promoting complete or partial differentiation of selected cells, methods for identifying complete or partially differentiated cell types, and methods for implanting complete or partially differentiated cells are described below. 
     Cells suitable for use in the present invention include, but are not limited to, cells that are capable of differentiating completely or partially into a mature cell of the inner ear, e.g., a hair cell (e.g., an inner and/or outer hair cell), when contacted, e.g., in vitro, with one or more of the compounds described herein. Exemplary cells that are capable of differentiating into a hair cell include, but are not limited to stem cells (e.g., inner ear stem cells, adult stem cells, bone marrow derived stem cells, embryonic stem cells, mesenchymal stem cells, skin stem cells, iPS cells, and fat derived stem cells), progenitor cells (e.g., inner ear progenitor cells), support cells (e.g., Deiters&#39; cells, pillar cells, inner phalangeal cells, tectal cells and Hensen&#39;s cells), and/or germ cells. The use of stem cells for the replacement of inner ear sensory cells is described in Li et al., (U.S. Publication No. 2005/0287127) and Li et al., (U.S. application Ser. No. 11/953,797). The use of bone marrow derived stem cells for the replacement of inner ear sensory cells is described in Edge et al., PCT/US2007/084654. iPS cells are described, e.g., at Takahashi et al., Cell, Volume 131, Issue 5, Pages 861-872 (2007); Takahashi and Yamanaka, Cell 126, 663-76 (2006); Okita et al., Nature 448, 260-262 (2007); Yu, J. et al., Science 318(5858):1917-1920 (2007); Nakagawa et al., Nat. Biotechnol. 26:101-106 (2008); and Zaehres and Scholer, Cell 131(5):834-835 (2007). Such suitable cells can be identified by analyzing (e.g., qualitatively or quantitatively) the presence of one or more tissue specific genes. For example, gene expression can be detected by detecting the protein product of one or more tissue-specific genes. Protein detection techniques involve staining proteins (e.g., using cell extracts or whole cells) using antibodies against the appropriate antigen. In this case, the appropriate antigen is the protein product of the tissue-specific gene expression. Although, in principle, a first antibody (i.e., the antibody that binds the antigen) can be labeled, it is more common (and improves the visualization) to use a second antibody directed against the first (e.g., an anti-IgG). This second antibody is conjugated either with fluorochromes, or appropriate enzymes for colorimetric reactions, or gold beads (for electron microscopy), or with the biotin-avidin system, so that the location of the primary antibody, and thus the antigen, can be recognized. 
     The CRISPR Cas molecules of the present invention may be delivered to the ear by direct application of pharmaceutical composition to the outer ear, with compositions modified from US Published application, 20110142917. In some embodiments the pharmaceutical composition is applied to the ear canal. Delivery to the ear may also be referred to as aural or otic delivery. 
     In some embodiments, the CRISPR-Cas systems or components thereof and/or vectors or vector systems can be delivered to ear via a transfection to the inner ear through the intact round window by a novel proteidic delivery technology which may be applied to the nucleic acid-targeting system of the present invention (see, e.g., Qi et al., Gene Therapy (2013), 1-9). About 40 μl of 10 mM RNA may be contemplated as the dosage for administration to the ear. 
     According to Rejali et al. (Hear Res. 2007 June; 228(1-2):180-7), cochlear implant function can be improved by good preservation of the spiral ganglion neurons, which are the target of electrical stimulation by the implant and brain derived neurotrophic factor (BDNF) has previously been shown to enhance spiral ganglion survival in experimentally deafened ears. Rejali et al. tested a modified design of the cochlear implant electrode that includes a coating of fibroblast cells transduced by a viral vector with a BDNF gene insert. To accomplish this type of ex vivo gene transfer, Rejali et al. transduced guinea pig fibroblasts with an adenovirus with a BDNF gene cassette insert, and determined that these cells secreted BDNF and then attached BDNF-secreting cells to the cochlear implant electrode via an agarose gel, and implanted the electrode in the scala tympani. Rejali et al. determined that the BDNF expressing electrodes were able to preserve significantly more spiral ganglion neurons in the basal turns of the cochlea after 48 days of implantation when compared to control electrodes and demonstrated the feasibility of combining cochlear implant therapy with ex vivo gene transfer for enhancing spiral ganglion neuron survival. Such a system may be applied to the nucleic acid-targeting system of the present invention for delivery to the ear. 
     In some embodiments, the system set forth in Mukherj ea et al. (Antioxidants &amp; Redox Signaling, Volume 13, Number 5, 2010) can be adapted for transtympanic administration of the CRISPR-Cas system or component thereof to the ear. In some embodiments, a dosage of about 2 mg to about 4 mg of CRISPR Cas for administration to a human. 
     In some embodiments, the system set forth in [Jung et al. (Molecular Therapy, vol. 21 no. 4, 834-841 April 2013) can be adapted for vestibular epithelial delivery of the CRISPR-Cas system or component thereof to the ear. In some embodiments, a dosage of about 1 to about 30 mg of CRISPR Cas for administration to a human. 
     Treating Diseases in Non-Dividing Cells 
     In some embodiments, the gene or transcript to be corrected is in a non-dividing cell. Exemplary non-dividing cells are muscle cells or neurons. Non-dividing (especially non-dividing, fully differentiated) cell types present issues for gene targeting or genome engineering, for example because homologous recombination (HR) is generally suppressed in the G1 cell-cycle phase. However, while studying the mechanisms by which cells control normal DNA repair systems, Durocher discovered a previously unknown switch that keeps HR “off” in non-dividing cells and devised a strategy to toggle this switch back on. Orthwein et al. (Daniel Durocher&#39;s lab at the Mount Sinai Hospital in Ottawa, Canada) recently reported (Nature 16142, published online 9 Dec. 2015) have shown that the suppression of HR can be lifted and gene targeting successfully concluded in both kidney (293T) and osteosarcoma (U20S) cells. Tumor suppressors, BRCA1, PALB2 and BRAC2 are known to promote DNA DSB repair by HR. They found that formation of a complex of BRCA1 with PALB2-BRAC2 is governed by a ubiquitin site on PALB2, such that action on the site by an E3 ubiquitin ligase. This E3 ubiquitin ligase is composed of KEAP1 (a PALB2-interacting protein) in complex with cullin-3 (CUL3)-RBX1. PALB2 ubiquitylation suppresses its interaction with BRCA1 and is counteracted by the deubiquitylase USP11, which is itself under cell cycle control. Restoration of the BRCA1-PALB2 interaction combined with the activation of DNA-end resection is sufficient to induce homologous recombination in G1, as measured by a number of methods including a CRISPR-Cas9-based gene-targeting assay directed at USP11 or KEAP1 (expressed from a pX459 vector). However, when the BRCA1-PALB2 interaction was restored in resection-competent G1 cells using either KEAP1 depletion or expression of the PALB2-KR mutant, a robust increase in gene-targeting events was detected. These teachings can be adapted for and/or applied to the Cas CRISPR-Cas systems described herein. 
     Thus, reactivation of HR in cells, especially non-dividing, fully differentiated cell types is preferred, in some embodiments. In some embodiments, promotion of the BRCA1-PALB2 interaction is preferred in some embodiments. In some embodiments, the target ell is a non-dividing cell. In some embodiments, the target cell is a neuron or muscle cell. In some embodiments, the target cell is targeted in vivo. In some embodiments, the cell is in G1 and HR is suppressed. In some embodiments, use of KEAP1 depletion, for example inhibition of expression of KEAP1 activity, is preferred. KEAP1 depletion may be achieved through siRNA, for example as shown in Orthwein et al. Alternatively, expression of the PALB2-KR mutant (lacking all eight Lys residues in the BRCA1-interaction domain is preferred, either in combination with KEAP1 depletion or alone. PALB2-KR interacts with BRCA1 irrespective of cell cycle position. Thus, promotion or restoration of the BRCA1-PALB2 interaction, especially in G1 cells, is preferred in some embodiments, especially where the target cells are non-dividing, or where removal and return (ex vivo gene targeting) is problematic, for example neuron or muscle cells. KEAP1 siRNA is available from ThermoFischer. In some embodiments, a BRCA1-PALB2 complex may be delivered to the G1 cell. In some embodiments, PALB2 deubiquitylation may be promoted for example by increased expression of the deubiquitylase USP11, so it is envisaged that a construct may be provided to promote or up-regulate expression or activity of the deubiquitylase USP11. 
     Treating Diseases of the Eye 
     In some embodiments, the disease to be treated is a disease that affects the eyes. Thus, in some embodiments, the CRISPR-Cas system or component thereof described herein is delivered to one or both eyes. 
     The CRISPR-Cas system can be used to correct ocular defects that arise from several genetic mutations further described in Genetic Diseases of the Eye, Second Edition, edited by Elias I. Traboulsi, Oxford University Press, 2012. 
     In some embodiments, the condition to be treated or targeted is an eye disorder. In some embodiments, the eye disorder may include glaucoma. In some embodiments, the eye disorder includes a retinal degenerative disease. In some embodiments, the retinal degenerative disease is selected from Stargardt disease, Bardet-Biedl Syndrome, Best disease, Blue Cone Monochromacy, Choroidermia, Cone-rod dystrophy, Congenital Stationary Night Blindness, Enhanced S-Cone Syndrome, Juvenile X-Linked Retinoschisis, Leber Congenital Amaurosis, Malattia Leventinesse, Norrie Disease or X-linked Familial Exudative Vitreoretinopathy, Pattern Dystrophy, Sorsby Dystrophy, Usher Syndrome, Retinitis Pigmentosa, Achromatopsia or Macular dystrophies or degeneration, Retinitis Pigmentosa, Achromatopsia, and age related macular degeneration. In some embodiments, the retinal degenerative disease is Leber Congenital Amaurosis (LCA) or Retinitis Pigmentosa. Other exemplary eye diseases are described in greater detail elsewhere herein. 
     In some embodiments, the CRISPR-Cas system is delivered to the eye, optionally via intravitreal injection or subretinal injection. Intraocular injections may be performed with the aid of an operating microscope. For subretinal and intravitreal injections, eyes may be prolapsed by gentle digital pressure and fundi visualized using a contact lens system consisting of a drop of a coupling medium solution on the cornea covered with a glass microscope slide coverslip. For subretinal injections, the tip of a 10-mm 34-gauge needle, mounted on a 5-μl Hamilton syringe may be advanced under direct visualization through the superior equatorial sclera tangentially towards the posterior pole until the aperture of the needle was visible in the subretinal space. Then, 2 μl of vector suspension may be injected to produce a superior bullous retinal detachment, thus confirming subretinal vector administration. This approach creates a self-sealing sclerotomy allowing the vector suspension to be retained in the subretinal space until it is absorbed by the RPE, usually within 48 h of the procedure. This procedure may be repeated in the inferior hemisphere to produce an inferior retinal detachment. This technique results in the exposure of approximately 70% of neurosensory retina and RPE to the vector suspension. For intravitreal injections, the needle tip may be advanced through the sclera 1 mm posterior to the corneoscleral limbus and 2 μl of vector suspension injected into the vitreous cavity. For intracameral injections, the needle tip may be advanced through a corneoscleral limbal paracentesis, directed towards the central cornea, and 2 μl of vector suspension may be injected. For intracameral injections, the needle tip may be advanced through a corneoscleral limbal paracentesis, directed towards the central cornea, and 2 μl of vector suspension may be injected. These vectors may be injected at titers of either 1.0-1.4×10 10  or 1.0-1.4×10 9  transducing units (TU)/ml. 
     In some embodiments, for administration to the eye, lentiviral vectors. In some embodiments, the lentiviral vector is an equine infectious anemia virus (EIAV) vector. Exemplary EIAV vectors for eye delivery are described in Balagaan, J Gene Med 2006; 8: 275-285, Published online 21 Nov. 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jgm.845; Binley et al., HUMAN GENE THERAPY 23:980-991 (September 2012), which can be adapted for use with the CRISPR-Cas system described herein. In some embodiments, the dosage can be 1.1×10 5  transducing units per eye (TU/eye) in a total volume of 100 μl. 
     Other viral vectors can also be used for delivery to the eye, such as AAV vectors, such as those described in Campochiaro et al., Human Gene Therapy 17:167-176 (February 2006), Millington-Ward et al. (Molecular Therapy, vol. 19 no. 4, 642-649 April 2011; Dalkara et al. (Sci Transl Med 5, 189ra76 (2013)), which can be adapted for use with the CRISPR-Cas system described herein. In some embodiments, the dose can range from about 10 6  to 10 9.5  particle units. In the context of the Millington-Ward AAV vectors, a dose of about 2×10 11  to about 6×10 13  virus particles can be administered. In the context of Dalkara vectors, a dose of about 1×10 15  to about 1×10 16  vg/ml administered to a human. 
     In some embodiments, the sd-rxRNA® system of RXi Pharmaceuticals may be used/and or adapted for delivering CRISPR-Cas system to the eye. In this system, a single intravitreal administration of 3 μg of sd-rxRNA results in sequence-specific reduction of PPIB mRNA levels for 14 days. The sd-rxRNA® system may be applied to the nucleic acid-targeting system of the present invention, contemplating a dose of about 3 to 20 mg of CRISPR administered to a human. 
     In other embodiments, the methods of US Patent Publication No. 20130183282, which is directed to methods of cleaving a target sequence from the human rhodopsin gene, may also be modified to the nucleic acid-targeting system of the present invention. 
     In other embodiments, the methods of US Patent Publication No. 20130202678 for treating retinopathies and sight-threatening ophthalmologic disorders relating to delivering of the Puf-A gene (which is expressed in retinal ganglion and pigmented cells of eye tissues and displays a unique anti-apoptotic activity) to the sub-retinal or intravitreal space in the eye. In particular, desirable targets are zgc:193933, prdm1a, spata2, tex10, rbb4, ddx3, zp2.2, Blimp-1 and HtrA2, all of which may be targeted by the CRISPR-Cas system of the present invention. 
     Wu (Cell Stem Cell, 13:659-62, 2013) designed a guide RNA that led Cas9 to a single base pair mutation that causes cataracts in mice, where it induced DNA cleavage. Then using either the other wild-type allele or oligos given to the zygotes repair mechanisms corrected the sequence of the broken allele and corrected the cataract-causing genetic defect in mutant mouse. This approach can be adapted to and/or applied to the CRISPR-Cas systems described herein. 
     US Patent Publication No. 20120159653, describes use of zinc finger nucleases to genetically modify cells, animals and proteins associated with macular degeneration (MD), the teachings of which can be applied to and/or adapted for the CRISPR-Cas systems described herein. 
     One aspect of US Patent Publication No. 20120159653 relates to editing of any chromosomal sequences that encode proteins associated with MD which may be applied to the nucleic acid-targeting system of the present invention. 
     Treating Muscle Diseases and Cardiovascular Diseases 
     In some embodiments, the CRISPR-Cas system can be used to treat and/or prevent a muscle disease and associated circulatory or cardiovascular disease or disorder. The present invention also contemplates delivering the CRISPR-Cas system described herein, e.g. Cas effector protein systems, to the heart. For the heart, a myocardium tropic adeno-associated virus (AAVM) is preferred, in particular AAVM41 which showed preferential gene transfer in the heart (see, e.g., Lin-Yanga et al., PNAS, Mar. 10, 2009, vol. 106, no. 10). Administration may be systemic or local. A dosage of about 1-10×10 14  vector genomes are contemplated for systemic administration. See also, e.g., Eulalio et al. (2012) Nature 492: 376 and Somasuntharam et al. (2013) Biomaterials 34: 7790, the teachings of which can be adapted for and/or applied to the CRISPR-Cas systems described herein. 
     For example, US Patent Publication No. 20110023139, the teachings of which can be adapted for and/or applied to the CRISPR-Cas systems described herein describes use of zinc finger nucleases to genetically modify cells, animals and proteins associated with cardiovascular disease. Cardiovascular diseases generally include high blood pressure, heart attacks, heart failure, and stroke and TIA. Any chromosomal sequence involved in cardiovascular disease or the protein encoded by any chromosomal sequence involved in cardiovascular disease may be utilized in the methods described in this disclosure. The cardiovascular-related proteins are typically selected based on an experimental association of the cardiovascular-related protein to the development of cardiovascular disease. For example, the production rate or circulating concentration of a cardiovascular-related protein may be elevated or depressed in a population having a cardiovascular disorder relative to a population lacking the cardiovascular disorder. Differences in protein levels may be assessed using proteomic techniques including but not limited to Western blot, immunohistochemical staining, enzyme linked immunosorbent assay (ELISA), and mass spectrometry. Alternatively, the cardiovascular-related proteins may be identified by obtaining gene expression profiles of the genes encoding the proteins using genomic techniques including but not limited to DNA microarray analysis, serial analysis of gene expression (SAGE), and quantitative real-time polymerase chain reaction (Q-PCR). Exemplary chromosomal sequences can be found in Table 12. 
     The CRISPR-Cas systems herein can be used for treating diseases of the muscular system. The present invention also contemplates delivering the CRISPR-Cas system described herein, e.g. Cas (e.g. Cas9 and/or Cas12) effector protein systems, to muscle(s). 
     In some embodiments, the muscle disease to be treated is a muscle dystrophy such as DMD. In some embodiments, the CRISPR-Cas system, such as a system capable of RNA modification, described herein can be used to achieve exon skipping to achieve correction of the diseased gene. As used herein, the term “exon skipping” refers to the modification of pre-mRNA splicing by the targeting of splice donor and/or acceptor sites within a pre-mRNA with one or more complementary antisense oligonucleotide(s) (AONs). By blocking access of a spliceosome to one or more splice donor or acceptor site, an AON may prevent a splicing reaction thereby causing the deletion of one or more exons from a fully-processed mRNA. Exon skipping may be achieved in the nucleus during the maturation process of pre-mRNAs. In some examples, exon skipping may include the masking of key sequences involved in the splicing of targeted exons by using a CRISPR-Cas system described herein capable of RNA modification. In some embodiments, exon skipping can be achieved in dystrophin mRNA. In some embodiments, the CRISPR-Cas system can induce exon skipping at exon 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 45, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or any combination thereof of the dystrophin mRNA. In some embodiments, the CRISPR-Cas system can induce exon skipping at exon 43, 44, 50, 51, 52, 55, or any combination thereof of the dystrophin mRNA. Mutations in these exons, can also be corrected using non-exon skipping polynucleotide modification methods. 
     In some embodiments, for treatment of a muscle disease, the method of Bortolanza et al. Molecular Therapy vol. 19 no. 11, 2055-264 November 2011) may be applied to an AAV expressing CRISPR Cas and injected into humans at a dosage of about 2×10 15  or 2×10 16  vg of vector. The teachings of Bortolanza et al., can be adapted for and/or applied to the CRISPR-Cas systems described herein. 
     In some embodiments, the method of Dumonceaux et al. (Molecular Therapy vol. 18 no. 5, 881-887 May 2010) may be applied to an AAV expressing CRISPR Cas and injected into humans, for example, at a dosage of about 10 14  to about 10 15  vg of vector. The teachings of Dumonceaux described herein can be adapted for and/or applied to the CRISPR-Cas systems described herein. 
     In some embodiments, the method of Kinouchi et al. (Gene Therapy (2008) 15, 1126-1130) may be applied to CRISPR Cas systems described herein and injected into a human, for example, at a dosage of about 500 to 1000 ml of a 40 μM solution into the muscle. 
     In some embodiments, the method of Hagstrom et al. (Molecular Therapy Vol. 10, No. 2, August 2004) can be adapted for and/or applied to the CRISPR-Cas systems herein and injected at a dose of about 15 to about 50 mg into the great saphenous vein of a human. 
     Treating Diseases of the Liver and Kidney 
     In some embodiments, the CRISPR-Cas system or component thereof described herein can be used to treat a disease of the kidney or liver. Thus, in some embodiments, delivery of the CRISRP-Cas system or component thereof described herein is to the liver or kidney. 
     Delivery strategies to induce cellular uptake of the therapeutic nucleic acid include physical force or vector systems such as viral-, lipid- or complex-based delivery, or nanocarriers. From the initial applications with less possible clinical relevance, when nucleic acids were addressed to renal cells with hydrodynamic high-pressure injection systemically, a wide range of gene therapeutic viral and non-viral carriers have been applied already to target posttranscriptional events in different animal kidney disease models in vivo (Csaba Révész and Peter Hamar (2011). Delivery Methods to Target RNAs in the Kidney, Gene Therapy Applications, Prof. Chunsheng Kang (Ed.), ISBN: 978-953-307-541-9, InTech, Available from: www.intechopen.com/books/gene-therapy-applications/delivery-methods-to-target-rnas-inthe-kidney). Delivery methods to the kidney may include those in Yuan et al. (Am J Physiol Renal Physiol 295: F605-F617, 2008). The method of Yuang et al. may be applied to the CRISPR Cas system of the present invention contemplating a 1-2 g subcutaneous injection of CRISPR Cas conjugated with cholesterol to a human for delivery to the kidneys. In some embodiments, the method of Molitoris et al. (J Am Soc Nephrol 20: 1754-1764, 2009) can be adapted to the CRISRP-Cas system of the present invention and a cumulative dose of 12-20 mg/kg to a human can be used for delivery to the proximal tubule cells of the kidneys. In some embodiments, the methods of Thompson et al. (Nucleic Acid Therapeutics, Volume 22, Number 4, 2012) can be adapted to the CRISRP-Cas system of the present invention and a dose of up to 25 mg/kg can be delivered via i.v. administration. In some embodiments, the method of Shimizu et al. (J Am Soc Nephrol 21: 622-633, 2010) can be adapted to the CRISRP-Cas system of the present invention and a dose of about of 10-20 μmol CRISPR Cas complexed with nanocarriers in about 1-2 liters of a physiologic fluid for i.p. administration can be used. 
     Other various delivery vehicles can be used to deliver the CRISPR-Cas system to the kidney such as viral, hydrodynamic, lipid, polymer nanoparticles, aptamers and various combinations thereof (see e.g. Larson et al., Surgery, (August 2007), Vol. 142, No. 2, pp. (262-269); Hamar et al., Proc Natl Acad Sci, (October 2004), Vol. 101, No. 41, pp. (14883-14888); Zheng et al., Am J Pathol, (October 2008), Vol. 173, No. 4, pp. (973-980); Feng et al., Transplantation, (May 2009), Vol. 87, No. 9, pp. (1283-1289); Q. Zhang et al., PloS ONE, (July 2010), Vol. 5, No. 7, e11709, pp. (1-13); Kushibikia et al., J Controlled Release, (July 2005), Vol. 105, No. 3, pp. (318-331); Wang et al., Gene Therapy, (July 2006), Vol. 13, No. 14, pp. (1097-1103); Kobayashi et al., Journal of Pharmacology and Experimental Therapeutics, (February 2004), Vol. 308, No. 2, pp. (688-693); Wolfrum et al., Nature Biotechnology, (September 2007), Vol. 25, No. 10, pp. (1149-1157); Molitoris et al., J Am Soc Nephrol, (August 2009), Vol. 20, No. 8 pp. (1754-1764); Mikhaylova et al., Cancer Gene Therapy, (March 2011), Vol. 16, No. 3, pp. (217-226); Y. Zhang et al., J Am Soc Nephrol, (April 2006), Vol. 17, No. 4, pp. (1090-1101); Singhal et al., Cancer Res, (May 2009), Vol. 69, No. 10, pp. (4244-4251); Malek et al., Toxicology and Applied Pharmacology, (April 2009), Vol. 236, No. 1, pp. (97-108); Shimizu et al., J Am Soc Nephrology, (April 2010), Vol. 21, No. 4, pp. (622-633); Jiang et al., Molecular Pharmaceutics, (May-June 2009), Vol. 6, No. 3, pp. (727-737); Cao et al, J Controlled Release, (June 2010), Vol. 144, No. 2, pp. (203-212); Ninichuk et al., Am J Pathol, (March 2008), Vol. 172, No. 3, pp. (628-637); Purschke et al., Proc Natl Acad Sci, (March 2006), Vol. 103, No. 13, pp. (5173-5178). 
     In some embodiments, delivery is to liver cells. In some embodiments, the liver cell is a hepatocyte. Delivery of the CRISPR protein, such as Cas effector (e.g. Cas9 and/or Cas12) herein may be via viral vectors, especially AAV (and in particular AAV2/6) vectors. These can be administered by intravenous injection. A preferred target for the liver, whether in vitro or in vivo, is the albumin gene. This is a so-called ‘safe harbor” as albumin is expressed at very high levels and so some reduction in the production of albumin following successful gene editing is tolerated. It is also preferred as the high levels of expression seen from the albumin promoter/enhancer allows for useful levels of correct or transgene production (from the inserted donor template) to be achieved even if only a small fraction of hepatocytes are edited. See sites identified by Wechsler et al. (reported at the 57th Annual Meeting and Exposition of the American Society of Hematology—abstract available online at https://ash.confex.com/ash/2015/webprogram/Paper86495.html and presented on 6th December 2015) which can be adapted for use with the CRISPR-Cas systems herein. 
     Exemplary liver and kidney diseases that can be treated and/or prevented are described elsewhere herein. 
     Treating Epithelial and Lung Diseases 
     In some embodiments, the disease treated or prevented by the CRISPR-Cas system described herein can be a lung or epithelial disease. The CRISPR-Cas systems described herein can be used for treating epithelial and/or lung diseases. The present invention also contemplates delivering the CRISPR-Cas system described herein, e.g. Cas (e.g. Cas9 and/or Cas12) effector systems, to one or both lungs. 
     In some embodiments, as viral vector can be used to deliver the CRISPR-Cas system or component thereof to the lungs. In some embodiments, the AAV is an AAV-1, AAV-2, AAV-5, AAV-6, and/or AAV-9 for delivery to the lungs. (see, e.g., Li et al., Molecular Therapy, vol. 17 no. 12, 2067-277 December 2009). In some embodiments, the MOI can vary from 1×10 3  to 4×10 5  vector genomes/cell. In some embodiments, the delivery vector can be an RSV vector as in Zamora et al. (Am J Respir Crit Care Med Vol 183. pp 531-538, 2011. The method of Zamora et al. may be applied to the nucleic acid-targeting system of the present invention and an aerosolized CRISPR Cas, for example with a dosage of 0.6 mg/kg, may be contemplated for the present invention. 
     Subjects treated for a lung disease may for example receive pharmaceutically effective amount of aerosolized AAV vector system per lung endobronchially delivered while spontaneously breathing. As such, aerosolized delivery is preferred for AAV delivery in general. An adenovirus or an AAV particle may be used for delivery. Suitable gene constructs, each operably linked to one or more regulatory sequences, may be cloned into the delivery vector. In this instance, the following constructs are provided as examples: Cbh or EF1a promoter for Cas (Cas (e.g. Cas9 and/or Cas12)), U6 or H1 promoter for guide RNA). A preferred arrangement is to use a CFTRdelta508 targeting guide, a repair template for deltaF508 mutation and a codon optimized Cas (e.g. Cas9 and/or Cas12) enzyme, with optionally one or more nuclear localization signal or sequence(s) (NLS(s)), e.g., two (2) NLSs. 
     Treating Diseases of the Skin 
     The CRISPR-Cas systems described herein can be used for the treatment of skin diseases. The present invention also contemplates delivering the CRISPR-Cas system described herein, e.g. Cas (e.g. Cas9 and/or Cas12) effector protein systems, to the skin. 
     In some embodiments, delivery to the skin (intradermal delivery) of the CRISPR-Cas system or component thereof can be via one or more microneedles or microneedle containing device. For example, in some embodiments the device and methods of Hickerson et al. (Molecular Therapy—Nucleic Acids (2013) 2, e129) can be used and/or adapted to deliver the CRISPR-Cas system described herein, for example, at a dosage of up to 300 μl of 0.1 mg/ml CRISPR-Cas (e.g. Cas9 and/or Cas12) system to the skin. 
     In some embodiments, the methods and techniques of Leachman et al. (Molecular Therapy, vol. 18 no. 2, 442-446 February 2010) can be used and/or adapted for delivery of a CIRPSR-Cas system described herein to the skin. 
     In some embodiments, the methods and techniques of Zheng et al. (PNAS, Jul. 24, 2012, vol. 109, no. 30, 11975-11980) can be used and/or adapted for nanoparticle delivery of a CIRPSR-Cas system described herein to the skin. In some embodiments, as dosage of about 25 nM applied in a single application can achieve gene knockdown in the skin. 
     Treating Cancer 
     The CRISPR-Cas systems described herein can be used for the treatment of cancer. The present invention also contemplates delivering the CRISPR-Cas system described herein, e.g. Cas (e.g. Cas9 and/or Cas12) effector protein systems, to a cancer cell. Also, as is described elsewhere herein the CRISPR-Cas systems can be used to modify an immune cell, such as a CAR or CAR T cell, which can then in turn be used to treat and/or prevent cancer. This is also described in WO2015161276, the disclosure of which is hereby incorporated by reference and described herein below. 
     Target genes suitable for the treatment or prophylaxis of cancer can include those set forth in Tables 12 and 13. In some embodiments, target genes for cancer treatment and prevention can also include those described in WO2015048577 the disclosure of which is hereby incorporated by reference and can be adapted for and/or applied to the CRISPR-Cas system described herein. 
     Diseases 
     Genetic Diseases and Diseases with a Genetic and/or Epigenetic Aspect 
     The CRISPR-Cas systems or components thereof can be used to treat and/or prevent a genetic disease or a disease with a genetic and/or epigenetic aspect. The genes and conditions exemplified herein are not exhaustive. In some embodiments, a method of treating and/or preventing a genetic disease can include administering a CRISPR-Cas system and/or one or more components thereof to a subject, where the CRISPR-Cas system and/or one or more components thereof is capable of modifying one or more copies of one or more genes associated with the genetic disease or a disease with a genetic and/or epigenetic aspect in one or more cells of the subject. In some embodiments, modifying one or more copies of one or more genes associated with a genetic disease or a disease with a genetic and/or epigenetic aspect in the subject can eliminate a genetic disease or a symptom thereof in the subject. In some embodiments, modifying one or more copies of one or more genes associated with a genetic disease or a disease with a genetic and/or epigenetic aspect in the subject can decrease the severity of a genetic disease or a symptom thereof in the subject. In some embodiments, the CRISPR-Cas systems or components thereof can modify one or more genes or polynucleotides associated with one or more diseases, including genetic diseases and/or those having a genetic aspect and/or epigenetic aspect, including but not limited to, any one or more set forth in Table 12. It will be appreciated that those diseases and associated genes listed herein are non-exhaustive and non-limiting. Further some genes play roles in the development of multiple diseases. 
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 Exemplary Genetic and Other Diseases and Associated Genes 
               
            
           
           
               
               
               
               
            
               
                   
                 Primary 
                 Additional 
                   
               
               
                   
                 Tissues or 
                 Tissues/ 
               
               
                   
                 System 
                 Systems 
               
               
                 Disease Name 
                 Affected 
                 Affected 
                 Genes 
               
               
                   
               
               
                 Achondroplasia 
                 Bone and 
                   
                 fibroblast growth factor receptor 3 
               
               
                   
                 Muscle 
                   
                 (FGFR3) 
               
               
                 Achromatopsia 
                 eye 
                   
                 CNGA3, CNGB3, GNAT2, PDE6C, 
               
               
                   
                   
                   
                 PDE6H, ACHM2, ACHM3, 
               
               
                 Acute Renal Injury 
                 kidney 
                   
                 NFkappaB, AATF, p85alpha, FAS, 
               
               
                   
                   
                   
                 Apoptosis cascade elements (e.g. 
               
               
                   
                   
                   
                 FASR, Caspase 2, 3, 4, 6, 7, 8, 9, 10, 
               
               
                   
                   
                   
                 AKT, TNF alpha, IGF1, IGF1R, 
               
               
                   
                   
                   
                 RIPK1), p53 
               
               
                 Age Related Macular 
                 eye 
                   
                 Abcr; CCL2; CC2; CP 
               
               
                 Degeneration 
                   
                   
                 (ceruloplasmin); Timp3; cathepsinD; 
               
               
                   
                   
                   
                 VLDLR, CCR2 
               
               
                 AIDS 
                 Immune System 
                   
                 KIR3DL1, NKAT3, NKB1, AMB11, 
               
               
                   
                   
                   
                 KIR3DS1, IFNG, CXCL12, SDF1 
               
               
                 Albinism (including 
                 Skin, hair, eyes, 
                   
                 TYR, OCA2, TYRP1, and SLC45A2, 
               
               
                 oculocutaneous albinism (types 
                   
                   
                 SLC24A5 and C10orf11 
               
               
                 1-7) and ocular albinism) 
               
               
                 Alkaptonuria 
                 Metabolism of 
                 Tissues/organs 
                 HGD 
               
               
                   
                 amino acids 
                 where 
               
               
                   
                   
                 homogentisic 
               
               
                   
                   
                 acid 
               
               
                   
                   
                 accumulates, 
               
               
                   
                   
                 particularly 
               
               
                   
                   
                 cartilage (joints), 
               
               
                   
                   
                 heart valves, 
               
               
                   
                   
                 kidneys 
               
               
                 alpha-1 antitrypsin deficiency 
                 Lung 
                 Liver, skin, 
                 SERPINA1, those set forth in 
               
               
                 (AATD or A1AD) 
                   
                 vascular system, 
                 WO2017165862, PiZ allele 
               
               
                   
                   
                 kidneys, GI 
               
               
                 ALS 
                 CNS 
                   
                 SOD1; ALS2; ALS3; ALS5; 
               
               
                   
                   
                   
                 ALS7; STEX; FUS; TARDBP; VEGF 
               
               
                   
                   
                   
                 (VEGF-a; 
               
               
                   
                   
                   
                 VEGF-b; VEGF-c); DPP6; NEFH, 
               
               
                   
                   
                   
                 PTGS1, SLC1A2, TNFRSF10B, 
               
               
                   
                   
                   
                 PRPH, HSP90AA1, CRIA2, IFNG, 
               
               
                   
                   
                   
                 AMPA2 S100B, FGF2, AOX1, CS, 
               
               
                   
                   
                   
                 TXN, RAPHJ1, MAP3K5, NBEAL1, 
               
               
                   
                   
                   
                 GPX1, ICA1L, RAC1, MAPT, ITPR2, 
               
               
                   
                   
                   
                 ALS2CR4, GLS, ALS2CR8, CNTFR, 
               
               
                   
                   
                   
                 ALS2CR11, FOLH1, FAM117B, 
               
               
                   
                   
                   
                 P4HB, CNTF, SQSTM1, STRADB, 
               
               
                   
                   
                   
                 NAIP, NLR, YWHAQ, SLC33A1, 
               
               
                   
                   
                   
                 TRAK2, SCA1, NIF3L1, NIF3, 
               
               
                   
                   
                   
                 PARD3B, COX8A, CDK15, HECW1, 
               
               
                   
                   
                   
                 HECT, C2, WW 15, NOS1, MET, 
               
               
                   
                   
                   
                 SOD2, HSPB1, NEFL, CTSB, ANG, 
               
               
                   
                   
                   
                 HSPA8, RNase A, VAPB, VAMP, 
               
               
                   
                   
                   
                 SNCA, alpha HGF, CAT, ACTB, 
               
               
                   
                   
                   
                 NEFM, TH, BCL2, FAS, CASP3, 
               
               
                   
                   
                   
                 CLU, SMN1, G6PD, BAX, HSF1, 
               
               
                   
                   
                   
                 RNF19A, JUN, ALS2CR12, HSPA5, 
               
               
                   
                   
                   
                 MAPK14, APEX1, TXNRD1, NOS2, 
               
               
                   
                   
                   
                 TIMP1, CASP9, XIAP, GLG1, EPO, 
               
               
                   
                   
                   
                 VEGFA, ELN, GDNF, NFE2L2, 
               
               
                   
                   
                   
                 SLC6A3, HSPA4, APOE, PSMB8, 
               
               
                   
                   
                   
                 DCTN2, TIMP3, KIFAP3, SLC1A1, 
               
               
                   
                   
                   
                 SMN2, CCNC, STUB1, ALS2, 
               
               
                   
                   
                   
                 PRDX6, SYP, CABIN1, CASP1, 
               
               
                   
                   
                   
                 GART, CDK5, ATXN3, RTN4, 
               
               
                   
                   
                   
                 C1QB, VEGFC, HTT, PARK7, XDH, 
               
               
                   
                   
                   
                 GFAP, MAP2, CYCS, FCGR3B, CCS, 
               
               
                   
                   
                   
                 UBL5, MMP9m SLC18A3, TRPM7, 
               
               
                   
                   
                   
                 HSPB2, AKT1, DEERL1, CCL2, 
               
               
                   
                   
                   
                 NGRN, GSR, TPPP3, APAF1, 
               
               
                   
                   
                   
                 BTBD10, GLUD1, CXCR4, S:C1A3, 
               
               
                   
                   
                   
                 FLT1, PON1, AR, LIF, ERBB3, :GA:S1, 
               
               
                   
                   
                   
                 CD44, TP53, TLR3, GRIA1, 
               
               
                   
                   
                   
                 GAPDH, AMPA, GRIK1, DES, 
               
               
                   
                   
                   
                 CHAT, FLT4, CHMP2B, BAG1, 
               
               
                   
                   
                   
                 CHRNA4, GSS, BAK1, KDR, GSTP1, 
               
               
                   
                   
                   
                 OGG1, IL6 
               
               
                 Alzheimer&#39;s Disease 
                 Brain 
                   
                 E1; CHIP; UCH; UBB; Tau; LRP; 
               
               
                   
                   
                   
                 PICALM; CLU; PS1; 
               
               
                   
                   
                   
                 SORL1; CR1; VLDLR; UBA1; 
               
               
                   
                   
                   
                 UBA3; CHIP28; AQP1; UCHL1; 
               
               
                   
                   
                   
                 UCHL3; APP, AAA, CVAP, AD1, 
               
               
                   
                   
                   
                 APOE, AD2, DCP1, ACE1, MPO, 
               
               
                   
                   
                   
                 PACIP1, PAXIP1L, PTIP, A2M, 
               
               
                   
                   
                   
                 BLMH, BMH, PSEN1, AD3, ALAS2, 
               
               
                   
                   
                   
                 ABCA1, BIN1, BDNF, BTNL8, 
               
               
                   
                   
                   
                 C1ORF49, CDH4, CHRNB2, 
               
               
                   
                   
                   
                 CKLFSF2, CLEC4E, CR1L, CSF3R, 
               
               
                   
                   
                   
                 CST3, CYP2C, DAPK1, ESR1, 
               
               
                   
                   
                   
                 FCAR, FCGR3B, FFA2, FGA, GAB2, 
               
               
                   
                   
                   
                 GALP, GAPDHS, GMPB, HP, HTR7, 
               
               
                   
                   
                   
                 IDE, IF127, IFI6, IFIT2, IL1RN, IL- 
               
               
                   
                   
                   
                 1RA, IL8RA, IL8RB, JAG1, KCNJ15, 
               
               
                   
                   
                   
                 LRP6, MAPT, MARK4, MPHOSPH1, 
               
               
                   
                   
                   
                 MTHFR, NBN, NCSTN, NIACR2, 
               
               
                   
                   
                   
                 NMNAT3, NTM, ORM1, P2RY13, 
               
               
                   
                   
                   
                 PBEF1, PCK1, PICALM, PLAU, 
               
               
                   
                   
                   
                 PLXNC1, PRNP, PSEN1, PSEN2, 
               
               
                   
                   
                   
                 PTPRA, RALGPS2, RGSL2, 
               
               
                   
                   
                   
                 SELENBP1, SLC25A37, SORL1, 
               
               
                   
                   
                   
                 Mitoferrin-1, TF, TFAM, TNF, 
               
               
                   
                   
                   
                 TNFRSF10C, UBE1C 
               
               
                 Amyloidosis 
                   
                   
                 APOA1, APP, AAA, CVAP, AD1, 
               
               
                   
                   
                   
                 GSN, FGA, LYZ, TTR, PALB 
               
               
                 Amyloid neuropathy 
                   
                   
                 TTR, PALB 
               
               
                 Anemia 
                 Blood 
                   
                 CDAN1, CDA1, RPS19, DBA, PKLR, 
               
               
                   
                   
                   
                 PK1, NT5C3, UMPH1, PSN1, RHAG, 
               
               
                   
                   
                   
                 RH50A, NRAMP2, SPTB, ALAS2, 
               
               
                   
                   
                   
                 ANH1, ASB, ABCB7, ABC7, ASAT 
               
               
                 Angelman Syndrome 
                 Nervous system, 
                   
                 UBE3A 
               
               
                   
                 brain 
               
               
                 Attention Deficit Hyperactivity 
                 Brain 
                   
                 PTCHD1 
               
               
                 Disorder (ADHD) 
               
               
                 Autoimmune lymphoproliferative 
                 Immune system 
                   
                 TNFRSF6, APT1, FAS, CD95, 
               
               
                 syndrome 
                   
                   
                 ALPS1A 
               
               
                 Autism, Autism spectrum 
                 Brain 
                   
                 PTCHD1; Mecp2; BZRAP1; MDGA2; 
               
               
                 disorders (ASDs), including 
                   
                   
                 Sema5A; Neurexin 1; GLO1, RTT, 
               
               
                 Asperger&#39;s and a general 
                   
                   
                 PPMX, MRX16, RX79, NLGN3, 
               
               
                 diagnostic category called 
                   
                   
                 NLGN4, KIAA1260, AUTSX2, 
               
               
                 Pervasive Developmental 
                   
                   
                 FMRI, FMR2; FXR1; FXR2; 
               
               
                 Disorders (PDDs) 
                   
                   
                 MGLUR5, ATP10C, CDH10, GRM6, 
               
               
                   
                   
                   
                 MGLUR6, CDH9, CNTN4, NLGN2, 
               
               
                   
                   
                   
                 CNTNAP2, SEMA5A, DHCR7, 
               
               
                   
                   
                   
                 NLGN4X, NLGN4Y, DPP6, NLGN5, 
               
               
                   
                   
                   
                 EN2, NRCAM, MDGA2, NRXN1, 
               
               
                   
                   
                   
                 FMR2, AFF2, FOXP2, OR4M2, 
               
               
                   
                   
                   
                 OXTR, FXR1, FXR2, PAH, 
               
               
                   
                   
                   
                 GABRA1, PTEN, GABRA5, PTPRZ1, 
               
               
                   
                   
                   
                 GABRB3, GABRG1, HIRIP3, 
               
               
                   
                   
                   
                 SEZ6L2, HOXA1, SHANK3, IL6, 
               
               
                   
                   
                   
                 SHBZRAP1, LAMB1, SLC6A4, 
               
               
                   
                   
                   
                 SERT, MAPK3, TAS2R1, MAZ, 
               
               
                   
                   
                   
                 TSC1, MDGA2, TSC2, MECP2, 
               
               
                   
                   
                   
                 UBE3A, WNT2, see also 
               
               
                   
                   
                   
                 20110023145 
               
               
                 autosomal dominant polycystic 
                 kidney 
                 liver 
                 PKD1, PKD2 
               
               
                 kidney disease (ADPKD) - 
               
               
                 (includes diseases such as von 
               
               
                 Hippel-Lindau disease and 
               
               
                 tubreous sclerosis complex 
               
               
                 disease) 
               
               
                 Autosomal Recessive Polycystic 
                 kidney 
                 liver 
                 PKDH1 
               
               
                 Kidney Disease (ARPKD) 
               
               
                 Ataxia-Telangiectasia (a.k.a 
                 Nervous system, 
                 various 
                 ATM 
               
               
                 Louis Bar syndrome) 
                 immune system 
               
               
                 B-Cell Non-Hodgkin Lymphoma 
                   
                   
                 BCL7A, BCL7 
               
               
                 Bardet-Biedl syndrome 
                 Eye, 
                 Liver, ear, 
                 ARL6, BBS1, BBS2, BBS4, BBS5, 
               
               
                   
                 musculoskeletal 
                 gastrointestinal 
                 BBS7, BBS9, BBS10, BBS12, 
               
               
                   
                 system, kidney, 
                 system, brain 
                 CEP290, INPP5E, LZTFL1, MKKS, 
               
               
                   
                 reproductive 
                   
                 MKS1, SDCCAG8, TRIM32, TTC8 
               
               
                   
                 organs 
               
               
                 Bare Lymphocyte Syndrome 
                 blood 
                   
                 TAPBP, TPSN, TAP2, ABCB3, PSF2, 
               
               
                   
                   
                   
                 RING11, MHC2TA, C2TA, RFX5, 
               
               
                   
                   
                   
                 RFXAP, RFX5 
               
               
                 Barter&#39;s Syndrome (types I, II, 
                 kidney 
                   
                 SLC12A1 (type I), KCNJ1 (type II), 
               
               
                 III, IVA and B, and V) 
                   
                   
                 CLCNKB (type III), BSND (type IV 
               
               
                   
                   
                   
                 A), or both the CLCNKA CLCNKB 
               
               
                   
                   
                   
                 genes (type IV B), CASR (type V). 
               
               
                 Becker muscular dystrophy 
                 Muscle 
                   
                 DMD, BMD, MYF6 
               
               
                 Best Disease (Vitelliform 
                 eye 
                   
                 VMD2 
               
               
                 Macular Dystrophy type 2) 
               
               
                 Bleeding Disorders 
                 blood 
                   
                 TBXA2R, P2RX1, P2X1 
               
               
                 Blue Cone Monochromacy 
                 eye 
                   
                 OPN1LW, OPN1MW, and LCR 
               
               
                 Breast Cancer 
                 Breast tissue 
                   
                 BRCA1, BRCA2, COX-2 
               
               
                 Bruton&#39;s Disease (aka X-linked 
                 Immune system, 
                   
                 BTK 
               
               
                 Agammglobulinemia) 
                 specifically B 
               
               
                   
                 cells 
               
               
                 Cancers (e.g., lymphoma, chronic 
                 Various 
                   
                 FAS, BID, CTLA4, PDCD1, CBLB, 
               
               
                 lymphocytic leukemia (CLL), B 
                   
                   
                 PTPN6, TRAC, TRBC, those 
               
               
                 cell acute lymphocytic leukemia 
                   
                   
                 described in WO2015048577 
               
               
                 (B-ALL), acute lymphoblastic 
               
               
                 leukemia, acute myeloid 
               
               
                 leukemia, non-Hodgkin&#39;s 
               
               
                 lymphoma (NHL), diffuse large 
               
               
                 cell lymphoma (DLCL), multiple 
               
               
                 myeloma, renal cell carcinoma 
               
               
                 (RCC), neuroblastoma, colorectal 
               
               
                 cancer, breast cancer, ovarian 
               
               
                 cancer, melanoma, sarcoma, 
               
               
                 prostate cancer, lung cancer, 
               
               
                 esophageal cancer, hepatocellular 
               
               
                 carcinoma, pancreatic cancer, 
               
               
                 astrocytoma, mesothelioma, head 
               
               
                 and neck cancer, and 
               
               
                 medulloblastoma 
               
               
                 Cardiovascular Diseases 
                 heart 
                 Vascular system 
                 IL1B, XDH, TP53, PTGS, MB, IL4, 
               
               
                   
                   
                   
                 ANGPT1, ABCGu8, CTSK, PTGIR, 
               
               
                   
                   
                   
                 KCNJ11, INS, CRP, PDGFRB, 
               
               
                   
                   
                   
                 CCNA2, PDGFB, KCNJ5, KCNN3, 
               
               
                   
                   
                   
                 CAPN10, ADRA2B, ABCG5, 
               
               
                   
                   
                   
                 PRDX2, CPAN5, PARP14, MEX3C, 
               
               
                   
                   
                   
                 ACE, RNF, IL6, TNF, STN, 
               
               
                   
                   
                   
                 SERPINE1, ALB, ADIPOQ, APOB, 
               
               
                   
                   
                   
                 APOE, LEP, MTHFR, APOA1, 
               
               
                   
                   
                   
                 EDN1, NPPB, NOS3, PPARG, PLAT, 
               
               
                   
                   
                   
                 PTGS2, CETP, AGTR1, HMGCR, 
               
               
                   
                   
                   
                 IGF1, SELE, REN, PPARA, PON1, 
               
               
                   
                   
                   
                 KNG1, CCL2, LPL, VWF, F2, 
               
               
                   
                   
                   
                 ICAM1, TGFB, NPPA, IL10, EPO, 
               
               
                   
                   
                   
                 SOD1, VCAM1, IFNG, LPA, MPO, 
               
               
                   
                   
                   
                 ESR1, MAPK, HP, F3, CST3, COG2, 
               
               
                   
                   
                   
                 MMP9, SERPINC1, F8, HMOX1, 
               
               
                   
                   
                   
                 APOC3, IL8, PROL1, CBS, NOS2, 
               
               
                   
                   
                   
                 TLR4, SELP, ABCA1, AGT, LDLR, 
               
               
                   
                   
                   
                 GPT, VEGFA, NR3C2, IL18, NOS1, 
               
               
                   
                   
                   
                 NR3C1, FGB, HGF, ILIA, AKT1, 
               
               
                   
                   
                   
                 LIPC, HSPD1, MAPK14, SPP1, 
               
               
                   
                   
                   
                 ITGB3, CAT, UTS2, THBD, F10, CP, 
               
               
                   
                   
                   
                 TNFRSF11B, EGFR, MMP2, PLG, 
               
               
                   
                   
                   
                 NPY, RHOD, MAPK8, MYC, FN1, 
               
               
                   
                   
                   
                 CMA1, PLAU, GNB3, ADRB2, 
               
               
                   
                   
                   
                 SOD2, F5, VDR, ALOX5, HLA- 
               
               
                   
                   
                   
                 DRB1, PARP1, CD40LG, PON2, 
               
               
                   
                   
                   
                 AGER, IRS1, PTGS1, ECE1, F7, 
               
               
                   
                   
                   
                 IRMN, EPHX2, IGFBP1, MAPK10, 
               
               
                   
                   
                   
                 FAS, ABCB1, JUN, IGFBP3, CD14, 
               
               
                   
                   
                   
                 PDE5A, AGTR2, CD40, LCAT, 
               
               
                   
                   
                   
                 CCR5, MMP1, TIMP1, ADM, 
               
               
                   
                   
                   
                 DYT10, STAT3, MMP3, ELN, USF1, 
               
               
                   
                   
                   
                 CFH, HSPA4, MMP12, MME, F2R, 
               
               
                   
                   
                   
                 SELL, CTSB, ANXA5, ADRB1, 
               
               
                   
                   
                   
                 CYBA, FGA, GGT1, LIPG, HIF1A, 
               
               
                   
                   
                   
                 CXCR4, PROC, SCARB1, CD79A, 
               
               
                   
                   
                   
                 PLTP, ADD1, FGG, SAA1, KCNH2, 
               
               
                   
                   
                   
                 DPP4, NPR1, VTN, KIAA0101, FOS, 
               
               
                   
                   
                   
                 TLR2, PPIG, IL1R1, AR, CYP1A1, 
               
               
                   
                   
                   
                 SERPINA1, MTR, RBP4, APOA4, 
               
               
                   
                   
                   
                 CDKN2A, FGF2, EDNRB, ITGA2, 
               
               
                   
                   
                   
                 VLA-2, CABIN1, SHBG, HMGB1, 
               
               
                   
                   
                   
                 HSP90B2P, CYP3A4, GJA1, CAV1, 
               
               
                   
                   
                   
                 ESR2, LTA, GDF15, BDNF, 
               
               
                   
                   
                   
                 CYP2D6, NGF, SP1, TGIF1, SRC, 
               
               
                   
                   
                   
                 EGF, PIK3CG, HLA-A, KCNQ1, 
               
               
                   
                   
                   
                 CNR1, FBN1, CHKA, BEST1, 
               
               
                   
                   
                   
                 CTNNB1, IL2, CD36, PRKAB1, TPO, 
               
               
                   
                   
                   
                 ALDH7A1, CX3CR1, TH, F9, CH1, 
               
               
                   
                   
                   
                 TF, HFE, IL17A, PTEN, GSTM1, 
               
               
                   
                   
                   
                 DMD, GATA4, F13A1, TTR, FABP4, 
               
               
                   
                   
                   
                 PON3, APOC1, INSR, TNFRSF1B, 
               
               
                   
                   
                   
                 HTR2A, CSF3, CYP2C9, TXN, 
               
               
                   
                   
                   
                 CYP11B2, PTH, CSF2, KDR, 
               
               
                   
                   
                   
                 PLA2G2A, THBS1, GCG, RHOA, 
               
               
                   
                   
                   
                 ALDH2, TCF7L2, NFE2L2, 
               
               
                   
                   
                   
                 NOTCH1, UGT1A1, IFNA1, PPARD, 
               
               
                   
                   
                   
                 SIRT1, GNHR1, PAPPA, ARR3, 
               
               
                   
                   
                   
                 NPPC, AHSP, PTK2, IL13, MTOR, 
               
               
                   
                   
                   
                 ITGB2, GSTT1, IL6ST, CPB2, 
               
               
                   
                   
                   
                 CYP1A2, HNF4A, SLC64A, 
               
               
                   
                   
                   
                 PLA2G6, TNFSF11, SLC8A1, F2RL1, 
               
               
                   
                   
                   
                 AKR1A1, ALDH9A1, BGLAP, 
               
               
                   
                   
                   
                 MTTP, MTRR, SULT1A3, RAGE, 
               
               
                   
                   
                   
                 C4B, P2RY12, RNLS, CREB1, 
               
               
                   
                   
                   
                 POMC, RAC1, LMNA, CD59, 
               
               
                   
                   
                   
                 SCM5A, CYP1B1, MIF, MMP13, 
               
               
                   
                   
                   
                 TIMP2, CYP19A1, CUP21A2, 
               
               
                   
                   
                   
                 PTPN22, MYH14, MBL2, SELPLG, 
               
               
                   
                   
                   
                 AOC3, CTSL1, PCNA, IGF2, ITGB1, 
               
               
                   
                   
                   
                 CAST, CXCL12, IGHE, KCNE1, 
               
               
                   
                   
                   
                 TFRC, COL1A1, COL1A2, IL2RB, 
               
               
                   
                   
                   
                 PLA2G10, ANGPT2, PROCR, NOX4, 
               
               
                   
                   
                   
                 HAMP, PTPN11, SLCA1, IL2RA, 
               
               
                   
                   
                   
                 CCL5, IRF1, CF:AR, CA:CA, EIF4E, 
               
               
                   
                   
                   
                 GSTP1, JAK2, CYP3A5, HSPG2, 
               
               
                   
                   
                   
                 CCL3, MYD88, VIP, SOAT1, 
               
               
                   
                   
                   
                 ADRBK1, NR4A2, MMP8, NPR2, 
               
               
                   
                   
                   
                 GCH1, EPRS, PPARGC1A, F12, 
               
               
                   
                   
                   
                 PECAM1, CCL4, CERPINA34, 
               
               
                   
                   
                   
                 CASR, FABP2, TTF2, PROS1, CTF1, 
               
               
                   
                   
                   
                 SGCB, YME1L1, CAMP, ZC3H12A, 
               
               
                   
                   
                   
                 AKR1B1, MMP7, AHR, CSF1, 
               
               
                   
                   
                   
                 HDAC9, CTGF, KCNMA1, UGT1A, 
               
               
                   
                   
                   
                 PRKCA, COMT, S100B, EGR1, PRL, 
               
               
                   
                   
                   
                 IL15, DRD4, CAMK2G, SLC22A2, 
               
               
                   
                   
                   
                 CCL11, PGF, THPO, GP6, TACR1, 
               
               
                   
                   
                   
                 NTS, HNF1A, SST, KCDN1, 
               
               
                   
                   
                   
                 LOC646627, TBXAS1, CUP2J2, 
               
               
                   
                   
                   
                 TBXA2R, ADH1C, ALOX12, AHSG, 
               
               
                   
                   
                   
                 BHMT, GJA4, SLC25A4, ACLY, 
               
               
                   
                   
                   
                 ALOX5AP, NUMA1, CYP27B1, 
               
               
                   
                   
                   
                 CYSLTR2, SOD3, LTC4S, UCN, 
               
               
                   
                   
                   
                 GHRL, APOC2, CLEC4A, 
               
               
                   
                   
                   
                 KBTBD10, TNC, TYMS, SHC1, 
               
               
                   
                   
                   
                 LRP1, SOCS3, ADH1B, KLK3, 
               
               
                   
                   
                   
                 HSD11B1, VKORC1, SERPINB2, 
               
               
                   
                   
                   
                 TNS1, RNF19A, EPOR, ITGAM, 
               
               
                   
                   
                   
                 PITX2, MAPK7, FCGR3A, LEEPR, 
               
               
                   
                   
                   
                 ENG, GPX1, GOT2, HRH1, NR112, 
               
               
                   
                   
                   
                 CRH, HTR1A, VDAC1, HPSE, 
               
               
                   
                   
                   
                 SFTPD, TAP2, RMF123, PTK2Bm 
               
               
                   
                   
                   
                 NTRK2, IL6R, ACHE, GLP1R, GHR, 
               
               
                   
                   
                   
                 GSR, NQO1, NR5A1, GJB2, 
               
               
                   
                   
                   
                 SLC9A1, MAOA, PCSK9, FCGR2A, 
               
               
                   
                   
                   
                 SERPINF1, EDN3, UCP2, TFAP2A, 
               
               
                   
                   
                   
                 C4BPA, SERPINF2, TYMP, ALPP, 
               
               
                   
                   
                   
                 CXCR2, SLC3A3, ABCG2, ADA, 
               
               
                   
                   
                   
                 JAK3, HSPA1A, FASN, FGF1, F11, 
               
               
                   
                   
                   
                 ATP7A, CR1, GFPA, ROCK1, 
               
               
                   
                   
                   
                 MECP2, MYLK, BCHE, LIPE, 
               
               
                   
                   
                   
                 ADORA1, WRN, CXCR3, CD81, 
               
               
                   
                   
                   
                 SMAD7, LAMC2, MAP3K5, CHGA, 
               
               
                   
                   
                   
                 IAPP, RHO, ENPP1, PTHLH, NRG1, 
               
               
                   
                   
                   
                 VEGFC, ENPEP, CEBPB, NAGLU,. 
               
               
                   
                   
                   
                 F2RL3, CX3CL1, BDKRB1, 
               
               
                   
                   
                   
                 ADAMTS13, ELANE, ENPP2, CISH, 
               
               
                   
                   
                   
                 GAST, MYOC, ATP1A2, NF1, GJB1, 
               
               
                   
                   
                   
                 MEF2A, VCL, BMPR2, TUBB, 
               
               
                   
                   
                   
                 CDC42, KRT18, HSF1, MYB, 
               
               
                   
                   
                   
                 PRKAA2, ROCK2, TFP1, PRKG1, 
               
               
                   
                   
                   
                 BMP2, CTNND1, CTH, CTSS, 
               
               
                   
                   
                   
                 VAV2, NPY2R, IGFBP2, CD28, 
               
               
                   
                   
                   
                 GSTA1, PPIA, APOH, S100A8, IL11, 
               
               
                   
                   
                   
                 ALOX15, FBLN1, NR1H3, SCD, GIP, 
               
               
                   
                   
                   
                 CHGB, PRKCB, SRD5A1,HSD11B2, 
               
               
                   
                   
                   
                 CALCRL, GALNT2, ANGPTL4, 
               
               
                   
                   
                   
                 KCNN4, PIK3C2A, HBEGF, 
               
               
                   
                   
                   
                 CYP7A1, HLA-DRB5, BNIP3, 
               
               
                   
                   
                   
                 GCKR, S100A12, PADI4, HSPA14, 
               
               
                   
                   
                   
                 CXCR1, H19, KRTAP19-3, IDDM2, 
               
               
                   
                   
                   
                 RAC2, YRY1, CLOCK, NGFR, DBH, 
               
               
                   
                   
                   
                 CHRNA4, CACNA1C, PRKAG2, 
               
               
                   
                   
                   
                 CHAT, PTGDS, NR1H2, TEK, 
               
               
                   
                   
                   
                 VEGFB, MEF2C, MAPKAPK2, 
               
               
                   
                   
                   
                 TNFRSF11A, HSPA9, CYSLTR1, 
               
               
                   
                   
                   
                 MATIA, OPRL1, IMPA1, CLCN2, 
               
               
                   
                   
                   
                 DLD, PSMA6, PSMB8, CHI3L1, 
               
               
                   
                   
                   
                 ALDH1B1, PARP2,STAR, LBP, 
               
               
                   
                   
                   
                 ABCC6, RGS2, EFNB2, GJB6, 
               
               
                   
                   
                   
                 APOA2, AMPD1, DYSF, 
               
               
                   
                   
                   
                 FDFT1, EMD2, CCR6, GJB3, IL1RL1, 
               
               
                   
                   
                   
                 ENTPD1, BBS4, CELSR2, F11R, 
               
               
                   
                   
                   
                 RAPGEF3, HYAL1, ZNF259, 
               
               
                   
                   
                   
                 ATOX1, ATF6, KHK, SAT1, GGH, 
               
               
                   
                   
                   
                 TIMP4, SLC4A4, PDE2A, PDE3B, 
               
               
                   
                   
                   
                 FADS1, FADS2, TMSB4X, TXNIP, 
               
               
                   
                   
                   
                 LIMS1, RHOB, LY96, FOXO1, 
               
               
                   
                   
                   
                 PNPLA2,TRH, GJC1, S:C17A5, FTO, 
               
               
                   
                   
                   
                 GJD2, PRSC1, CASP12, GPBAR1, 
               
               
                   
                   
                   
                 PXK, IL33, TRIB1, PBX4, NUPR1, 
               
               
                   
                   
                   
                 15-SEP, CILP2, TERC, GGT2, 
               
               
                   
                   
                   
                 MTCO1, UOX, AVP 
               
               
                 Cataract 
                 eye 
                   
                 CRYAA, CRYA1, CRYBB2, CRYB2, 
               
               
                   
                   
                   
                 PITX3, BFSP2, CP49, CP47, CRYAA, 
               
               
                   
                   
                   
                 CRYA1, PAX6, AN2, MGDA, 
               
               
                   
                   
                   
                 CRYBA1, CRYB1, CRYGC, CRYG3, 
               
               
                   
                   
                   
                 CCL, LIM2, MP19, CRYGD, CRYG4, 
               
               
                   
                   
                   
                 BFSP2, CP49, CP47, HSF4, CTM, 
               
               
                   
                   
                   
                 HSF4, CTM, MIP, AQP0, CRYAB, 
               
               
                   
                   
                   
                 CRYA2, CTPP2, CRYBB1, CRYGD, 
               
               
                   
                   
                   
                 CRYG4, CRYBB2, CRYB2, CRYGC, 
               
               
                   
                   
                   
                 CRYG3, CCL, CRYAA, CRYA1, 
               
               
                   
                   
                   
                 GJA8, CX50, CAE1, GJA3, CX46, 
               
               
                   
                   
                   
                 CZP3, CAE3, CCM1, CAM, KRIT1 
               
               
                 CDKL-5 Deficiencies or 
                 Brain, CNS 
                   
                 CDKL5 
               
               
                 Mediated Diseases 
               
               
                 Charcot-Marie-Tooth (CMT) 
                 Nervous system 
                 Muscles 
                 PMP22 (CMT1A and E), MPZ 
               
               
                 disease (Types 1, 2, 3, 4,) 
                   
                 (dystrophy) 
                 (CMT1B), LITAF (CMT1C), EGR2 
               
               
                   
                   
                   
                 (CMT1D), NEFL (CMT1F), GJB1 
               
               
                   
                   
                   
                 (CMT1X), MFN2 (CMT2A), KIF1B 
               
               
                   
                   
                   
                 (CMT2A2B), RAB7A (CMT2B), 
               
               
                   
                   
                   
                 TRPV4 (CMT2C), GARS (CMT2D), 
               
               
                   
                   
                   
                 NEFL (CMT2E), GAPD1 (CMT2K), 
               
               
                   
                   
                   
                 HSPB8 (CMT2L), DYNC1H1, 
               
               
                   
                   
                   
                 CMT20), LRSAM1 (CMT2P), 
               
               
                   
                   
                   
                 IGHMBP2 (CMT2S), MORC2 
               
               
                   
                   
                   
                 (CMT2Z), GDAP1 (CMT4A), 
               
               
                   
                   
                   
                 MTMR2 or SBF2/MTMR13 
               
               
                   
                   
                   
                 (CMT4B), SH3TC2 (CMT4C), 
               
               
                   
                   
                   
                 NDRG1 (CMT4D), PRX (CMT4F), 
               
               
                   
                   
                   
                 FIG4 (CMT4J), NT-3 
               
               
                 Chédiak-Higashi Syndrome 
                 Immune system 
                 Skin, hair, eyes, 
                 LYST 
               
               
                   
                   
                 neurons 
               
               
                 Choroidermia 
                   
                   
                 CHM, REP1, 
               
               
                 Chorioretinal atrophy 
                 eye 
                   
                 PRDM13, RGR, TEAD1 
               
               
                 Chronic Granulomatous Disease 
                 Immune system 
                   
                 CYBA, CYBB, NCF1, NCF2, NCF4 
               
               
                 Chronic Mucocutaneous 
                 Immune system 
                   
                 AIRE, CARD9, CLEC7A IL12B, 
               
               
                 Candidiasis 
                   
                   
                 IL12B1, IL1F, IL17RA, IL17RC, 
               
               
                   
                   
                   
                 RORC, STAT1, STAT3, TRAF31P2 
               
               
                 Cirrhosis 
                 liver 
                   
                 KRT18, KRT8, CIRH1A, NAIC, 
               
               
                   
                   
                   
                 TEX292, KIAA1988 
               
               
                 Colon cancer (Familial 
                 Gastrointestinal 
                   
                 FAP: APC HNPCC: 
               
               
                 adenomatous polyposis (FAP) 
                   
                   
                 MSH2, MLH1, PMS2, SH6, PMS1 
               
               
                 and hereditary nonpolyposis 
               
               
                 colon cancer (HNPCC)) 
               
               
                 Combined Immunodeficiency 
                 Immune System 
                   
                 IL2RG, SCIDX1, SCIDX, IMD4); 
               
               
                   
                   
                   
                 HIV-1 (CCL5, SCYA5, D17S136E, 
               
               
                   
                   
                   
                 TCP228 
               
               
                 Cone(-rod) dystrophy 
                 eye 
                   
                 AIPL1, CRX, GUA1A, GUCY2D, 
               
               
                   
                   
                   
                 PITPM3, PROM1, PRPH2, RIMS1, 
               
               
                   
                   
                   
                 SEMA4A, ABCA4, ADAM9, ATF6, 
               
               
                   
                   
                   
                 C21ORF2, C8ORF37, CACNA2D4, 
               
               
                   
                   
                   
                 CDHR1, CERKL, CNGA3, CNGB3, 
               
               
                   
                   
                   
                 CNNM4, CNAT2, IFT81, KCNV2, 
               
               
                   
                   
                   
                 PDE6C, PDE6H, POC1B, RAX2, 
               
               
                   
                   
                   
                 RDH5, RPGRIP1, TTLL5, RetCG1, 
               
               
                   
                   
                   
                 GUCY2E 
               
               
                 Congenital Stationary Night 
                 eye 
                   
                 CABP4, CACNA1F, CACNA2D4, 
               
               
                 Blindness 
                   
                   
                 GNAT1, CPR179, GRK1, GRM6, 
               
               
                   
                   
                   
                 LRIT3, NYX, PDE6B, RDH5, RHO, 
               
               
                   
                   
                   
                 RLBP1, RPE65, SAG, SLC24A1, 
               
               
                   
                   
                   
                 TRPM1, 
               
               
                 Congenital Fructose Intolerance 
                 Metabolism 
                   
                 ALDOB 
               
               
                 Cori&#39;s Disease (Glycogen Storage 
                 Various- 
                   
                 AGL 
               
               
                 Disease Type III) 
                 wherever 
               
               
                   
                 glycogen 
               
               
                   
                 accumulates, 
               
               
                   
                 particularly 
               
               
                   
                 liver, heart, 
               
               
                   
                 skeletal muscle 
               
               
                 Corneal clouding and dystrophy 
                 eye 
                   
                 APOA1, TGFBI, CSD2, CDGG1, 
               
               
                   
                   
                   
                 CSD, BIGH3, CDG2, TACSTD2, 
               
               
                   
                   
                   
                 TROP2, M1S1, VSX1, RINX, PPCD, 
               
               
                   
                   
                   
                 PPD, KTCN, COL8A2, FECD, 
               
               
                   
                   
                   
                 PPCD2, PIP5K3, CFD 
               
               
                 Cornea plana congenital 
                   
                   
                 KERA, CNA2 
               
               
                 Cri du chat Syndrome, also 
                   
                   
                 Deletions involving only band 5p15.2 
               
               
                 known as 5p syndrome and cat 
                   
                   
                 to the entire short arm of chromosome 
               
               
                 cry syndrome 
                   
                   
                 5, e.g. CTNND2, TERT, 
               
               
                 Cystic Fibrosis (CF) 
                 Lungs and 
                 Pancreas, liver, 
                 CTFR, ABCC7, CF, MRP7, SCNN1A, 
               
               
                   
                 respiratory 
                 digestive 
                 those described in WO2015157070 
               
               
                   
                 system 
                 system, 
               
               
                   
                   
                 reproductive 
               
               
                   
                   
                 system, 
               
               
                   
                   
                 exocrine, glands, 
               
               
                 Diabetic nephropathy 
                 kidney 
                   
                 Gremlin, 12/15- lipoxygenase, TIM44, 
               
               
                 Dent Disease (Types 1 and 2) 
                 Kidney 
                   
                 Type 1: CLCN5, Type 2: ORCL 
               
               
                 Dentatorubro-Pallidoluysian 
                 CNS, brain, 
                   
                 Atrophin-1 and Atn1 
               
               
                 Atrophy (DRPLA) (aka Haw 
                 muscle 
               
               
                 River and Naito-Oyanagi 
               
               
                 Disease) 
               
               
                 Down Syndrome 
                 various 
                   
                 Chromosome 21 trisomy 
               
               
                 Drug Addiction 
                 Brain 
                   
                 Prkce; Drd2; Drd4; ABAT; 
               
               
                   
                   
                   
                 GRIA2;Grm5; Grin1; Htr1b; Grin2a; 
               
               
                   
                   
                   
                 Drd3; Pdyn; Gria1 
               
               
                 Duane syndrome (Types 1, 2, and 
                 eye 
                   
                 CHN1, indels on chromosomes 4 and 8 
               
               
                 3, including subgroups A, B and 
               
               
                 C). Other names for this 
               
               
                 condition include: Duane&#39;s 
               
               
                 Retraction Syndrome (or DR 
               
               
                 syndrome), Eye Retraction 
               
               
                 Syndrome, Retraction Syndrome, 
               
               
                 Congenital retraction syndrome 
               
               
                 and Stilling-Turk-Duane 
               
               
                 Syndrome 
               
               
                 Duchenne muscular dystrophy 
                 muscle 
                 Cardiovascular, 
                 DMD, BMD, dystrophin gene, intron 
               
               
                 (DMD) 
                   
                 respiratory 
                 flanking exon 51 of DMD gene, exon 
               
               
                   
                   
                   
                 51 mutations in DMD gene, see also 
               
               
                   
                   
                   
                 WO2013163628 and US Pat. Pub. 
               
               
                   
                   
                   
                 20130145487 
               
               
                 Edward&#39;s Syndrome 
                   
                   
                 Complete or partial trisomy of 
               
               
                 (Trisomy 18) 
                   
                   
                 chromosome 18 
               
               
                 Ehlers-Danlos Syndrome (Types 
                 Various 
                   
                 COL5A1, COL5A2, COL1A1, 
               
               
                 I-VI) 
                 depending on 
                   
                 COL3A1, TNXB, PLOD1, COL1A2, 
               
               
                   
                 type: including 
                   
                 FKBP14 and ADAMTS2 
               
               
                   
                 musculoskeletal, 
               
               
                   
                 eye, vasculature, 
               
               
                   
                 immune, and 
               
               
                   
                 skin 
               
               
                 Emery-Dreifuss muscular 
                 muscle 
                   
                 LMNA, LMN1, EMD2, FPLD, 
               
               
                 dystrophy 
                   
                   
                 CMD1A, HGPS, LGMD1B, LMNA, 
               
               
                   
                   
                   
                 LMN1, EMD2, FPLD, CMD1A 
               
               
                 Enhanced S-Cone Syndrome 
                 eye 
                   
                 NR2E3, NRL 
               
               
                 Fabry&#39;s Disease 
                 Various - 
                   
                 GLA 
               
               
                   
                 including skin, 
               
               
                   
                 eyes, and 
               
               
                   
                 gastrointestinal 
               
               
                   
                 system, kidney, 
               
               
                   
                 heart, brain, 
               
               
                   
                 nervous system 
               
               
                 Facioscapulohumeral muscular 
                 muscles 
                   
                 FSHMD1A, FSHD1A, FRG1, 
               
               
                 dystrophy 
               
               
                 Factor H and Factor H-like 1 
                 blood 
                   
                 HF1, CFH, HUS 
               
               
                 Factor V Leiden thrombophilia 
                 blood 
                   
                 Factor V (F5) 
               
               
                 and Factor V deficiency 
               
               
                 Factor V and Factor VII 
                 blood 
                   
                 MCFD2 
               
               
                 deficiency 
               
               
                 Factor VII deficiency 
                 blood 
                   
                 F7 
               
               
                 Factor X deficiency 
                 blood 
                   
                 F10 
               
               
                 Factor XI deficiency 
                 blood 
                   
                 F11 
               
               
                 Factor XII deficiency 
                 blood 
                   
                 F12, HAF 
               
               
                 Factor XIIIA deficiency 
                 blood 
                   
                 F13A1, F13A 
               
               
                 Factor XIIIB deficiency 
                 blood 
                   
                 F13B 
               
               
                 Familial Hypercholestereolemia 
                 Cardiovascular 
                   
                 APOB, LDLR, PCSK9 
               
               
                   
                 system 
               
               
                 Familial Mediterranean Fever 
                 Various- 
                 Heart, kidney, 
                 MEFV 
               
               
                 (FMF) also called recurrent 
                 organs/tissues 
                 brain/CNS, 
               
               
                 polyserositis or familial 
                 with serous or 
                 reproductive 
               
               
                 paroxysmal polyserositis 
                 synovial 
                 organs 
               
               
                   
                 membranes, 
               
               
                   
                 skin, joints 
               
               
                 Fanconi Anemia 
                 Various - blood 
                   
                 FANCA, FACA, FA1, FA, FAA, 
               
               
                   
                 (anemia), 
                   
                 FAAP95, FAAP90, FLJ34064, 
               
               
                   
                 immune system, 
                   
                 FANCC, FANCG, RAD51, BRCA1, 
               
               
                   
                 cognitive, 
                   
                 BRCA2, BRIP1, BACH1, FANCJ, 
               
               
                   
                 kidneys, eyes, 
                   
                 FANCB, FANCD1, FANCD2, 
               
               
                   
                 musculoskeletal 
                   
                 FANCD, FAD, FANCE, FACE, 
               
               
                   
                   
                   
                 FANCF, FANCI, ERCC4, FANCL, 
               
               
                   
                   
                   
                 FANCM, PALB2, RAD51C, SLX4, 
               
               
                   
                   
                   
                 UBE2T, FANCB, XRCC9, PHF9, 
               
               
                   
                   
                   
                 KIAA1596 
               
               
                 Fanconi Syndrome Types I 
                 kidneys 
                   
                 FRTS1, GATM 
               
               
                 (Childhood onset) and II (Adult 
               
               
                 Onset) 
               
               
                 Fragile X syndrome and related 
                 brain 
                   
                 FMR1, FMR2; FXR1; FXR2; 
               
               
                 disorders 
                   
                   
                 mGLUR5 
               
               
                 Fragile XE Mental Retardation 
                 Brain, nervous 
                   
                 FMR1 
               
               
                 (aka Martin Bell syndrome) 
                 system 
               
               
                 Friedreich Ataxia (FRDA) 
                 Brain, nervous 
                 heart 
                 FXN/X25 
               
               
                   
                 system 
               
               
                 Fuchs endothelial corneal 
                 Eye 
                   
                 TCF4; COL8A2 
               
               
                 dystrophy 
               
               
                 Galactosemia 
                 Carbohydrate 
                 Various-where 
                 GALT, GALK1, and GALE 
               
               
                   
                 metabolism 
                 galactose 
               
               
                   
                 disorder 
                 accumulates - 
               
               
                   
                   
                 liver, brain, eyes 
               
               
                 Gastrointestinal Epithelial 
                   
                   
                 CISH 
               
               
                 Cancer, GI cancer 
               
               
                 Gaucher Disease (Types 1, 2, and 
                 Fat metabolism 
                 Various-liver, 
                 GBA 
               
               
                 3, as well as other unusual forms 
                 disorder 
                 spleen, blood, 
               
               
                 that may not fit into these types) 
                   
                 CNS, skeletal 
               
               
                   
                   
                 system 
               
               
                 Griscelli syndrome 
               
               
                 Glaucoma 
                 eye 
                   
                 MYOC, TIGR, GLC1A, JOAG, 
               
               
                   
                   
                   
                 GPOA, OPTN, GLC1E, FIP2, HYPL, 
               
               
                   
                   
                   
                 NRP, CYP1B1, GLC3A, OPA1, NTG, 
               
               
                   
                   
                   
                 NPG, CYP1B1, GLC3A, those 
               
               
                   
                   
                   
                 described in WO2015153780 
               
               
                 Glomerulo sclerosis 
                 kidney 
                   
                 CC chemokine ligand 2 
               
               
                 Glycogen Storage Diseases 
                 Metabolism 
                   
                 SLC2A2, GLUT2, G6PC, G6PT, 
               
               
                 Types I-VI -See also Cori&#39;s 
                 Diseases 
                   
                 G6PT1, GAA, LAMP2, LAMPB, 
               
               
                 Disease, Pompe&#39;s Disease, 
                   
                   
                 AGL, GDE, GBE1, GYS2, PYGL, 
               
               
                 McArdle&#39;s disease, Hers Disease, 
                   
                   
                 PFKM, see also Cori&#39;s Disease, 
               
               
                 and Von Gierke&#39;s disease 
                   
                   
                 Pompe&#39;s Disease, McArdle&#39;s disease, 
               
               
                   
                   
                   
                 Hers Disease, and Von Gierke&#39;s 
               
               
                   
                   
                   
                 disease 
               
               
                 RBC Glycolytic enzyme 
                 blood 
                   
                 any mutations in a gene for an enzyme 
               
               
                 deficiency 
                   
                   
                 in the glycolysis pathway including 
               
               
                   
                   
                   
                 mutations in genes for hexokinases I 
               
               
                   
                   
                   
                 and II, glucokinase, phosphoglucose 
               
               
                   
                   
                   
                 isomerase, phosphofructokinase, 
               
               
                   
                   
                   
                 aldolase Bm triosephosphate 
               
               
                   
                   
                   
                 isomerease, glyceraldehydee-3- 
               
               
                   
                   
                   
                 phosphate dehydrogenase, 
               
               
                   
                   
                   
                 phosphoglycerokinase, 
               
               
                   
                   
                   
                 phosphoglycerate mutase, enolase I, 
               
               
                   
                   
                   
                 pyruvate kinase 
               
               
                 Hartnup&#39;s disease 
                 Malabsorption 
                 Various- brain, 
                 SLC6A19 
               
               
                   
                 disease 
                 gastrointestinal, 
               
               
                   
                   
                 skin, 
               
               
                 Hearing Loss 
                 ear 
                   
                 NOX3, Hes5, BDNF, 
               
               
                 Hemochromatosis (HH) 
                 Iron absorption 
                 Various- 
                 HFE and H63D 
               
               
                   
                 regulation 
                 wherever iron 
               
               
                   
                 disease 
                 accumulates, 
               
               
                   
                   
                 liver, heart, 
               
               
                   
                   
                 pancreas, joints, 
               
               
                   
                   
                 pituitary gland 
               
               
                 Hemophagocytic 
                 blood 
                   
                 PRF1, HPLH2, UNC13D, MUNC13- 
               
               
                 lymphohistiocytosis disorders 
                   
                   
                 4, HPLH3, HLH3, FHL3 
               
               
                 Hemorrhagic disorders 
                 blood 
                   
                 PI, ATT, F5 
               
               
                 Hers disease (Glycogen storage 
                 liver 
                 muscle 
                 PYGL 
               
               
                 disease Type VI) 
               
               
                 Hereditary angioedema (HAE) 
                   
                   
                 kalikrein B1 
               
               
                 Hereditary Hemorrhagic 
                 Skin and 
                   
                 ACVRL1, ENG and SMAD4 
               
               
                 Telangiectasia (Osler-Weber- 
                 mucous 
               
               
                 Rendu Syndrome) 
                 membranes 
               
               
                 Hereditary Spherocytosis 
                 blood 
                   
                 NK1, EPB42, SLC4A1, SPTA1, and 
               
               
                   
                   
                   
                 SPTB 
               
               
                 Hereditary Persistence of Fetal 
                 blood 
                   
                 HBG1, HBG2, BCL11A, promoter 
               
               
                 Hemoglobin 
                   
                   
                 region of HBG 1 and/or 2 (in the 
               
               
                   
                   
                   
                 CCAAT box) 
               
               
                 Hemophilia (hemophilia A 
                 blood 
                   
                 A: FVIII, F8C, HEMA 
               
               
                 (Classic) a B (aka Christmas 
                   
                   
                 B: FVIX, HEMB 
               
               
                 disease) and C) 
                   
                   
                 C: F9, F11 
               
               
                 Hepatic adenoma 
                 liver 
                   
                 TCF1, HNF1A, MODY3 
               
               
                 Hepatic failure, early onset, and 
                 liver 
                   
                 SCOD1, SCO1 
               
               
                 neurologic disorder 
               
               
                 Hepatic lipase deficiency 
                 liver 
                   
                 LIPC 
               
               
                 Hepatoblastoma, cancer and 
                 liver 
                   
                 CTNNB1, PDGFRL, PDGRL, PRLTS, 
               
               
                 carcinomas 
                   
                   
                 AXIN1, AXIN, CTNNB1, TP53, P53, 
               
               
                   
                   
                   
                 LFS1, IGF2R, MPRI, MET, CASP8, 
               
               
                   
                   
                   
                 MCH5 
               
               
                 Hermansky-Pudlak syndrome 
                 Skin, eyes, 
                   
                 HPS1, HPS3, HPS4, HPS5, HPS6, 
               
               
                   
                 blood, lung, 
                   
                 HPS7, DTNBP1, BLOC1, BLOC1S2, 
               
               
                   
                 kidneys, 
                   
                 BLOC3 
               
               
                   
                 intestine 
               
               
                 HIV susceptibility or infection 
                 Immune system 
                   
                 IL10, CSIF, CMKBR2, CCR2, 
               
               
                   
                   
                   
                 CMKBR5, CCCKR5 (CCR5), those in 
               
               
                   
                   
                   
                 WO2015148670A1 
               
               
                 Holoprosencephaly (HPE) 
                 brain 
                   
                 ACVRL1, ENG, SMAD4 
               
               
                 (Alobar, Semilobar, and Lobar) 
               
               
                 Homocystinuria 
                 Metabolic 
                 Various- 
                 CBS, MTHFR, MTR, MTRR, and 
               
               
                   
                 disease 
                 connective 
                 MMADHC 
               
               
                   
                   
                 tissue, muscles, 
               
               
                   
                   
                 CNS, 
               
               
                   
                   
                 cardiovascular 
               
               
                   
                   
                 system 
               
               
                 HPV 
                   
                   
                 HPV16 and HPV18 E6/E7 
               
               
                 HSV1, HSV2, and related 
                 eye 
                   
                 HSV1 genes (immediate early and late 
               
               
                 keratitis 
                   
                   
                 HSV-1 genes (UL1, 1.5, 5, 6, 8, 9, 12, 
               
               
                   
                   
                   
                 15, 16, 18, 19, 22, 23, 26, 26.5, 27, 28, 
               
               
                   
                   
                   
                 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 
               
               
                   
                   
                   
                 42, 48, 49.5, 50, 52, 54, S6, RL2, RS1, 
               
               
                   
                   
                   
                 those described in WO2015153789, 
               
               
                   
                   
                   
                 WO2015153791 
               
               
                 Hunter&#39;s Syndrome (aka 
                 Lysosomal 
                 Various- liver, 
                 IDS 
               
               
                 Mucopolysaccharidosis type II) 
                 storage disease 
                 spleen, eye, 
               
               
                   
                   
                 joint, heart, 
               
               
                   
                   
                 brain, skeletal 
               
               
                 Huntington&#39;s disease (HD) and 
                 Brain, nervous 
                   
                 HD, HTT, IT15, PRNP, PRIP, JPH3, 
               
               
                 HD-like disorders 
                 system 
                   
                 JP3, HDL2, TBP, SCA17, PRKCE; 
               
               
                   
                   
                   
                 IGF1; EP300; RCOR1; PRKCZ; 
               
               
                   
                   
                   
                 HDAC4; and TGM2, and those 
               
               
                   
                   
                   
                 described in WO2013130824, 
               
               
                   
                   
                   
                 WO2015089354 
               
               
                 Hurler&#39;s Syndrome (aka 
                 Lysosomal 
                 Various- liver, 
                 IDUA, α-L-iduronidase 
               
               
                 mucopolysaccharidosis type I H, 
                 storage disease 
                 spleen, eye, 
               
               
                 MPS IH) 
                   
                 joint, heart, 
               
               
                   
                   
                 brain, skeletal 
               
               
                 Hurler-Scheie syndrome (aka 
                 Lysosomal 
                 Various- liver, 
                 IDUA, α-L-iduronidase 
               
               
                 mucopolysaccharidosis type I H- 
                 storage disease 
                 spleen, eye, 
               
               
                 S, MPS I H-S) 
                   
                 joint, heart, 
               
               
                   
                   
                 brain, skeletal 
               
               
                 hyaluronidase deficiency (aka 
                 Soft and 
                   
                 HYAL1 
               
               
                 MPS IX) 
                 connective 
               
               
                   
                 tissues 
               
               
                 Hyper IgM syndrome 
                 Immune system 
                   
                 CD40L 
               
               
                 Hyper- tension caused renal 
                 kidney 
                   
                 Mineral corticoid receptor 
               
               
                 damage 
               
               
                 Immunodeficiencies 
                 Immune System 
                   
                 CD3E, CD3G, AICDA, AID, HIGM2, 
               
               
                   
                   
                   
                 TNFRSF5, CD40, UNG, DGU, 
               
               
                   
                   
                   
                 HIGM4, TNFSF5, CD40LG, HIGM1, 
               
               
                   
                   
                   
                 IGM, FOXP3, IPEX, AIID, XPID, 
               
               
                   
                   
                   
                 PIDX, TNFRSF14B, TACI 
               
               
                 Inborn errors of metabolism: 
                 Metabolism 
                 Various organs 
                 See also: Carbohydrate metabolism 
               
               
                 including urea cycle disorders, 
                 diseases, liver 
                 and cells 
                 disorders (e.g. galactosemia), Amino 
               
               
                 organic acidemias), fatty acid 
                   
                   
                 acid Metabolism disorders (e.g. 
               
               
                 oxidation defects, amino 
                   
                   
                 phenylketonuria), Fatty acid 
               
               
                 acidopathies, carbohydrate 
                   
                   
                 metabolism (e.g. MCAD deficiency), 
               
               
                 disorders, mitochondrial 
                   
                   
                 Urea Cycle disorders (e.g. 
               
               
                 disorders 
                   
                   
                 Citrullinemia), Organic acidemias (e.g. 
               
               
                   
                   
                   
                 Maple Syrup Urine disease), 
               
               
                   
                   
                   
                 Mitochondrial disorders (e.g. 
               
               
                   
                   
                   
                 MELAS), peroxisomal disorders (e.g. 
               
               
                   
                   
                   
                 Zellweger syndrome) 
               
               
                 Inflammation 
                 Various 
                   
                 IL-10; IL-1 (IL-1a; IL-1b); IL-13; IL- 
               
               
                   
                   
                   
                 17 (IL-17a (CTLA8); IL- 
               
               
                   
                   
                   
                 17b; IL-17c; IL-17d; IL-17f); II-23; 
               
               
                   
                   
                   
                 Cx3cr1; ptpn22; TNFa; 
               
               
                   
                   
                   
                 NOD2/CARD15 for IBD; IL-6; IL-12 
               
               
                   
                   
                   
                 (IL-12a; IL-12b); 
               
               
                   
                   
                   
                 CTLA4; Cx3cl1 
               
               
                 Inflammatory Bowel Diseases 
                 Gastrointestinal 
                 Joints, skin 
                 NOD2, IRGM, LRRK2, ATG5, 
               
               
                 (e.g. Ulcerative Colitis and 
                   
                   
                 ATG16L1, IRGM, GATM, ECM1, 
               
               
                 Chron&#39;s Disease) 
                   
                   
                 CDH1, LAMB1, HNF4A, GNA12, 
               
               
                   
                   
                   
                 IL10, CARD9/15. CCR6, IL2RA, 
               
               
                   
                   
                   
                 MST1, TNFSF15, REL, STAT3, 
               
               
                   
                   
                   
                 IL23R, IL12B, FUT2 
               
               
                 Interstitial renal fibrosis 
                 kidney 
                   
                 TGF-β type II receptor 
               
               
                 Job&#39;s Syndrome (aka Hyper IgE 
                 Immune System 
                   
                 STAT3, DOCK8 
               
               
                 Syndrome) 
               
               
                 Juvenile Retinoschisis 
                 eye 
                   
                 RS1, XLRS1 
               
               
                 Kabuki Syndrome 1 
                   
                   
                 MLL4, KMT2D 
               
               
                 Kennedy Disease (aka 
                 Muscles, brain, 
                   
                 SBMA/SMAX1/AR 
               
               
                 Spinobulbar Muscular Atrophy) 
                 nervous system 
               
               
                 Klinefelter syndrome 
                 Various- 
                   
                 Extra X chromosome in males 
               
               
                   
                 particularly 
               
               
                   
                 those involved 
               
               
                   
                 in development 
               
               
                   
                 of male 
               
               
                   
                 characteristics 
               
               
                 Lafora Disease 
                 Brain, CNS 
                   
                 EMP2A and EMP2B 
               
               
                 Leber Congenital Amaurosis 
                 eye 
                   
                 CRB1, RP12, CORD2, CRD, CRX, 
               
               
                   
                   
                   
                 IMPDH1, OTX2, AIPL1, CABP4, 
               
               
                   
                   
                   
                 CCT2, CEP290, CLUAP1, CRB1, 
               
               
                   
                   
                   
                 CRX, DTHD1, GDF6, GUCY2D, 
               
               
                   
                   
                   
                 IFT140, IQCB1, KCNJ13, LCA5, 
               
               
                   
                   
                   
                 LRAT, NMNAT1, PRPH2, RD3, 
               
               
                   
                   
                   
                 RDH12, RPE65, RP20, RPGRIP1, 
               
               
                   
                   
                   
                 SPATA7, TULP1, LCA1, LCA4, 
               
               
                   
                   
                   
                 GUC2D, CORD6, LCA3, 
               
               
                 Lesch-Nyhan Syndrome 
                 Metabolism 
                 Various - joints, 
                 HPRT1 
               
               
                   
                 disease 
                 cognitive, brain, 
               
               
                   
                   
                 nervous system 
               
               
                 Leukocyte deficiencies and 
                 blood 
                   
                 ITGB2, CD18, LCAMB, LAD, 
               
               
                 disorders 
                   
                   
                 EIF2B1, EIF2BA, EIF2B2, EIF2B3, 
               
               
                   
                   
                   
                 EIF2B5, LVWM, CACH, CLE, 
               
               
                   
                   
                   
                 EIF2B4 
               
               
                 Leukemia 
                 Blood 
                   
                 TAL1, TCL5, SCL, TAL2, FLT3, 
               
               
                   
                   
                   
                 NBS1, NBS, ZNFN1A1, IK1, LYF1, 
               
               
                   
                   
                   
                 HOXD4, HOX4B, BCR, CML, PHL, 
               
               
                   
                   
                   
                 ALL, ARNT, KRAS2, RASK2, 
               
               
                   
                   
                   
                 GMPS, AF10, ARHGEF12, LARG, 
               
               
                   
                   
                   
                 KIAA0382, CALM, CLTH, CEBPA, 
               
               
                   
                   
                   
                 CEBP, CHIC2, BTL, FLT3, KIT, 
               
               
                   
                   
                   
                 PBT, LPP, NPM1, NUP214, D9S46E, 
               
               
                   
                   
                   
                 CAN, CAIN, RUNX1, CBFA2, 
               
               
                   
                   
                   
                 AML1, WHSC1L1, NSD3, FLT3, 
               
               
                   
                   
                   
                 AF1Q, NPM1, NUMA1, ZNF145, 
               
               
                   
                   
                   
                 PLZF, PML, MYL, STAT5B, AF10, 
               
               
                   
                   
                   
                 CALM, CLTH, ARL11, ARLTS1, 
               
               
                   
                   
                   
                 P2RX7, P2X7, BCR, CML, PHL, 
               
               
                   
                   
                   
                 ALL, GRAF, NF1, VRNF, WSS, 
               
               
                   
                   
                   
                 NFNS, PTPN11, PTP2C, SHP2, NS1, 
               
               
                   
                   
                   
                 BCL2, CCND1, PRAD1, BCL1, 
               
               
                   
                   
                   
                 TCRA, GATA1, GF1, ERYF1, NFE1, 
               
               
                   
                   
                   
                 ABL1, NQO1, DIA4, NMOR1, 
               
               
                   
                   
                   
                 NUP214, D9S46E, CAN, CAIN 
               
               
                 Limb-girdle muscular dystrophy 
                 muscle 
                   
                 LGMD 
               
               
                 diseases 
               
               
                 Lowe syndrome 
                 brain, eyes, 
                   
                 OCRL 
               
               
                   
                 kidneys 
               
               
                 Lupus glomerulo- nephritis 
                 kidney 
                   
                 MAPK1 
               
               
                 Machado- 
                 Brain, CNS, 
                   
                 ATX3 
               
               
                 Joseph&#39;s Disease (also known as 
                 muscle 
               
               
                 Spinocerebellar ataxia Type 3) 
               
               
                 Macular degeneration 
                 eye 
                   
                 ABC4, CBC1, CHM1, APOE, 
               
               
                   
                   
                   
                 C1QTNF5, C2, C3, CCL2, CCR2, 
               
               
                   
                   
                   
                 CD36, CFB, CFH, CFHR1, CFHR3, 
               
               
                   
                   
                   
                 CNGB3, CP, CRP, CST3, CTSD, 
               
               
                   
                   
                   
                 CX3CR1, ELOVL4, ERCC6, FBLN5, 
               
               
                   
                   
                   
                 FBLN6, FSCN2, HMCN1, HIRAI, 
               
               
                   
                   
                   
                 IL6, IL8, PLEKHA1, PROM1, 
               
               
                   
                   
                   
                 PRPH2, RPGR, SERPING1, TCOF1, 
               
               
                   
                   
                   
                 TIMP3, TLR3 
               
               
                 Macular Dystrophy 
                 eye 
                   
                 BEST1, C1QTNF5, CTNNA1, 
               
               
                   
                   
                   
                 EFEMP1, ELOVL4, FSCN2, 
               
               
                   
                   
                   
                 GUCA1B, HMCN1, IMPG1, OTX2, 
               
               
                   
                   
                   
                 PRDM13, PROM1, PRPH2, RP1L1, 
               
               
                   
                   
                   
                 TIMP3, ABCA4, CFH, DRAM2, 
               
               
                   
                   
                   
                 IMG1, MFSD8, ADMD, STGD2, 
               
               
                   
                   
                   
                 STGD3, RDS, RP7, PRPH, AVMD, 
               
               
                   
                   
                   
                 AOFMD, VMD2 
               
               
                 Malattia Leventinesse 
                 eye 
                   
                 EFEMP1, FBLN3 
               
               
                 Maple Syrup Urine Disease 
                 Metabolism 
                   
                 BCKDHA, BCKDHB, and DBT 
               
               
                   
                 disease 
               
               
                 Marfan syndrome 
                 Connective 
                 Musculoskeletal 
                 FBN1 
               
               
                   
                 tissue 
               
               
                 Maroteaux-Lamy Syndrome (aka 
                 Musculoskeletal 
                 Liver, spleen 
                 ARSB 
               
               
                 MPS VI) 
                 system, nervous 
               
               
                   
                 system 
               
               
                 McArdle&#39;s Disease (Glycogen 
                 Glycogen 
                 muscle 
                 PYGM 
               
               
                 Storage Disease Type V) 
                 storage disease 
               
               
                 Medullary cystic kidney disease 
                 kidney 
                   
                 UMOD, HNFJ, FJHN, MCKD2, 
               
               
                   
                   
                   
                 ADMCKD2 
               
               
                 Metachromatic leukodystrophy 
                 Lysosomal 
                 Nervous system 
                 ARSA 
               
               
                   
                 storage disease 
               
               
                 Methylmalonic acidemia (MMA) 
                 Metabolism 
                   
                 MMAA, MMAB, MUT, MMACHC, 
               
               
                   
                 disease 
                   
                 MMADHC, LMBRD1 
               
               
                 Morquio Syndrome (aka MPS IV 
                 Connective 
                 heart 
                 GALNS 
               
               
                 A and B) 
                 tissue, skin, 
               
               
                   
                 bone, eyes 
               
               
                 Mucopolysaccharidosis diseases 
                 Lysosomal 
                   
                 See also Hurler/Scheie syndrome, 
               
               
                 (Types I H/S, I H, II, III A B and 
                 storage disease - 
                   
                 Hurler disease, Sanfillipo syndrome, 
               
               
                 C, I S, IVA and B, IX, VII, and 
                 affects various 
                   
                 Scheie syndrome, Morquio syndrome, 
               
               
                 VI) 
                 organs/tissues 
                   
                 hyaluronidase deficiency, Sly 
               
               
                   
                   
                   
                 syndrome, and Maroteaux-Lamy 
               
               
                   
                   
                   
                 syndrome 
               
               
                 Muscular Atrophy 
                 muscle 
                   
                 VAPB, VAPC, ALS8, SMN1, SMA1, 
               
               
                   
                   
                   
                 SMA2, SMA3, SMA4, BSCL2, 
               
               
                   
                   
                   
                 SPG17, GARS, SMAD1, CMT2D, 
               
               
                   
                   
                   
                 HEXB, IGHMBP2, SMUBP2, 
               
               
                   
                   
                   
                 CATF1, SMARD1 
               
               
                 Muscular dystrophy 
                 muscle 
                   
                 FKRP, MDC1C, LGMD2I, LAMA2, 
               
               
                   
                   
                   
                 LAMM, LARGE, KIAA0609, 
               
               
                   
                   
                   
                 MDC1D, FCMD, TTID, MYOT, 
               
               
                   
                   
                   
                 CAPN3, CANP3, DYSF, LGMD2B, 
               
               
                   
                   
                   
                 SGCG, LGMD2C, DMDA1, SCG3, 
               
               
                   
                   
                   
                 SGCA, ADL, DAG2, LGMD2D, 
               
               
                   
                   
                   
                 DMDA2, SGCB, LGMD2E, SGCD, 
               
               
                   
                   
                   
                 SGD, LGMD2F, CMD1L, TCAP, 
               
               
                   
                   
                   
                 LGMD2G, CMD1N, TRIM32, HT2A, 
               
               
                   
                   
                   
                 LGMD2H, FKRP, MDC1C, LGMD2I, 
               
               
                   
                   
                   
                 TTN, CMD1G, TMD, LGMD2J, 
               
               
                   
                   
                   
                 POMT1, CAV3, LGMD1C, SEPN1, 
               
               
                   
                   
                   
                 SELN, RSMD1, PLEC1, PLTN, EBS1 
               
               
                 Myotonic dystrophy (Type 1 and 
                 Muscles 
                 Eyes, heart, 
                 CNBP (Type 2) and DMPK (Type 1) 
               
               
                 Type 2) 
                   
                 endocrine 
               
               
                 Neoplasia 
                   
                   
                 PTEN; ATM; ATR; EGFR; ERBB2; 
               
               
                   
                   
                   
                 ERBB3; ERBB4; 
               
               
                   
                   
                   
                 Notch1; Notch2; Notch3; Notch4; 
               
               
                   
                   
                   
                 AKT; AKT2; AKT3; HIF; 
               
               
                   
                   
                   
                 HIF1a; HIF3a; Met; HRG; Bcl2; 
               
               
                   
                   
                   
                 PPAR alpha; PPAR 
               
               
                   
                   
                   
                 gamma; WT1 (Wilms Tumor); FGF 
               
               
                   
                   
                   
                 Receptor Family 
               
               
                   
                   
                   
                 members (5 members: 1, 2, 3, 4, 5); 
               
               
                   
                   
                   
                 CDKN2a; APC; RB 
               
               
                   
                   
                   
                 (retinoblastoma); MEN1; VHL; 
               
               
                   
                   
                   
                 BRCA1; BRCA2; AR 
               
               
                   
                   
                   
                 (Androgen Receptor); TSG101; IGF; 
               
               
                   
                   
                   
                 IGF Receptor; Igf1 (4 
               
               
                   
                   
                   
                 variants); Igf2 (3 variants); Igf 1 
               
               
                   
                   
                   
                 Receptor; Igf 2 Receptor; 
               
               
                   
                   
                   
                 Bax; Bcl2; caspases family (9 
               
               
                   
                   
                   
                 members: 
               
               
                   
                   
                   
                 1, 2, 3, 4, 6, 7, 8, 9, 12); Kras; Apc 
               
               
                 Neurofibromatosis (NF) (NF1, 
                 brain, spinal 
                   
                 NF1, NF2 
               
               
                 formerly Recklinghausen&#39;s NF, 
                 cord, nerves, 
               
               
                 and NF2) 
                 and skin 
               
               
                 Niemann-Pick Lipidosis (Types 
                 Lysosomal 
                 Various- where 
                 Types A and B: SMPD1; Type C: 
               
               
                 A, B, and C) 
                 Storage Disease 
                 sphingomyelin 
                 NPC1 or NPC2 
               
               
                   
                   
                 accumulates, 
               
               
                   
                   
                 particularly 
               
               
                   
                   
                 spleen, liver, 
               
               
                   
                   
                 blood, CNS 
               
               
                 Noonan Syndrome 
                 Various - 
                   
                 PTPN11, SOS1, RAF1 and KRAS 
               
               
                   
                 musculoskeletal, 
               
               
                   
                 heart, eyes, 
               
               
                   
                 reproductive 
               
               
                   
                 organs, blood 
               
               
                 Norrie Disease or X-linked 
                 eye 
                   
                 NDP 
               
               
                 Familial Exudative 
               
               
                 Vitreoretinopathy 
               
               
                 North Carolina Macular 
                 eye 
                   
                 MCDR1 
               
               
                 Dystrophy 
               
               
                 Osteogenesis imperfecta (OI) 
                 bones, 
                   
                 COL1A1, COL1A2, CRTAP, P3H 
               
               
                 (Types I, II, III, IV, V, VI, VII) 
                 musculoskeletal 
               
               
                 Osteopetrosis 
                 bones 
                   
                 LRP5, BMND1, LRP7, LR3, OPPG, 
               
               
                   
                   
                   
                 VBCH2, CLCN7, CLC7, OPTA2, 
               
               
                   
                   
                   
                 OSTM1, GL, TCIRG1, TIRC7, 
               
               
                   
                   
                   
                 OC116, OPTB1 
               
               
                 Patau&#39;s Syndrome 
                 Brain, heart, 
                   
                 Additional copy of chromosome 13 
               
               
                 (Trisomy 13) 
                 skeletal system 
               
               
                 Parkinson&#39;s disease (PD) 
                 Brain, nervous 
                   
                 SNCA (PARK1), UCHL1 (PARK 5), 
               
               
                   
                 system 
                   
                 and LRRK2 (PARK8), (PARK3), 
               
               
                   
                   
                   
                 PARK2, PARK4, PARK7 (PARK7), 
               
               
                   
                   
                   
                 PINK1 (PARK6); x-Synuclein, DJ-1, 
               
               
                   
                   
                   
                 Parkin, NR4A2, NURR1, NOT, 
               
               
                   
                   
                   
                 TINUR, SNCAIP, TBP, SCA17, 
               
               
                   
                   
                   
                 NCAP, PRKN, PDJ, DBH, NDUFV2 
               
               
                 Pattern Dystrophy of the RPE 
                 eye 
                   
                 RDS/peripherin 
               
               
                 Phenylketonuria (PKU) 
                 Metabolism 
                 Various due to 
                 PAH, PKU1, QDPR, DHPR, PTS 
               
               
                   
                 disorder 
                 build-up of 
               
               
                   
                   
                 phenylalanine, 
               
               
                   
                   
                 phenyl ketones 
               
               
                   
                   
                 in tissues and 
               
               
                   
                   
                 CNS 
               
               
                 Polycystic kidney and hepatic 
                 Kidney, liver 
                   
                 FCYT, PKHD1, ARPKD, PKD1, 
               
               
                 disease 
                   
                   
                 PKD2, PKD4, PKDTS, PRKCSH, 
               
               
                   
                   
                   
                 G19P1, PCLD, SEC63 
               
               
                 Pompe&#39;s Disease 
                 Glycogen 
                 Various - heart, 
                 GAA 
               
               
                   
                 storage disease 
                 liver, spleen 
               
               
                 Porphyria (actually refers to a 
                 Various- 
                   
                 ALAD, ALAS2, CPOX, FECH, 
               
               
                 group of different diseases all 
                 wherever heme 
                   
                 HMBS, PPOX, UROD, or UROS 
               
               
                 having a specific heme 
                 precursors 
               
               
                 production process abnormality) 
                 accumulate 
               
               
                 posterior polymorphous corneal 
                 eyes 
                   
                 TCF4; COL8A2 
               
               
                 dystrophy 
               
               
                 Primary Hyperoxaluria (e.g. type 
                 Various - eyes, 
                   
                 LDHA (lactate dehydrogenase A) and 
               
               
                 1) 
                 heart, kidneys, 
                   
                 hydroxyacid oxidase 1 (HAO1) 
               
               
                   
                 skeletal system 
               
               
                 Primary Open Angle Glaucoma 
                 eyes 
                   
                 MYOC 
               
               
                 (POAG) 
               
               
                 Primary sclerosing cholangitis 
                 Liver, 
                   
                 TCF4; COL8A2 
               
               
                   
                 gallbladder 
               
               
                 Progeria (also called Hutchinson- 
                 All 
                   
                 LMNA 
               
               
                 Gilford progeria syndrome) 
               
               
                 Prader-Willi Syndrome 
                 Musculoskeletal 
                   
                 Deletion of region of short arm of 
               
               
                   
                 system, brain, 
                   
                 chromosome 15, including UBE3A 
               
               
                   
                 reproductive 
               
               
                   
                 and endocrine 
               
               
                   
                 system 
               
               
                 Prostate Cancer 
                 prostate 
                   
                 HOXB13, MSMB, GPRC6A, TP53 
               
               
                 Pyruvate Dehydrogenase 
                 Brain, nervous 
                   
                 PDHA1 
               
               
                 Deficiency 
                 system 
               
               
                 Kidney/Renal carcinoma 
                 kidney 
                   
                 RLIP76, VEGF 
               
               
                 Rett Syndrome 
                 Brain 
                   
                 MECP2, RTT, PPMX, MRX16, 
               
               
                   
                   
                   
                 MRX79, CDKL5, STK9, MECP2, 
               
               
                   
                   
                   
                 RTT, PPMX, MRX16, MRX79, x- 
               
               
                   
                   
                   
                 Synuclein, DJ-1 
               
               
                 Retinitis pigmentosa (RP) 
                 eye 
                   
                 ADIPOR1, ABCA4, AGBL5, 
               
               
                   
                   
                   
                 ARHGEF18, ARL2BP, ARL3, ARL6, 
               
               
                   
                   
                   
                 BEST1, BBS1, BBS2, C2ORF71, 
               
               
                   
                   
                   
                 C8ORF37, CA4, CERKL, CLRN1, 
               
               
                   
                   
                   
                 CNGA1, CMGB1, CRB1, CRX, 
               
               
                   
                   
                   
                 CYP4V2, DHDDS, DHX38, EMC1, 
               
               
                   
                   
                   
                 EYS, FAM161A, FSCN2, GPR125, 
               
               
                   
                   
                   
                 GUCA1B, HK1, HPRPF3, HGSNAT, 
               
               
                   
                   
                   
                 IDH3B, IMPDH1, IMPG2, IFT140, 
               
               
                   
                   
                   
                 IFT172, KLHL7, KIAA1549, KIZ, 
               
               
                   
                   
                   
                 LRAT, MAK, MERTK, MVK, NEK2, 
               
               
                   
                   
                   
                 NUROD1, NR2E3, NRL, OFD1, 
               
               
                   
                   
                   
                 PDE6A, PDE6B, PDE6G, POMGNT1, 
               
               
                   
                   
                   
                 PRCD, PROM1, PRPF3, PRPF4, 
               
               
                   
                   
                   
                 PRPF6, PRPF8, PRPF31, PRPH2, 
               
               
                   
                   
                   
                 RPB3, RDH12, REEP6, RP39, RGR, 
               
               
                   
                   
                   
                 RHO, RLBP1, ROM1, RP1, RP1L1, 
               
               
                   
                   
                   
                 RPY, RP2, RP9, RPE65, RPGR, 
               
               
                   
                   
                   
                 SAMD11, SAG, SEMA4A, SLC7A14, 
               
               
                   
                   
                   
                 SNRNP200, SPP2, SPATA7, TRNT1, 
               
               
                   
                   
                   
                 TOPORS, TTC8, TULP1, USH2A, 
               
               
                   
                   
                   
                 ZFN408, ZNF513, see also 
               
               
                   
                   
                   
                 20120204282 
               
               
                 Scheie syndrome (also known as 
                 Various- liver, 
                   
                 IDUA, α-L-iduronidase 
               
               
                 mucopolysaccharidosis type I 
                 spleen, eye, 
               
               
                 S(MPS I-S)) 
               
               
                   
                 joint, heart, 
               
               
                   
                 brain, skeletal 
               
               
                 Schizophrenia 
                 Brain 
                   
                 Neuregulin1 (Nrg1); Erb4 (receptor for 
               
               
                   
                   
                   
                 Neuregulin); 
               
               
                   
                   
                   
                 Complexin1 (Cplx1); Tph1 
               
               
                   
                   
                   
                 Tryptophan hydroxylase; Tph2 
               
               
                   
                   
                   
                 Tryptophan hydroxylase 2; Neurexin 
               
               
                   
                   
                   
                 1; GSK3; GSK3a; 
               
               
                   
                   
                   
                 GSK3b; 5-HTT (Slc6a4); COMT; 
               
               
                   
                   
                   
                 DRD (Drd1a); SLC6A3; DAOA; 
               
               
                   
                   
                   
                 DTNBP1; Dao (Dao1); TCF4; 
               
               
                   
                   
                   
                 COL8A2 
               
               
                 Secretase Related Disorders 
                 Various 
                   
                 APH-1 (alpha and beta); PSEN1; 
               
               
                   
                   
                   
                 NCSTN; PEN-2; Nos1, Parp1, Nat1, 
               
               
                   
                   
                   
                 Nat2, CTSB, APP, APH1B, PSEN2, 
               
               
                   
                   
                   
                 PSENEN, BACE1, ITM2B, CTSD, 
               
               
                   
                   
                   
                 NOTCH1, TNF, INS, DYT10, 
               
               
                   
                   
                   
                 ADAM17, APOE, ACE, STN, TP53, 
               
               
                   
                   
                   
                 IL6, NGFR, IL1B, ACHE, CTNNB1, 
               
               
                   
                   
                   
                 IGF1, IFNG, NRG1, CASP3, MAPK1, 
               
               
                   
                   
                   
                 CDH1, APBB1, HMGCR, CREB1, 
               
               
                   
                   
                   
                 PTGS2, HES1, CAT, TGFB1, ENO2, 
               
               
                   
                   
                   
                 ERBB4, TRAPPC10, MAOB, NGF, 
               
               
                   
                   
                   
                 MMP12, JAG1, CD40LG, PPARG, 
               
               
                   
                   
                   
                 FGF2, LRP1, NOTCH4, MAPK8, 
               
               
                   
                   
                   
                 PREP, NOTCH3, PRNP, CTSG, EGF, 
               
               
                   
                   
                   
                 REN, CD44, SELP, GHR, ADCYAP1, 
               
               
                   
                   
                   
                 INSR, GFAP, MMP3, MAPK10, SP1, 
               
               
                   
                   
                   
                 MYC, CTSE, PPARA, JUN, TIMP1, 
               
               
                   
                   
                   
                 IL5, IL1A, MMP9, HTR4, HSPG2, 
               
               
                   
                   
                   
                 KRAS, CYCS, SMG1, IL1R1, 
               
               
                   
                   
                   
                 PROK1, MAPK3, NTRK1, IL13, 
               
               
                   
                   
                   
                 MME, TKT, CXCR2, CHRM1, 
               
               
                   
                   
                   
                 ATXN1, PAWR, NOTCJ2, M6PR, 
               
               
                   
                   
                   
                 CYP46A1, CSNK1D, MAPK14, 
               
               
                   
                   
                   
                 PRG2, PRKCA, L1 CAM, CD40, 
               
               
                   
                   
                   
                 NR1I2, JAG2, CTNND1, CMA1, 
               
               
                   
                   
                   
                 SORT1, DLK1, THEM4, JUP, CD46, 
               
               
                   
                   
                   
                 CCL11, CAV3, RNASE3, HSPA8, 
               
               
                   
                   
                   
                 CASP9, CYP3A4, CCR3, TFAP2A, 
               
               
                   
                   
                   
                 SCP2, CDK4, JOF1A, TCF7L2, 
               
               
                   
                   
                   
                 B3GALTL, MDM2, RELA, CASP7, 
               
               
                   
                   
                   
                 IDE, FANP4, CASK, ADCYAP1R1, 
               
               
                   
                   
                   
                 ATF4, PDGFA, C21ORF33, SCG5, 
               
               
                   
                   
                   
                 RMF123, NKFB1, ERBB2, CAV1, 
               
               
                   
                   
                   
                 MMP7, TGFA, RXRA, STX1A, 
               
               
                   
                   
                   
                 PSMC4, P2RY2, TNFRSF21, DLG1, 
               
               
                   
                   
                   
                 NUMBL, SPN, PLSCR1, UBQLN2, 
               
               
                   
                   
                   
                 UBQLN1, PCSK7, SPON1, SILV, 
               
               
                   
                   
                   
                 QPCT, HESS, GCC1 
               
               
                 Selective IgA Deficiency 
                 Immune system 
                   
                 Type 1: MSH5; Type 2: TNFRSF13B 
               
               
                 Severe Combined 
                 Immune system 
                   
                 JAK3, JAKL, DCLRE1C, ARTEMIS, 
               
               
                 Immunodeficiency (SCID) and 
                   
                   
                 SCIDA, RAG1, RAG2, ADA, PTPRC, 
               
               
                 SCID-χI, and ADA-SCID 
                   
                   
                 CD45, LCA, IL7R, CD3D, T3D, 
               
               
                   
                   
                   
                 IL2RG, SCIDX1, SCIDX, IMD4, 
               
               
                   
                   
                   
                 those identified in US Pat. App. Pub. 
               
               
                   
                   
                   
                 20110225664, 20110091441, 
               
               
                   
                   
                   
                 20100229252, 20090271881 and 
               
               
                   
                   
                   
                 20090222937; 
               
               
                 Sickle cell disease 
                 blood 
                   
                 HBB, BCL11A, BCL11Ae, cis- 
               
               
                   
                   
                   
                 regulatory elements of the B-globin 
               
               
                   
                   
                   
                 locus, HBG 1/2 promoter, HBG distal 
               
               
                   
                   
                   
                 CCAAT box region between -92 and - 
               
               
                   
                   
                   
                 130 of the HBG Transcription Start 
               
               
                   
                   
                   
                 Site, those described in 
               
               
                   
                   
                   
                 WO2015148863, WO 2013/126794, 
               
               
                   
                   
                   
                 US Pat. Pub. 20110182867 
               
               
                 Sly Syndrome (aka MPS VII) 
                   
                   
                 GUSB 
               
               
                 Spinocerebellar Ataxias (SCA 
                   
                   
                 ATXN1, ATXN2, ATX3 
               
               
                 types 1, 2, 3, 6, 7, 8, 12 and 17) 
               
               
                 Sorsby Fundus Dystrophy 
                 eye 
                   
                 TIMP3 
               
               
                 Stargardt disease 
                 eye 
                   
                 ABCR, ELOVL4, ABCA4, PROM1 
               
               
                 Tay-Sachs Disease 
                 Lysosomal 
                 Various - CNS, 
                 HEX-A 
               
               
                   
                 Storage disease 
                 brain, eye 
               
               
                 Thalassemia (Alpha, Beta, Delta) 
                 blood 
                   
                 HBA1, HBA2 (Alpha), HBB (Beta), 
               
               
                   
                   
                   
                 HBB and HBD (delta), LCRB, 
               
               
                   
                   
                   
                 BCL11A, BCL11Ae, cis-regulatory 
               
               
                   
                   
                   
                 elements of the B-globin locus, HBG 
               
               
                   
                   
                   
                 ½ promoter, those described in 
               
               
                   
                   
                   
                 WO2015148860, US Pat. Pub. 
               
               
                   
                   
                   
                 20110182867, 2015/148860 
               
               
                 Thymic Aplasia (DiGeorge 
                 Immune system, 
                   
                 deletion of 30 to 40 genes in the 
               
               
                 Syndrome; 22q11.2 deletion 
                 thymus 
                   
                 middle of chromosome 22 at 
               
               
                 syndrome) 
                   
                   
                 a location known as 22q11.2, including 
               
               
                   
                   
                   
                 TBX1, DGCR8 
               
               
                 Transthyretin amyloidosis 
                 liver 
                   
                 TTR (transthyretin) 
               
               
                 (ATTR) 
               
               
                 trimethylaminuria 
                 Metabolism 
                   
                 FMO3 
               
               
                   
                 disease 
               
               
                 Trinucleotide Repeat Disorders 
                 Various 
                   
                 HTT; SBMA/SMAX1/AR; 
               
               
                 (generally) 
                   
                   
                 FXN/X25 ATX3; 
               
               
                   
                   
                   
                 ATXN1; ATXN2; 
               
               
                   
                   
                   
                 DMPK; Atrophin-1 and Atn1 
               
               
                   
                   
                   
                 (DRPLA Dx); CBP (Creb-BP - global 
               
               
                   
                   
                   
                 instability); VLDLR; Atxn7; Atxn10; 
               
               
                   
                   
                   
                 FEN1, TNRC6A, PABPN1, JPH3, 
               
               
                   
                   
                   
                 MED15, ATXN1, ATXN3, TBP, 
               
               
                   
                   
                   
                 CACNA1A, ATXN80S, PPP2R2B, 
               
               
                   
                   
                   
                 ATXN7, TNRC6B, TNRC6C, CELF3, 
               
               
                   
                   
                   
                 MAB21L1, MSH2, TMEM185A, 
               
               
                   
                   
                   
                 SIX5, CNPY3, RAXE, GNB2, RPL14, 
               
               
                   
                   
                   
                 ATXN8, ISR, TTR, EP400, GIGYF2, 
               
               
                   
                   
                   
                 OGG1, STC1, CNDP1, C10ORF2, 
               
               
                   
                   
                   
                 MAML3, DKC1, PAXIP1, CASK, 
               
               
                   
                   
                   
                 MAPT, SP1, POLG, AFF2, THBS1, 
               
               
                   
                   
                   
                 TP53, ESR1, CGGBP1, ABT1, KLK3, 
               
               
                   
                   
                   
                 PRNP, JUN, KCNN3, BAX, FRAXA, 
               
               
                   
                   
                   
                 KBTBD10, MBNL1, RAD51, 
               
               
                   
                   
                   
                 NCOA3, ERDA1, TSC1, COMP, 
               
               
                   
                   
                   
                 GGLC, RRAD, MSH3, DRD2, CD44, 
               
               
                   
                   
                   
                 CTCF, CCND1, CLSPN, MEF2A, 
               
               
                   
                   
                   
                 PTPRU, GAPDH, TRIM22, WT1, 
               
               
                   
                   
                   
                 AHR, GPX1, TPMT, NDP, ARX, 
               
               
                   
                   
                   
                 TYR, EGR1, UNG, NUMBL, FABP2, 
               
               
                   
                   
                   
                 EN2, CRYGC, SRP14, CRYGB, 
               
               
                   
                   
                   
                 PDCD1, HOXA1, ATXN2L, PMS2, 
               
               
                   
                   
                   
                 GLA, CBL, FTH1, IL12RB2, OTX2, 
               
               
                   
                   
                   
                 HOXA5, POLG2, DLX2, AHRR, 
               
               
                   
                   
                   
                 MANF, RMEM158, see also 
               
               
                   
                   
                   
                 20110016540 
               
               
                 Turner&#39;s Syndrome (XO) 
                 Various - 
                   
                 Monosomy X 
               
               
                   
                 reproductive 
               
               
                   
                 organs, and sex 
               
               
                   
                 characteristics, 
               
               
                   
                 vasculature 
               
               
                 Tuberous Sclerosis 
                 CNS, heart, 
                   
                 TSC1, TSC2 
               
               
                   
                 kidneys 
               
               
                 Usher syndrome (Types I, II, and 
                 Ears, eyes 
                   
                 ABHD12, CDH23, CIB2, CLRN1, 
               
               
                 III) 
                   
                   
                 DFNB31, GPR98, HARS, MYO7A, 
               
               
                   
                   
                   
                 PCDH15, USH1C, USH1G, USH2A, 
               
               
                   
                   
                   
                 USH11A, those described in 
               
               
                   
                   
                   
                 WO2015134812A1 
               
               
                 Velocardiofacial syndrome (aka 
                 Various - 
                   
                 Many genes are deleted, COM, TBX1, 
               
               
                 22q11.2 deletion syndrome, 
                 skeletal, heart, 
                   
                 and other are associated with 
               
               
                 DiGeorge syndrome, conotruncal 
                 kidney, immune 
                   
                 symptoms 
               
               
                 anomaly face syndrome (CTAF), 
                 system, brain 
               
               
                 autosomal dominant Opitz G/BB 
               
               
                 syndrome or Cayler cardiofacial 
               
               
                 syndrome) 
               
               
                 Von Gierke&#39;s Disease (Glycogen 
                 Glycogen 
                 Various - liver, 
                 G6PC and SLC37A4 
               
               
                 Storage Disease type I) 
                 Storage disease 
                 kidney 
               
               
                 Von Hippel-Lindau Syndrome 
                 Various - cell 
                 CNS, Kidney, 
                 VHL 
               
               
                   
                 growth 
                 Eye, visceral 
               
               
                   
                 regulation 
                 organs 
               
               
                   
                 disorder 
               
               
                 Von Willebrand Disease (Types 
                 blood 
                   
                 VWF 
               
               
                 I, II and III) 
               
               
                 Wilson Disease 
                 Various - 
                 Liver, brains, 
                 ATP7B 
               
               
                   
                 Copper Storage 
                 eyes, other 
               
               
                   
                 Disease 
                 tissues where 
               
               
                   
                   
                 copper builds up 
               
               
                 Wiskott-Aldrich Syndrome 
                 Immune System 
                   
                 WAS 
               
               
                 Xeroderma Pigmentosum 
                 Skin 
                 Nervous system 
                 POLH 
               
               
                 XXX Syndrome 
                 Endocrine, brain 
                   
                 X chromosome trisomy 
               
               
                   
               
            
           
         
       
     
     In some embodiments, the CRISPR-Cas systems or components thereof can be used treat or prevent a disease in a subject by modifying one or more genes associated with one or more cellular functions, such as any one or more of those in Table 13. In some embodiments, the disease is a genetic disease or disorder. In some of embodiments, the CRISPR-Cas system or component thereof can modify one or more genes or polynucleotides associated with one or more genetic diseases such as any set forth in Table 13. 
     
       
         
           
               
             
               
                 TABLE 13 
               
             
            
               
                   
               
               
                 Exemplary Genes controlling Cellular Functions 
               
            
           
           
               
               
            
               
                 CELLULAR FUNCTION 
                 GENES 
               
               
                   
               
               
                 PI3K/AKT Signaling 
                 PRKCE; ITGAM; ITGA5; IRAK1; PRKAA2; EIF2AK2; 
               
               
                   
                 PTEN; EIF4E; PRKCZ; GRK6; MAPK1; TSC1; PLK1; 
               
               
                   
                 AKT2; IKBKB; PIK3CA; CDK8; CDKN1B; NFKB2; BCL2; 
               
               
                   
                 PIK3CB; PPP2R1A; MAPK8; BCL2L1; MAPK3; TSC2; 
               
               
                   
                 ITGA1; KRAS; EIF4EBP1; RELA; PRKCD; NOS3; 
               
               
                   
                 PRKAA1; MAPK9; CDK2; PPP2CA; PIM1; ITGB7; 
               
               
                   
                 YWHAZ; ILK; TP53; RAF1; IKBKG; RELB; DYRK1A; 
               
               
                   
                 CDKN1A; ITGB1; MAP2K2; JAK1; AKT1; JAK2; PIK3R1; 
               
               
                   
                 CHUK; PDPK1; PPP2R5C; CTNNB1; MAP2K1; NFKB1; 
               
               
                   
                 PAK3; ITGB3; CCND1; GSK3A; FRAP1; SFN; ITGA2; 
               
               
                   
                 TTK; CSNK1A1; BRAF; GSK3B; AKT3; FOXO1; SGK; 
               
               
                   
                 HSP90AA1; RPS6KB1 
               
               
                 ERK/MAPK Signaling 
                 PRKCE; ITGAM; ITGA5; HSPB1; IRAK1; PRKAA2; 
               
               
                   
                 EIF2AK2; RAC1; RAP1A; TLN1; EIF4E; ELK1; GRK6; 
               
               
                   
                 MAPK1; RAC2; PLK1; AKT2; PIK3CA; CDK8; CREB1; 
               
               
                   
                 PRKCI; PTK2; FOS; RPS6KA4; PIK3CB; PPP2R1A; 
               
               
                   
                 PIK3C3; MAPK8; MAPK3; ITGA1; ETS1; KRAS; MYCN; 
               
               
                   
                 EIF4EBP1; PPARG; PRKCD; PRKAA1; MAPK9; SRC; 
               
               
                   
                 CDK2; PPP2CA; PIM1; PIK3C2A; ITGB7; YWHAZ; 
               
               
                   
                 PPP1CC; KSR1; PXN; RAF1; FYN; DYRK1A; ITGB1; 
               
               
                   
                 MAP2K2; PAK4; PIK3R1; STAT3; PPP2R5C; MAP2K1; 
               
               
                   
                 PAK3; ITGB3; ESR1; ITGA2; MYC; TTK; CSNK1A1; 
               
               
                   
                 CRKL; BRAF; ATF4; PRKCA; SRF; STAT1; SGK 
               
               
                 Glucocorticoid Receptor 
                 RAC1; TAF4B; EP300; SMAD2; TRAF6; PCAF; ELK1; 
               
               
                 Signaling 
                 MAPK1; SMAD3; AKT2; IKBKB; NCOR2; UBE2I; 
               
               
                   
                 PIK3CA; CREB1; FOS; HSPA5; NFKB2; BCL2; 
               
               
                   
                 MAP3K14; STAT5B; PIK3CB; PIK3C3; MAPK8; BCL2L1; 
               
               
                   
                 MAPK3; TSC22D3; MAPK10; NRIP1; KRAS; MAPK13; 
               
               
                   
                 RELA; STAT5A; MAPK9; NOS2A; PBX1; NR3C1; 
               
               
                   
                 PIK3C2A; CDKN1C; TRAF2; SERPINE1; NCOA3; 
               
               
                   
                 MAPK14; TNF; RAF1; IKBKG; MAP3K7; CREBBP; 
               
               
                   
                 CDKN1A; MAP2K2; JAK1; IL8; NCOA2; AKT1; JAK2; 
               
               
                   
                 PIK3R1; CHUK; STAT3; MAP2K1; NFKB1; TGFBR1; 
               
               
                   
                 ESR1; SMAD4; CEBPB; JUN; AR; AKT3; CCL2; MMP1; 
               
               
                   
                 STAT1; IL6; HSP90AA1 
               
               
                 Axonal Guidance Signaling 
                 PRKCE; ITGAM; ROCK1; ITGA5; CXCR4; ADAM12; 
               
               
                   
                 IGF1; RAC1; RAP1A; EIF4E; PRKCZ; NRP1; NTRK2; 
               
               
                   
                 ARHGEF7; SMO; ROCK2; MAPK1; PGF; RAC2; 
               
               
                   
                 PTPN11; GNAS; AKT2; PIK3CA; ERBB2; PRKCI; PTK2; 
               
               
                   
                 CFL1; GNAQ; PIK3CB; CXCL12; PIK3C3; WNT11; 
               
               
                   
                 PRKD1; GNB2L1; ABL1; MAPK3; ITGA1; KRAS; RHOA; 
               
               
                   
                 PRKCD; PIK3C2A; ITGB7; GLI2; PXN; VASP; RAF1; 
               
               
                   
                 FYN; ITGB1; MAP2K2; PAK4; ADAM17; AKT1; PIK3R1; 
               
               
                   
                 GLI1; WNT5A; ADAM10; MAP2K1; PAK3; ITGB3; 
               
               
                   
                 CDC42; VEGFA; ITGA2; EPHA8; CRKL; RND1; GSK3B; 
               
               
                   
                 AKT3; PRKCA 
               
               
                 Ephrin Receptor Signaling 
                 PRKCE; ITGAM; ROCK1; ITGA5; CXCR4; IRAK1; 
               
               
                 Actin Cytoskeleton 
                 PRKAA2; EIF2AK2; RAC1; RAP1A; GRK6; ROCK2; 
               
               
                 Signaling 
                 MAPK1; PGF; RAC2; PTPN11; GNAS; PLK1; AKT2; 
               
               
                   
                 DOK1; CDK8; CREB1; PTK2; CFL1; GNAQ; MAP3K14; 
               
               
                   
                 CXCL12; MAPK8; GNB2L1; ABL1; MAPK3; ITGA1; 
               
               
                   
                 KRAS; RHOA; PRKCD; PRKAA1; MAPK9; SRC; CDK2; 
               
               
                   
                 PIM1; ITGB7; PXN; RAF1; FYN; DYRK1A; ITGB1; 
               
               
                   
                 MAP2K2; PAK4; AKT1; JAK2; STAT3; ADAM10; 
               
               
                   
                 MAP2K1; PAK3; ITGB3; CDC42; VEGFA; ITGA2; 
               
               
                   
                 EPHA8; TTK; CSNK1A1; CRKL; BRAF; PTPN13; ATF4; 
               
               
                   
                 AKT3; SGK 
               
               
                   
                 ACTN4; PRKCE; ITGAM; ROCK1; ITGA5; IRAK1; 
               
               
                   
                 PRKAA2; EIF2AK2; RAC1; INS; ARHGEF7; GRK6; 
               
               
                   
                 ROCK2; MAPK1; RAC2; PLK1; AKT2; PIK3CA; CDK8; 
               
               
                   
                 PTK2; CFL1; PIK3CB; MYH9; DIAPH1; PIK3C3; MAPK8; 
               
               
                   
                 F2R; MAPK3; SLC9A1; ITGA1; KRAS; RHOA; PRKCD; 
               
               
                   
                 PRKAA1; MAPK9; CDK2; PIM1; PIK3C2A; ITGB7; 
               
               
                   
                 PPP1CC; PXN; VIL2; RAF1; GSN; DYRK1A; ITGB1; 
               
               
                   
                 MAP2K2; PAK4; PIP5K1A; PIK3R1; MAP2K1; PAK3; 
               
               
                   
                 ITGB3; CDC42; APC; ITGA2; TTK; CSNK1A1; CRKL; 
               
               
                   
                 BRAF; VAV3; SGK 
               
               
                 Huntington&#39;s Disease 
                 PRKCE; IGF1; EP300; RCOR1; PRKCZ; HDAC4; TGM2; 
               
               
                 Signaling 
                 MAPK1; CAPNS1; AKT2; EGFR; NCOR2; SP1; CAPN2; 
               
               
                   
                 PIK3CA; HDAC5; CREB1; PRKCI; HSPA5; REST; 
               
               
                   
                 GNAQ; PIK3CB; PIK3C3; MAPK8; IGF1R; PRKD1; 
               
               
                   
                 GNB2L1; BCL2L1; CAPN1; MAPK3; CASP8; HDAC2; 
               
               
                   
                 HDAC7A; PRKCD; HDAC11; MAPK9; HDAC9; PIK3C2A; 
               
               
                   
                 HDAC3; TP53; CASP9; CREBBP; AKT1; PIK3R1; 
               
               
                   
                 PDPK1; CASP1; APAF1; FRAP1; CASP2; JUN; BAX; 
               
               
                   
                 ATF4; AKT3; PRKCA; CLTC; SGK; HDAC6; CASP3 
               
               
                 Apoptosis Signaling 
                 PRKCE; ROCK1; BID; IRAK1; PRKAA2; EIF2AK2; BAK1; 
               
               
                   
                 BIRC4; GRK6; MAPK1; CAPNS1; PLK1; AKT2; IKBKB; 
               
               
                   
                 CAPN2; CDK8; FAS; NFKB2; BCL2; MAP3K14; MAPK8; 
               
               
                   
                 BCL2L1; CAPN1; MAPK3; CASP8; KRAS; RELA; 
               
               
                   
                 PRKCD; PRKAA1; MAPK9; CDK2; PIM1; TP53; TNF; 
               
               
                   
                 RAF1; IKBKG; RELB; CASP9; DYRK1A; MAP2K2; 
               
               
                   
                 CHUK; APAF1; MAP2K1; NFKB1; PAK3; LMNA; CASP2; 
               
               
                   
                 BIRC2; TTK; CSNK1A1; BRAF; BAX; PRKCA; SGK; 
               
               
                   
                 CASP3; BIRC3; PARP1 
               
               
                 B Cell Receptor Signaling 
                 RAC1; PTEN; LYN; ELK1; MAPK1; RAC2; PTPN11; 
               
               
                   
                 AKT2; IKBKB; PIK3CA; CREB1; SYK; NFKB2; CAMK2A; 
               
               
                   
                 MAP3K14; PIK3CB; PIK3C3; MAPK8; BCL2L1; ABL1; 
               
               
                   
                 MAPK3; ETS1; KRAS; MAPK13; RELA; PTPN6; MAPK9; 
               
               
                   
                 EGR1; PIK3C2A; BTK; MAPK14; RAF1; IKBKG; RELB; 
               
               
                   
                 MAP3K7; MAP2K2; AKT1; PIK3R1; CHUK; MAP2K1; 
               
               
                   
                 NFKB1; CDC42; GSK3A; FRAP1; BCL6; BCL10; JUN; 
               
               
                   
                 GSK3B; ATF4; AKT3; VAV3; RPS6KB1 
               
               
                 Leukocyte Extravasation 
                 ACTN4; CD44; PRKCE; ITGAM; ROCK1; CXCR4; CYBA; 
               
               
                 Signaling 
                 RAC1; RAP1A; PRKCZ; ROCK2; RAC2; PTPN11; 
               
               
                   
                 MMP14; PIK3CA; PRKCI; PTK2; PIK3CB; CXCL12; 
               
               
                   
                 PIK3C3; MAPK8; PRKD1; ABL1; MAPK10; CYBB; 
               
               
                   
                 MAPK13; RHOA; PRKCD; MAPK9; SRC; PIK3C2A; BTK; 
               
               
                   
                 MAPK14; NOX1; PXN; VIL2; VASP; ITGB1; MAP2K2; 
               
               
                   
                 CTNND1; PIK3R1; CTNNB1; CLDN1; CDC42; F11R; ITK; 
               
               
                   
                 CRKL; VAV3; CTTN; PRKCA; MMP1; MMP9 
               
               
                 Integrin Signaling 
                 ACTN4; ITGAM; ROCK1; ITGA5; RAC1; PTEN; RAP1A; 
               
               
                   
                 TLN1; ARHGEF7; MAPK1; RAC2; CAPNS1; AKT2; 
               
               
                   
                 CAPN2; PIK3CA; PTK2; PIK3CB; PIK3C3; MAPK8; 
               
               
                   
                 CAV1; CAPN1; ABL1; MAPK3; ITGA1; KRAS; RHOA; 
               
               
                   
                 SRC; PIK3C2A; ITGB7; PPP1CC; ILK; PXN; VASP; 
               
               
                   
                 RAF1; FYN; ITGB1; MAP2K2; PAK4; AKT1; PIK3R1; 
               
               
                   
                 TNK2; MAP2K1; PAK3; ITGB3; CDC42; RND3; ITGA2; 
               
               
                   
                 CRKL; BRAF; GSK3B; AKT3 
               
               
                 Acute Phase Response 
                 IRAK1; SOD2; MYD88; TRAF6; ELK1; MAPK1; PTPN11; 
               
               
                 Signaling 
                 AKT2; IKBKB; PIK3CA; FOS; NFKB2; MAP3K14; 
               
               
                   
                 PIK3CB; MAPK8; RIPK1; MAPK3; IL6ST; KRAS; 
               
               
                   
                 MAPK13; IL6R; RELA; SOCS1; MAPK9; FTL; NR3C1; 
               
               
                   
                 TRAF2; SERPINE1; MAPK14; TNF; RAF1; PDK1; 
               
               
                   
                 IKBKG; RELB; MAP3K7; MAP2K2; AKT1; JAK2; PIK3R1; 
               
               
                   
                 CHUK; STAT3; MAP2K1; NFKB1; FRAP1; CEBPB; JUN; 
               
               
                   
                 AKT3; IL1R1; IL6 
               
               
                 PTEN Signaling 
                 ITGAM; ITGA5; RAC1; PTEN; PRKCZ; BCL2L11; 
               
               
                   
                 MAPK1; RAC2; AKT2; EGFR; IKBKB; CBL; PIK3CA; 
               
               
                   
                 CDKN1B; PTK2; NFKB2; BCL2; PIK3CB; BCL2L1; 
               
               
                   
                 MAPK3; ITGA1; KRAS; ITGB7; ILK; PDGFRB; INSR; 
               
               
                   
                 RAF1; IKBKG; CASP9; CDKN1A; ITGB1; MAP2K2; 
               
               
                   
                 AKT1; PIK3R1; CHUK; PDGFRA; PDPK1; MAP2K1; 
               
               
                   
                 NFKB1; ITGB3; CDC42; CCND1; GSK3A; ITGA2; 
               
               
                   
                 GSK3B; AKT3; FOXO1; CASP3; RPS6KB1 
               
               
                 p53 Signaling 
                 PTEN; EP300; BBC3; PCAF; FASN; BRCA1; GADD45A; 
               
               
                 Aryl Hydrocarbon Receptor 
                 BIRC5; AKT2; PIK3CA; CHEK1; TP53INP1; BCL2; 
               
               
                 Signaling 
                 PIK3CB; PIK3C3; MAPK8; THBS1; ATR; BCL2L1; E2F1; 
               
               
                   
                 PMAIP1; CHEK2; TNFRSF10B; TP73; RB1; HDAC9; 
               
               
                   
                 CDK2; PIK3C2A; MAPK14; TP53; LRDD; CDKN1A; 
               
               
                   
                 HIPK2; AKT1; PIK3R1; RRM2B; APAF1; CTNNB1; 
               
               
                   
                 SIRT1; CCND1; PRKDC; ATM; SFN; CDKN2A; JUN; 
               
               
                   
                 SNAI2; GSK3B; BAχ; AKT3 
               
               
                   
                 HSPB1; EP300; FASN; TGM2; RXRA; MAPK1; NQO1; 
               
               
                   
                 NCOR2; SP1; ARNT; CDKN1B; FOS; CHEK1; 
               
               
                   
                 SMARCA4; NFKB2; MAPK8; ALDH1A1; ATR; E2F1; 
               
               
                   
                 MAPK3; NRIP1; CHEK2; RELA; TP73; GSTP1; RB1; 
               
               
                   
                 SRC; CDK2; AHR; NFE2L2; NCOA3; TP53; TNF; 
               
               
                   
                 CDKN1A; NCOA2; APAF1; NFKB1; CCND1; ATM; ESR1; 
               
               
                   
                 CDKN2A; MYC; JUN; ESR2; BAX; IL6; CYP1B1; 
               
               
                   
                 HSP90AA1 
               
               
                 Xenobiotic Metabolism 
                 PRKCE; EP300; PRKCZ; RXRA; MAPK1; NQO1; 
               
               
                 Signaling 
                 NCOR2; PIK3CA; ARNT; PRKCI; NFKB2; CAMK2A; 
               
               
                   
                 PIK3CB; PPP2R1A; PIK3C3; MAPK8; PRKD1; 
               
               
                   
                 ALDH1A1; MAPK3; NRIP1; KRAS; MAPK13; PRKCD; 
               
               
                   
                 GSTP1; MAPK9; NOS2A; ABCB1; AHR; PPP2CA; FTL; 
               
               
                   
                 NFE2L2; PIK3C2A; PPARGC1A; MAPK14; TNF; RAF1; 
               
               
                   
                 CREBBP; MAP2K2; PIK3R1; PPP2R5C; MAP2K1; 
               
               
                   
                 NFKB1; KEAP1; PRKCA; EIF2AK3; IL6; CYP1B1; 
               
               
                   
                 HSP90AA1 
               
               
                 SAPK/JNK Signaling 
                 PRKCE; IRAK1; PRKAA2; EIF2AK2; RAC1; ELK1; 
               
               
                   
                 GRK6; MAPK1; GADD45A; RAC2; PLK1; AKT2; PIK3CA; 
               
               
                   
                 FADD; CDK8; PIK3CB; PIK3C3; MAPK8; RIPK1; 
               
               
                   
                 GNB2L1; IRS1; MAPK3; MAPK10; DAXX; KRAS; 
               
               
                   
                 PRKCD; PRKAA1; MAPK9; CDK2; PIM1; PIK3C2A; 
               
               
                   
                 TRAF2; TP53; LCK; MAP3K7; DYRK1A; MAP2K2; 
               
               
                   
                 PIK3R1; MAP2K1; PAK3; CDC42; JUN; TTK; CSNK1A1; 
               
               
                   
                 CRKL; BRAF; SGK 
               
               
                 PPAr/RXR Signaling 
                 PRKAA2; EP300; INS; SMAD2; TRAF6; PPARA; FASN; 
               
               
                   
                 RXRA; MAPK1; SMAD3; GNAS; IKBKB; NCOR2; 
               
               
                   
                 ABCA1; GNAQ; NFKB2; MAP3K14; STAT5B; MAPK8; 
               
               
                   
                 IRS1; MAPK3; KRAS; RELA; PRKAA1; PPARGC1A; 
               
               
                   
                 NCOA3; MAPK14; INSR; RAF1; IKBKG; RELB; MAP3K7; 
               
               
                   
                 CREBBP; MAP2K2; JAK2; CHUK; MAP2K1; NFKB1; 
               
               
                   
                 TGFBR1; SMAD4; JUN; IL1R1; PRKCA; IL6; HSP90AA1; 
               
               
                   
                 ADIPOQ 
               
               
                 NF-KB Signaling 
                 IRAK1; EIF2AK2; EP300; INS; MYD88; PRKCZ; TRAF6; 
               
               
                   
                 TBK1; AKT2; EGFR; IKBKB; PIK3CA; BTRC; NFKB2; 
               
               
                   
                 MAP3K14; PIK3CB; PIK3C3; MAPK8; RIPK1; HDAC2; 
               
               
                   
                 KRAS; RELA; PIK3C2A; TRAF2; TLR4; PDGFRB; TNF; 
               
               
                   
                 INSR; LCK; IKBKG; RELB; MAP3K7; CREBBP; AKT1; 
               
               
                   
                 PIK3R1; CHUK; PDGFRA; NFKB1; TLR2; BCL10; 
               
               
                   
                 GSK3B; AKT3; TNFAIP3; IL1R1 
               
               
                 Neuregulin Signaling 
                 ERBB4; PRKCE; ITGAM; ITGA5; PTEN; PRKCZ; ELK1; 
               
               
                 Wnt &amp; Beta catenin 
                 MAPK1; PTPN11; AKT2; EGFR; ERBB2; PRKCI; 
               
               
                 Signaling 
                 CDKN1B; STAT5B; PRKD1; MAPK3; ITGA1; KRAS; 
               
               
                   
                 PRKCD; STAT5A; SRC; ITGB7; RAF1; ITGB1; MAP2K2; 
               
               
                   
                 ADAM17; AKT1; PIK3R1; PDPK1; MAP2K1; ITGB3; 
               
               
                   
                 EREG; FRAP1; PSEN1; ITGA2; MYC; NRG1; CRKL; 
               
               
                   
                 AKT3; PRKCA; HSP90AA1; RPS6KB1 
               
               
                   
                 CD44; EP300; LRP6; DVL3; CSNK1E; GJA1; SMO; 
               
               
                   
                 AKT2; PIN1; CDH1; BTRC; GNAQ; MARK2; PPP2R1A; 
               
               
                   
                 WNT11; SRC; DKK1; PPP2CA; SOX6; SFRP2; ILK; 
               
               
                   
                 LEF1; SOX9; TP53; MAP3K7; CREBBP; TCF7L2; AKT1; 
               
               
                   
                 PPP2R5C; WNT5A; LRP5; CTNNB1; TGFBR1; CCND1; 
               
               
                   
                 GSK3A; DVL1; APC; CDKN2A; MYC; CSNK1A1; GSK3B; 
               
               
                   
                 AKT3; SOX2 
               
               
                 Insulin Receptor Signaling 
                 PTEN; INS; EIF4E; PTPN1; PRKCZ; MAPK1; TSC1; 
               
               
                   
                 PTPN11; AKT2; CBL; PIK3CA; PRKCI; PIK3CB; PIK3C3; 
               
               
                   
                 MAPK8; IRS1; MAPK3; TSC2; KRAS; EIF4EBP1; 
               
               
                   
                 SLC2A4; PIK3C2A; PPP1CC; INSR; RAF1; FYN; 
               
               
                   
                 MAP2K2; JAK1; AKT1; JAK2; PIK3R1; PDPK1; MAP2K1; 
               
               
                   
                 GSK3A; FRAP1; CRKL; GSK3B; AKT3; FOXO1; SGK; 
               
               
                   
                 RPS6KB1 
               
               
                 IL-6 Signaling 
                 HSPB1; TRAF6; MAPKAPK2; ELK1; MAPK1; PTPN11; 
               
               
                   
                 IKBKB; FOS; NFKB2; MAP3K14; MAPK8; MAPK3; 
               
               
                   
                 MAPK10; IL6ST; KRAS; MAPK13; IL6R; RELA; SOCS1; 
               
               
                   
                 MAPK9; ABCB1; TRAF2; MAPK14; TNF; RAF1; IKBKG; 
               
               
                   
                 RELB; MAP3K7; MAP2K2; IL8; JAK2; CHUK; STAT3; 
               
               
                   
                 MAP2K1; NFKB1; CEBPB; JUN; IL1R1; SRF; IL6 
               
               
                 Hepatic Cholestasis 
                 PRKCE; IRAK1; INS; MYD88; PRKCZ; TRAF6; PPARA; 
               
               
                   
                 RXRA; IKBKB; PRKCI; NFKB2; MAP3K14; MAPK8; 
               
               
                   
                 PRKD1; MAPK10; RELA; PRKCD; MAPK9; ABCB1; 
               
               
                   
                 TRAF2; TLR4; TNF; INSR; IKBKG; RELB; MAP3K7; IL8; 
               
               
                   
                 CHUK; NR1H2; TJP2; NFKB1; ESR1; SREBF1; FGFR4; 
               
               
                   
                 JUN; IL1R1; PRKCA; IL6 
               
               
                 IGF-1 Signaling 
                 IGF1; PRKCZ; ELK1; MAPK1; PTPN11; NEDD4; AKT2; 
               
               
                   
                 PIK3CA; PRKCI; PTK2; FOS; PIK3CB; PIK3C3; MAPK8; 
               
               
                   
                 IGF1R; IRS1; MAPK3; IGFBP7; KRAS; PIK3C2A; 
               
               
                   
                 YWHAZ; PXN; RAF1; CASP9; MAP2K2; AKT1; PIK3R1; 
               
               
                   
                 PDPK1; MAP2K1; IGFBP2; SFN; JUN; CYR61; AKT3; 
               
               
                   
                 FOXO1; SRF; CTGF; RPS6KB1 
               
               
                 NRF2-mediated Oxidative 
                 PRKCE; EP300; SOD2; PRKCZ; MAPK1; SQSTM1; 
               
               
                 Stress Response 
                 NQO1; PIK3CA; PRKCI; FOS; PIK3CB; PIK3C3; MAPK8; 
               
               
                   
                 PRKD1; MAPK3; KRAS; PRKCD; GSTP1; MAPK9; FTL; 
               
               
                   
                 NFE2L2; PIK3C2A; MAPK14; RAF1; MAP3K7; CREBBP; 
               
               
                   
                 MAP2K2; AKT1; PIK3R1; MAP2K1; PPIB; JUN; KEAP1; 
               
               
                   
                 GSK3B; ATF4; PRKCA; EIF2AK3; HSP90AA1 
               
               
                 Hepatic Fibrosis/Hepatic 
                 EDN1; IGF1; KDR; FLT1; SMAD2; FGFR1; MET; PGF; 
               
               
                 Stellate Cell Activation 
                 SMAD3; EGFR; FAS; CSF1; NFKB2; BCL2; MYH9; 
               
               
                   
                 IGF1R; IL6R; RELA; TLR4; PDGFRB; TNF; RELB; IL8; 
               
               
                   
                 PDGFRA; NFKB1; TGFBR1; SMAD4; VEGFA; BAX; 
               
               
                   
                 IL1R1; CCL2; HGF; MMP1; STAT1; IL6; CTGF; MMP9 
               
               
                 PPAR Signaling 
                 EP300; INS; TRAF6; PPARA; RXRA; MAPK1; IKBKB; 
               
               
                   
                 NCOR2; FOS; NFKB2; MAP3K14; STAT5B; MAPK3; 
               
               
                   
                 NRIP1; KRAS; PPARG; RELA; STAT5A; TRAF2; 
               
               
                   
                 PPARGC1A; PDGFRB; TNF; INSR; RAF1; IKBKG; 
               
               
                   
                 RELB; MAP3K7; CREBBP; MAP2K2; CHUK; PDGFRA; 
               
               
                   
                 MAP2K1; NFKB1; JUN; IL1R1; HSP90AA1 
               
               
                 Fc Epsilon RI Signaling 
                 PRKCE; RAC1; PRKCZ; LYN; MAPK1; RAC2; PTPN11; 
               
               
                   
                 AKT2; PIK3CA; SYK; PRKCI; PIK3CB; PIK3C3; MAPK8; 
               
               
                   
                 PRKD1; MAPK3; MAPK10; KRAS; MAPK13; PRKCD; 
               
               
                   
                 MAPK9; PIK3C2A; BTK; MAPK14; TNF; RAF1; FYN; 
               
               
                   
                 MAP2K2; AKT1; PIK3R1; PDPK1; MAP2K1; AKT3; 
               
               
                   
                 VAV3; PRKCA 
               
               
                 G-Protein Coupled 
                 PRKCE; RAP1A; RGS16; MAPK1; GNAS; AKT2; IKBKB; 
               
               
                 Receptor Signaling 
                 PIK3CA; CREB1; GNAQ; NFKB2; CAMK2A; PIK3CB; 
               
               
                   
                 PIK3C3; MAPK3; KRAS; RELA; SRC; PIK3C2A; RAF1; 
               
               
                   
                 IKBKG; RELB; FYN; MAP2K2; AKT1; PIK3R1; CHUK; 
               
               
                   
                 PDPK1; STAT3; MAP2K1; NFKB1; BRAF; ATF4; AKT3; 
               
               
                   
                 PRKCA 
               
               
                 Inositol Phosphate 
                 PRKCE; IRAK1; PRKAA2; EIF2AK2; PTEN; GRK6; 
               
               
                 Metabolism 
                 MAPK1; PLK1; AKT2; PIK3CA; CDK8; PIK3CB; PIK3C3; 
               
               
                   
                 MAPK8; MAPK3; PRKCD; PRKAA1; MAPK9; CDK2; 
               
               
                   
                 PIM1; PIK3C2A; DYRK1A; MAP2K2; PIP5K1A; PIK3R1; 
               
               
                   
                 MAP2K1; PAK3; ATM; TTK; CSNK1A1; BRAF; SGK 
               
               
                 PDGF Signaling 
                 EIF2AK2; ELK1; ABL2; MAPK1; PIK3CA; FOS; PIK3CB; 
               
               
                   
                 PIK3C3; MAPK8; CAV1; ABL1; MAPK3; KRAS; SRC; 
               
               
                   
                 PIK3C2A; PDGFRB; RAF1; MAP2K2; JAK1; JAK2; 
               
               
                   
                 PIK3R1; PDGFRA; STAT3; SPHK1; MAP2K1; MYC; 
               
               
                   
                 JUN; CRKL; PRKCA; SRF; STAT1; SPHK2 
               
               
                 VEGF Signaling 
                 ACTN4; ROCK1; KDR; FLT1; ROCK2; MAPK1; PGF; 
               
               
                   
                 AKT2; PIK3CA; ARNT; PTK2; BCL2; PIK3CB; PIK3C3; 
               
               
                   
                 BCL2L1; MAPK3; KRAS; HIF1A; NOS3; PIK3C2A; PXN; 
               
               
                   
                 RAF1; MAP2K2; ELAVL1; AKT1; PIK3R1; MAP2K1; SFN; 
               
               
                   
                 VEGFA; AKT3; FOXO1; PRKCA 
               
               
                 Natural Killer Cell Signaling 
                 PRKCE; RAC1; PRKCZ; MAPK1; RAC2; PTPN11; 
               
               
                   
                 KIR2DL3; AKT2; PIK3CA; SYK; PRKCI; PIK3CB; 
               
               
                   
                 PIK3C3; PRKD1; MAPK3; KRAS; PRKCD; PTPN6; 
               
               
                   
                 PIK3C2A; LCK; RAF1; FYN; MAP2K2; PAK4; AKT1; 
               
               
                   
                 PIK3R1; MAP2K1; PAK3; AKT3; VAV3; PRKCA 
               
               
                 Cell Cycle: G1/S 
                 HDAC4; SMAD3; SUV39H1; HDAC5; CDKN1B; BTRC; 
               
               
                 Checkpoint Regulation 
                 ATR; ABL1; E2F1; HDAC2; HDAC7A; RB1; HDAC11; 
               
               
                   
                 HDAC9; CDK2; E2F2; HDAC3; TP53; CDKN1A; CCND1; 
               
               
                   
                 E2F4; ATM; RBL2; SMAD4; CDKN2A; MYC; NRG1; 
               
               
                   
                 GSK3B; RBL1; HDAC6 
               
               
                 T Cell Receptor Signaling 
                 RAC1; ELK1; MAPK1; IKBKB; CBL; PIK3CA; FOS; 
               
               
                   
                 NFKB2; PIK3CB; PIK3C3; MAPK8; MAPK3; KRAS; 
               
               
                   
                 RELA; PIK3C2A; BTK; LCK; RAF1; IKBKG; RELB; FYN; 
               
               
                   
                 MAP2K2; PIK3R1; CHUK; MAP2K1; NFKB1; ITK; BCL10; 
               
               
                   
                 JUN; VAV3 
               
               
                 Death Receptor Signaling 
                 CRADD; HSPB1; BID; BIRC4; TBK1; IKBKB; FADD; 
               
               
                   
                 FAS; NFKB2; BCL2; MAP3K14; MAPK8; RIPK1; CASP8; 
               
               
                   
                 DAXX; TNFRSF10B; RELA; TRAF2; TNF; IKBKG; RELB; 
               
               
                   
                 CASP9; CHUK; APAF1; NFKB1; CASP2; BIRC2; CASP3; 
               
               
                   
                 BIRC3 
               
               
                 FGF Signaling 
                 RAC1; FGFR1; MET; MAPKAPK2; MAPK1; PTPN11; 
               
               
                   
                 AKT2; PIK3CA; CREB1; PIK3CB; PIK3C3; MAPK8; 
               
               
                   
                 MAPK3; MAPK13; PTPN6; PIK3C2A; MAPK14; RAF1; 
               
               
                   
                 AKT1; PIK3R1; STAT3; MAP2K1; FGFR4; CRKL; ATF4; 
               
               
                   
                 AKT3; PRKCA; HGF 
               
               
                 GM-CSF Signaling 
                 LYN; ELK1; MAPK1; PTPN11; AKT2; PIK3CA; CAMK2A; 
               
               
                   
                 STAT5B; PIK3CB; PIK3C3; GNB2L1; BCL2L1; MAPK3; 
               
               
                   
                 ETS1; KRAS; RUNX1; PIM1; PIK3C2A; RAF1; MAP2K2; 
               
               
                   
                 AKT1; JAK2; PIK3R1; STAT3; MAP2K1; CCND1; AKT3; 
               
               
                   
                 STAT1 
               
               
                 Amyotrophic Lateral 
                 BID; IGF1; RAC1; BIRC4; PGF; CAPNS1; CAPN2; 
               
               
                 Sclerosis Signaling 
                 PIK3CA; BCL2; PIK3CB; PIK3C3; BCL2L1; CAPN1; 
               
               
                   
                 PIK3C2A; TP53; CASP9; PIK3R1; RAB5A; CASP1; 
               
               
                   
                 APAF1; VEGFA; BIRC2; BAχ; AKT3; CASP3; BIRC3 
               
               
                 JAK/Stat Signaling 
                 PTPN1; MAPK1; PTPN11; AKT2; PIK3CA; STAT5B; 
               
               
                   
                 PIK3CB; PIK3C3; MAPK3; KRAS; SOCS1; STAT5A; 
               
               
                   
                 PTPN6; PIK3C2A; RAF1; CDKN1A; MAP2K2; JAK1; 
               
               
                   
                 AKT1; JAK2; PIK3R1; STAT3; MAP2K1; FRAP1; AKT3; 
               
               
                   
                 STAT1 
               
               
                 Nicotinate and Nicotinamide 
                 PRKCE; IRAK1; PRKAA2; EIF2AK2; GRK6; MAPK1; 
               
               
                 Metabolism 
                 PLK1; AKT2; CDK8; MAPK8; MAPK3; PRKCD; PRKAA1; 
               
               
                   
                 PBEF1; MAPK9; CDK2; PIM1; DYRK1A; MAP2K2; 
               
               
                   
                 MAP2K1; PAK3; NT5E; TTK; CSNK1A1; BRAF; SGK 
               
               
                 Chemokine Signaling 
                 CXCR4; ROCK2; MAPK1; PTK2; FOS; CFL1; GNAQ; 
               
               
                   
                 CAMK2A; CXCL12; MAPK8; MAPK3; KRAS; MAPK13; 
               
               
                   
                 RHOA; CCR3; SRC; PPP1CC; MAPK14; NOX1; RAF1; 
               
               
                   
                 MAP2K2; MAP2K1; JUN; CCL2; PRKCA 
               
               
                 IL-2 Signaling 
                 ELK1; MAPK1; PTPN11; AKT2; PIK3CA; SYK; FOS; 
               
               
                   
                 STAT5B; PIK3CB; PIK3C3; MAPK8; MAPK3; KRAS; 
               
               
                   
                 SOCS1; STAT5A; PIK3C2A; LCK; RAF1; MAP2K2; 
               
               
                   
                 JAK1; AKT1; PIK3R1; MAP2K1; JUN; AKT3 
               
               
                 Synaptic Long Term 
                 PRKCE; IGF1; PRKCZ; PRDX6; LYN; MAPK1; GNAS; 
               
               
                 Depression 
                 PRKCI; GNAQ; PPP2R1A; IGF1R; PRKD1; MAPK3; 
               
               
                   
                 KRAS; GRN; PRKCD; NOS3; NOS2A; PPP2CA; 
               
               
                   
                 YWHAZ; RAF1; MAP2K2; PPP2R5C; MAP2K1; PRKCA 
               
               
                 Estrogen Receptor 
                 TAF4B; EP300; CARMI; PCAF; MAPK1; NCOR2; 
               
               
                 Signaling 
                 SMARCA4; MAPK3; NRIP1; KRAS; SRC; NR3C1; 
               
               
                   
                 HDAC3; PPARGC1A; RBM9; NCOA3; RAF1; CREBBP; 
               
               
                   
                 MAP2K2; NCOA2; MAP2K1; PRKDC; ESR1; ESR2 
               
               
                 Protein Ubiquitination 
                 TRAF6; SMURF1; BIRC4; BRCA1; UCHL1; NEDD4; 
               
               
                 Pathway 
                 CBL; UBE2I; BTRC; HSPA5; USP7; USP10; FBXW7; 
               
               
                   
                 USP9X; STUB1; USP22; B2M; BIRC2; PARK2; USP8; 
               
               
                   
                 USP1; VHL; HSP90AA1; BIRC3 
               
               
                 IL-10 Signaling 
                 TRAF6; CCR1; ELK1; IKBKB; SP1; FOS; NFKB2; 
               
               
                   
                 MAP3K14; MAPK8; MAPK13; RELA; MAPK14; TNF; 
               
               
                   
                 IKBKG; RELB; MAP3K7; JAK1; CHUK; STAT3; NFKB1; 
               
               
                   
                 JUN; IL1R1; IL6 
               
               
                 VDR/RXR Activation 
                 PRKCE; EP300; PRKCZ; RXRA; GADD45A; HES1; 
               
               
                   
                 NCOR2; SP1; PRKCI; CDKN1B; PRKD1; PRKCD; 
               
               
                   
                 RUNX2; KLF4; YY1; NCOA3; CDKN1A; NCOA2; SPP1; 
               
               
                   
                 LRP5; CEBPB; FOXO1; PRKCA 
               
               
                 TGF-beta Signaling 
                 EP300; SMAD2; SMURF1; MAPK1; SMAD3; SMAD1; 
               
               
                   
                 FOS; MAPK8; MAPK3; KRAS; MAPK9; RUNX2; 
               
               
                   
                 SERPINE1; RAF1; MAP3K7; CREBBP; MAP2K2; 
               
               
                   
                 MAP2K1; TGFBR1; SMAD4; JUN; SMAD5 
               
               
                 Toll-like Receptor Signaling 
                 IRAK1; EIF2AK2; MYD88; TRAF6; PPARA; ELK1; 
               
               
                   
                 IKBKB; FOS; NFKB2; MAP3K14; MAPK8; MAPK13; 
               
               
                   
                 RELA; TLR4; MAPK14; IKBKG; RELB; MAP3K7; CHUK; 
               
               
                   
                 NFKB1; TLR2; JUN 
               
               
                 p38 MAPK Signaling 
                 HSPB1; IRAK1; TRAF6; MAPKAPK2; ELK1; FADD; FAS; 
               
               
                   
                 CREB1; DDIT3; RPS6KA4; DAXX; MAPK13; TRAF2; 
               
               
                   
                 MAPK14; TNF; MAP3K7; TGFBR1; MYC; ATF4; IL1R1; 
               
               
                   
                 SRF; STAT1 
               
               
                 Neurotrophin/TRK Signaling 
                 NTRK2; MAPK1; PTPN11; PIK3CA; CREB1; FOS; 
               
               
                   
                 PIK3CB; PIK3C3; MAPK8; MAPK3; KRAS; PIK3C2A; 
               
               
                   
                 RAF1; MAP2K2; AKT1; PIK3R1; PDPK1; MAP2K1; 
               
               
                   
                 CDC42; JUN; ATF4 
               
               
                 FXR/RXR Activation 
                 INS; PPARA; FASN; RXRA; AKT2; SDC1; MAPK8; 
               
               
                   
                 APOB; MAPK10; PPARG; MTTP; MAPK9; PPARGC1A; 
               
               
                   
                 TNF; CREBBP; AKT1; SREBF1; FGFR4; AKT3; FOXO1 
               
               
                 Synaptic Long Term 
                 PRKCE; RAP1A; EP300; PRKCZ; MAPK1; CREB1; 
               
               
                 Potentiation 
                 PRKCI; GNAQ; CAMK2A; PRKD1; MAPK3; KRAS; 
               
               
                   
                 PRKCD; PPP1CC; RAF1; CREBBP; MAP2K2; MAP2K1; 
               
               
                   
                 ATF4; PRKCA 
               
               
                 Calcium Signaling 
                 RAP1A; EP300; HDAC4; MAPK1; HDAC5; CREB1; 
               
               
                   
                 CAMK2A; MYH9; MAPK3; HDAC2; HDAC7A; HDAC11; 
               
               
                   
                 HDAC9; HDAC3; CREBBP; CALR; CAMKK2; ATF4; 
               
               
                   
                 HDAC6 
               
               
                 EGF Signaling 
                 ELK1; MAPK1; EGFR; PIK3CA; FOS; PIK3CB; PIK3C3; 
               
               
                   
                 MAPK8; MAPK3; PIK3C2A; RAF1; JAK1; PIK3R1; 
               
               
                   
                 STAT3; MAP2K1; JUN; PRKCA; SRF; STAT1 
               
               
                 Hypoxia Signaling in the 
                 EDN1; PTEN; EP300; NQO1; UBE2I; CREB1; ARNT; 
               
               
                 Cardiovascular System 
                 HIF1A; SLC2A4; NOS3; TP53; LDHA; AKT1; ATM; 
               
               
                   
                 VEGFA; JUN; ATF4; VHL; HSP90AA1 
               
               
                 LPS/IL-1 Mediated Inhibition 
                 IRAK1; MYD88; TRAF6; PPARA; RXRA; ABCA1; 
               
               
                 of RXR Function 
                 MAPK8; ALDH1A1; GSTP1; MAPK9; ABCB1; TRAF2; 
               
               
                   
                 TLR4; TNF; MAP3K7; NR1H2; SREBF1; JUN; IL1R1 
               
               
                 LXR/RXR Activation 
                 FASN; RXRA; NCOR2; ABCA1; NFKB2; IRF3; RELA; 
               
               
                   
                 NOS2A; TLR4; TNF; RELB; LDLR; NR1H2; NFKB1; 
               
               
                   
                 SREBF1; IL1R1; CCL2; IL6; MMP9 
               
               
                 Amyloid Processing 
                 PRKCE; CSNK1E; MAPK1; CAPNS1; AKT2; CAPN2; 
               
               
                   
                 CAPN1; MAPK3; MAPK13; MAPT; MAPK14; AKT1; 
               
               
                   
                 PSEN1; CSNK1A1; GSK3B; AKT3; APP 
               
               
                 IL-4 Signaling 
                 AKT2; PIK3CA; PIK3CB; PIK3C3; IRS1; KRAS; SOCS1; 
               
               
                   
                 PTPN6; NR3C1; PIK3C2A; JAK1; AKT1; JAK2; PIK3R1; 
               
               
                   
                 FRAP1; AKT3; RPS6KB1 
               
               
                 Cell Cycle: G2/M DNA 
                 EP300; PCAF; BRCA1; GADD45A; PLK1; BTRC; 
               
               
                 Damage Checkpoint 
                 CHEK1; ATR; CHEK2; YWHAZ; TP53; CDKN1A; 
               
               
                 Regulation 
                 PRKDC; ATM; SFN; CDKN2A 
               
               
                 Nitric Oxide Signaling in the 
                 KDR; FLT1; PGF; AKT2; PIK3CA; PIK3CB; PIK3C3; 
               
               
                 Cardiovascular System 
                 CAV1; PRKCD; NOS3; PIK3C2A; AKT1; PIK3R1; 
               
               
                   
                 VEGFA; AKT3; HSP90AA1 
               
               
                 Purine Metabolism 
                 NME2; SMARCA4; MYH9; RRM2; ADAR; EIF2AK4; 
               
               
                   
                 PKM2; ENTPD1; RAD51; RRM2B; TJP2; RAD51C; 
               
               
                   
                 NT5E; POLDI; NME1 
               
               
                 cAMP-mediated Signaling 
                 RAP1A; MAPK1; GNAS; CREB1; CAMK2A; MAPK3; 
               
               
                   
                 SRC; RAF1; MAP2K2; STAT3; MAP2K1; BRAF; ATF4 
               
               
                 Mitochondrial Dysfunction 
                 SOD2; MAPK8; CASP8; MAPK10; MAPK9; CASP9; 
               
               
                 Notch Signaling 
                 PARK7; PSEN1; PARK2; APP; CASP3 
               
               
                   
                 HES1; JAG1; NUMB; NOTCH4; ADAM17; NOTCH2; 
               
               
                   
                 PSEN1; NOTCH3; NOTCH1; DLL4 
               
               
                 Endoplasmic Reticulum 
                 HSPA5; MAPK8; XBP1; TRAF2; ATF6; CASP9; ATF4; 
               
               
                 Stress Pathway 
                 EIF2AK3; CASP3 
               
               
                 Pyrimidine Metabolism 
                 NME2; AICDA; RRM2; EIF2AK4; ENTPD1; RRM2B; 
               
               
                   
                 NT5E; POLD1; NME1 
               
               
                 Parkinson&#39;s Signaling 
                 UCHL1; MAPK8; MAPK13; MAPK14; CASP9; PARK7; 
               
               
                   
                 PARK2; CASP3 
               
               
                 Cardiac &amp; Beta Adrenergic 
                 GNAS; GNAQ; PPP2R1A; GNB2L1; PPP2CA; PPP1CC; 
               
               
                 Signaling 
                 PPP2R5C 
               
               
                 Glycolysis/Gluconeogenesis 
                 HK2; GCK; GPI; ALDH1A1; PKM2; LDHA; HK1 
               
               
                 Interferon Signaling 
                 IRF1; SOCS1; JAK1; JAK2; IFITM1; STAT1; IFIT3 
               
               
                 Sonic Hedgehog Signaling 
                 ARRB2; SMO; GLI2; DYRK1A; GLI1; GSK3B; DYRK1B 
               
               
                 Glycerophospholipid 
                 PLD1; GRN; GPAM; YWHAZ; SPHK1; SPHK2 
               
               
                 Metabolism 
               
               
                 Phospholipid Degradation 
                 PRDX6; PLD1; GRN; YWHAZ; SPHK1; SPHK2 
               
               
                 Tryptophan Metabolism 
                 SIAH2; PRMT5; NEDD4; ALDH1A1; CYP1B1; SIAH1 
               
               
                 Lysine Degradation 
                 SUV39H1; EHMT2; NSD1; SETD7; PPP2R5C 
               
               
                 Nucleotide Excision Repair 
                 ERCC5; ERCC4; XPA; XPC; ERCC1 
               
               
                 Pathway 
               
               
                 Starch and Sucrose 
                 UCHL1; HK2; GCK; GPI; HK1 
               
               
                 Metabolism 
               
               
                 Aminosugars Metabolism 
                 NQO1; HK2; GCK; HK1 
               
               
                 Arachidonic Acid 
                 PRDX6; GRN; YWHAZ; CYP1B1 
               
               
                 Metabolism 
               
               
                 Circadian Rhythm Signaling 
                 CSNK1E; CREB1; ATF4; NR1D1 
               
               
                 Coagulation System 
                 BDKRB1; F2R; SERPINE1; F3 
               
               
                 Dopamine Receptor 
                 PPP2R1A; PPP2CA; PPP1CC; PPP2R5C 
               
               
                 Signaling 
               
               
                 Glutathione Metabolism 
                 IDH2; GSTP1; ANPEP; IDH1 
               
               
                 Glycerolipid Metabolism 
                 ALDH1A1; GPAM; SPHK1; SPHK2 
               
               
                 Linoleic Acid Metabolism 
                 PRDX6; GRN; YWHAZ; CYP1B1 
               
               
                 Methionine Metabolism 
                 DNMT1; DNMT3B; AHCY; DNMT3A 
               
               
                 Pyruvate Metabolism 
                 GLO1; ALDH1A1; PKM2; LDHA 
               
               
                 Arginine and Proline 
                 ALDH1A1; NOS3; NOS2A 
               
               
                 Metabolism 
               
               
                 Eicosanoid Signaling 
                 PRDX6; GRN; YWHAZ 
               
               
                 Fructose and Mannose 
                 HK2; GCK; HK1 
               
               
                 Metabolism 
               
               
                 Galactose Metabolism 
                 HK2; GCK; HK1 
               
               
                 Stilbene, Coumarine and 
                 PRDX6; PRDX1; TYR 
               
               
                 Lignin Biosynthesis 
               
               
                 Antigen Presentation 
                 CALR; B2M 
               
               
                 Pathway 
               
               
                 Biosynthesis of Steroids 
                 NQO1; DHCR7 
               
               
                 Butanoate Metabolism 
                 ALDH1A1; NLGN1 
               
               
                 Citrate Cycle 
                 IDH2; IDH1 
               
               
                 Fatty Acid Metabolism 
                 ALDH1A1; CYP1B1 
               
               
                 Glycerophospholipid 
                 PRDX6; CHKA 
               
               
                 Metabolism 
               
               
                 Histidine Metabolism 
                 PRMT5; ALDH1A1 
               
               
                 Inositol Metabolism 
                 EROIL; APEX1 
               
               
                 Metabolism of Xenobiotics 
                 GSTP1; CYP1B1 
               
               
                 by Cytochrome p450 
               
               
                 Methane Metabolism 
                 PRDX6; PRDX1 
               
               
                 Phenylalanine Metabolism 
                 PRDX6; PRDX1 
               
               
                 Propanoate Metabolism 
                 ALDH1A1; LDHA 
               
               
                 Selenoamino Acid 
                 PRMT5; AHCY 
               
               
                 Metabolism 
               
               
                 Sphingolipid Metabolism 
                 SPHK1; SPHK2 
               
               
                 Aminophosphonate 
                 PRMT5 
               
               
                 Metabolism 
               
               
                 Androgen and Estrogen 
                 PRMT5 
               
               
                 Metabolism 
               
               
                 Ascorbate and Aldarate 
                 ALDH1A1 
               
               
                 Metabolism 
               
               
                 Bile Acid Biosynthesis 
                 ALDH1A1 
               
               
                 Cysteine Metabolism 
                 LDHA 
               
               
                 Fatty Acid Biosynthesis 
                 FASN 
               
               
                 Glutamate Receptor 
                 GNB2L1 
               
               
                 Signaling 
               
               
                 NRF2-mediated Oxidative 
                 PRDX1 
               
               
                 Stress Response 
               
               
                 Pentose Phosphate 
                 GPI 
               
               
                 Pathway 
               
               
                 Pentose and Glucuronate 
                 UCHL1 
               
               
                 Interconversions 
               
               
                 Retinol Metabolism 
                 ALDH1A1 
               
               
                 Riboflavin Metabolism 
                 TYR 
               
               
                 Tyrosine Metabolism 
                 PRMT5, TYR 
               
               
                 Ubiquinone Biosynthesis 
                 PRMT5 
               
               
                 Valine, Leucine and 
                 ALDH1A1 
               
               
                 Isoleucine Degradation 
               
               
                 Glycine, Serine and 
                 CHKA 
               
               
                 Threonine Metabolism 
               
               
                 Lysine Degradation 
                 ALDH1A1 
               
               
                 Pain/Taste 
                 TRPM5; TRPA1 
               
               
                 Pain 
                 TRPM7; TRPC5; TRPC6; TRPC1; Cnr1; crn2; Grk2; 
               
               
                   
                 Trpa1; Pomc; Cgrp; Crf; Pka; Era; Nr2b; TRPM5; Prkaca; 
               
               
                   
                 Prkacb; Prkar1a; Prkar2a 
               
               
                 Mitochondrial Function 
                 AIF; CytC; SMAC (Diablo); Aifm-1; Aifm-2 
               
               
                 Developmental Neurology 
                 BMP-4; Chordin (Chrd); Noggin (Nog); WNT (Wnt2; 
               
               
                   
                 Wnt2b; Wnt3a; Wnt4; Wnt5a; Wnt6; Wnt7b; Wnt8b; 
               
               
                   
                 Wnt9a; Wnt9b; Wnt10a; Wnt10b; Wnt16); beta-catenin; 
               
               
                   
                 Dkk-1; Frizzled related proteins; Otx-2; Gbx2; FGF-8; 
               
               
                   
                 Reelin; Dab1; unc-86 (Pou4f1 orBm3a); Numb; Reln 
               
               
                   
               
            
           
         
       
     
     Further non-limiting examples of disease-associated genes and polynucleotides and disease specific information is available from McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.), available on the World Wide Web. 
     In an aspect, the invention provides a method of individualized or personalized treatment of a genetic disease in a subject in need of such treatment comprising: (a) introducing one or more mutations ex vivo in a tissue, organ or a cell line, or in vivo in a transgenic non-human mammal, comprising delivering to cell(s) of the tissue, organ, cell or mammal a composition comprising the particle delivery system or the delivery system or the virus particle of any one of the above embodiment or the cell of any one of the above embodiment, wherein the specific mutations or precise sequence substitutions are or have been correlated to the genetic disease; (b) testing treatment(s) for the genetic disease on the cells to which the vector has been delivered that have the specific mutations or precise sequence substitutions correlated to the genetic disease; and (c) treating the subject based on results from the testing of treatment(s) of step (b). 
     Infectious Diseases 
     In some embodiments, the CRISPR-Cas system(s) or component(s) thereof can be used to diagnose, prognose, treat, and/or prevent an infectious disease caused by a microorganism, such as bacteria, virus, fungi, parasites, or combinations thereof. 
     In some embodiments, the Cas system(s) or component(s) thereof can be capable of targeting specific microorganism within a mixed population. Exemplary methods of such techniques are described in e.g. Gomaa A A, Klumpe H E, Luo M L, Selle K, Barrangou R, Beisel C L. 2014. Programmable removal of bacterial strains by use of genome-targeting CRISPR-Cas systems. mBio 5:e00928-13; Citorik R J, Mimee M, Lu T K. 2014. Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases. Nat Biotechnol 32:1141-1145, the teachings of which can be adapted for use with the CRISPR-Cas systems and components thereof described herein. 
     In some embodiments, the CRISPR-Cas system(s) and/or components thereof can be capable of targeting pathogenic and/or drug-resistant microorganisms, such as bacteria, virus, parasites, and fungi. In some embodiments, the CRISPR-Cas system(s) and/or components thereof can be capable of targeting and modifying one or more polynucleotides in a pathogenic microorganism such that the microorganism is less virulent, killed, inhibited, or is otherwise rendered incapable of causing disease and/or infecting and/or replicating in a host cell. 
     In some embodiments, the pathogenic bacteria that can be targeted and/or modified by the CRISPR-Cas system(s) and/or component(s) thereof described herein include, but are not limited to, those of the genus  Actinomyces  (e.g.  A. israelii ),  Bacillus  (e.g.  B. anthracis, B. cereus ),  Bactereoides  (e.g.  B. fragilis ),  Bartonella  ( B. henselae, B. quintana ),  Bordetella  ( B. pertussis ),  Borrelia  (e.g.  B. burgdorferi, B. garinii, B. afzelii , and  B. recurreentis ),  Brucella  (e.g.  B. abortus, B. canis, B. melitensis , and  B. suis ),  Campylobacter  (e.g.  C. jejuni ),  Chlamydia  (e.g.  C. pneumoniae  and  C. trachomatis ),  Chlamydophila  (e.g.  C. psittaci ),  Clostridium  (e.g.  C. botulinum, C. difficile, C. perfringens. C. tetani ),  Corynebacterium  (e.g.  C. diptheriae ),  Enterococcus  (e.g. E.  Faecalis, E. faecium ),  Ehrlichia  ( E. canis  and  E. chaffensis )  Escherichia  (e.g.  E. coli ),  Francisella  (e.g.  F. tularensis ),  Haemophilus  (e.g.  H. influenzae ),  Helicobacter  ( H. pylori ),  Klebsiella  (E.g.  K. pneumoniae ),  Legionella  (e.g.  L. pneumophila ),  Leptospira  (e.g.  L. interrogans, L. santarosai, L. weilii, L. noguchii ),  Listereia  (e.g.  L. monocytogeenes ),  Mycobacterium  (e.g.  M. leprae, M. tuberculosis, M. ulcerans ),  Mycoplasma  ( M. pneumoniae ),  Neisseria  ( N. gonorrhoeae  and  N. menigitidis ),  Nocardia  (e.g.  N. asteeroides ),  Pseudomonas  ( P. aeruginosa ),  Rickettsia  ( R. rickettsia ),  Salmonella  ( S. typhi  and  S. typhimurium ),  Shigella  ( S. sonnei  and  S. dysenteriae ),  Staphylococcus  ( S. aureus, S. epidermidis , and  S. saprophyticus ), Streeptococcus (S. agalactiaee,  S. pneumoniae, S. pyogenes ),  Treponema  ( T. pallidum ),  Ureeaplasma  (e.g.  U. urealyticum ),  Vibrio  (e.g.  V. cholerae ),  Yersinia  (e.g.  Y pestis, Y, enteerocolitica , and  Y, pseudotuberculosis ). 
     In some embodiments, the pathogenic virus that can be targeted and/or modified by the CRISPR-Cas system(s) and/or component(s) thereof described herein include, but are not limited to, a double-stranded DNA virus, a partly double-stranded DNA virus, a single-stranded DNA virus, a positive single-stranded RNA virus, a negative single-stranded RNA virus, or a double stranded RNA virus. In some embodiments, the pathogenic virus can be from the family Adenoviridae (e.g. Adenovirus), Herpesviridae (e.g. Herpes simplex, type 1, Herpes simplex, type 2, Varicella-zoster virus, Epstein-Barr virus, Human cytomegalovirus, Human herpesvirus, type 8), Papillomaviridae (e.g. Human papillomavirus), Polyomaviridae (e.g. BK virus, JC virus), Poxviridae (e.g. smallpox), Hepadnaviridae (e.g. Hepatitis B), Parvoviridae (e.g. Parvovirus B19), Astroviridae (e.g. Human astrovirus), Caliciviridae (e.g. Norwalk virus), Picornaviridae (e.g. coxsackievirus, hepatitis A virus, poliovirus, rhinovirus), Coronaviridae (e.g. Severe acute respiratory syndrome-related coronavirus, strains: Severe acute respiratory syndrome virus, Severe acute respiratory syndrome coronavirus 2 (COVID-19)), Flaviviridae (e.g. Hepatitis C virus, yellow fever virus, dengue virus, West Nile virus, TBE virus), Togaviridae (e.g. Rubella virus), Hepeviridae (e.g. Hepatitis E virus), Retroviridae (Human immunodeficiency virus (HIV)), Orthomyxoviridae (e.g. Influenza virus), Arenaviridae (e.g. Lassa virus), Bunyaviridae (e.g. Crimean-Congo hemorrhagic fever virus, Hantaan virus), Filoviridae (e.g. Ebola virus and Marburg virus), Paramyxoviridae (e.g. Measles virus, Mumps virus, Parainfluenza virus, Respiratory syncytial virus), Rhabdoviridae (Rabies virus), Hepatitis D virus, Reoviridae (e.g. Rotavirus, Orbivirus, Coltivirus, Banna virus). 
     In some embodiments, the pathogenic fungi that can be targeted and/or modified by the CRISPR-Cas system(s) and/or component(s) thereof described herein include, but are not limited to, those of the genus  Candida  (e.g.  C. albicans ),  Aspergillus  (e.g.  A. fumigatus, A. flavus, A. clavatus ),  Cryptococcus  (e.g.  C. neoformans, C. gattii ),  Histoplasma  ( H. capsulatum ),  Pneumocystis  (e.g.  P. jiroveecii ),  Stachybotrys  (e.g.  S. chartarum ). 
     In some embodiments, the pathogenic parasites that can be targeted and/or modified by the CRISPR-Cas system(s) and/or component(s) thereof described herein include, but are not limited to, protozoa, helminths, and ectoparasites. In some embodiments, the pathogenic protozoa that can be targeted and/or modified by the CRISPR-Cas system(s) and/or component(s) thereof described herein include, but are not limited to, those from the groups Sarcodina (e.g. ameba such as  Entamoeba ),  Mastigophora  (e.g.  flagellates  such as  Giardia  and  Leishmania ), Cilophora (e.g. ciliates such as Balantidum), and sporozoa (e.g. plasmodium and cryptosporidium). In some embodiments, the pathogenic helminths that can be targeted and/or modified by the CRISPR-Cas system(s) and/or component(s) thereof described herein include, but are not limited to, flatworms (platyhelminths), thorny-headed worms (acanthoceephalins), and roundworms (nematodes). In some embodiments, the pathogenic ectoparasites that can be targeted and/or modified by the CRISPR-Cas system(s) and/or component(s) thereof described herein include, but are not limited to, ticks, fleas, lice, and mites. 
     In some embodiments, the pathogenic parasite that can be targeted and/or modified by the CRISPR-Cas system(s) and/or component(s) thereof described herein include, but are not limited to,  Acanthamoeba  spp.,  Balamuthia mandrillaris, Babesiosis  spp. (e.g.  Babesia B. divergens, B. bigemina, B. equi, B. microfti, B. duncani ),  Balantidiasis  spp. (e.g.  Balantidium coli ),  Blastocystis  spp.,  Cryptosporidium  spp.,  Cyclosporiasis  spp. (e.g.  Cyclospora cayetanensis ), Dientamoebiasis spp. (e.g.  Dientamoeba fragilis ),  Amoebiasis  spp. (e.g.  Entamoeba histolytica ),  Giardiasis  spp. (e.g.  Giardia lamblia ),  Isosporiasis  spp. (e.g.  Isospora belli ),  Leishmania  spp.,  Naegleria  spp. (e.g.  Naegleria fowleri ),  Plasmodium  spp. (e.g.  Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale curtisi, Plasmodium ovale wallikeri, Plasmodium malariae, Plasmodium knowlesi ),  Rhinosporidiosis  spp. (e.g.  Rhinosporidium seeberi ),  Sarcocystosis  spp. (e.g.  Sarcocystis bovihominis, Sarcocystis suihominis ),  Toxoplasma  spp. (e.g.  Toxoplasma gondii ),  Trichomonas  spp. (e.g.  Trichomonas vaginalis ),  Trypanosoma  spp. (e.g.  Trypanosoma brucei ),  Trypanosoma  spp. (e.g.  Trypanosoma cruzi ), Tapeworm (e.g. Cestoda,  Taenia multiceps, Taenia saginata, Taenia solium ),  Diphyllobothrium latum  spp.,  Echinococcus  spp. (e.g.  Echinococcus granulosus, Echinococcus multilocularis, E. vogeli, E. oligarthrus ),  Hymenolepis  spp. (e.g.  Hymenolepis nana, Hymenolepis diminuta ),  Bertiella  spp. (e.g.  Bertiella mucronata, Bertiella studeri ),  Spirometra  (e.g.  Spirometra erinaceieuropaei ),  Clonorchis  spp. (e.g.  Clonorchis sinensis; Clonorchis viverrini ),  Dicrocoelium  spp. (e.g.  Dicrocoelium dendriticum ),  Fasciola  spp. (e.g.  Fasciola hepatica, Fasciola gigantica ),  Fasciolopsis  spp. (e.g.  Fasciolopsis buski ),  Metagonimus  spp. (e.g.  Metagonimus yokogawai ),  Metorchis  spp. (e.g.  Metorchis  conjunctus),  Opisthorchis  spp. (e.g.  Opisthorchis viverrini, Opisthorchis felineus ),  Clonorchis  spp. (e.g.  Clonorchis sinensis ),  Paragonimus  spp. (e.g.  Paragonimus westermani; Paragonimus africanus; Paragonimus caliensis; Paragonimus kellicotti; Paragonimus skrjabini; Paragonimus uterobilateralis ),  Schistosoma  sp.,  Schistosoma  spp. (e.g.  Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicum, Schistosoma mekongi , and  Schistosoma intercalatum ),  Echinostoma  spp. (e.g.  E. echinatum ),  Trichobilharzia  spp. (e.g.  Trichobilharzia  regent),  Ancylostoma  spp. (e.g.  Ancylostoma duodenale ), Necator spp. (e.g.  Necator americanus ),  Angiostrongylus  spp.,  Anisakis  spp.,  Ascaris  spp. (e.g.  Ascaris lumbricoides ),  Baylisascaris  spp. (e.g.  Baylisascaris procyonis ),  Brugia  spp. (e.g.  Brugia malayi, Brugia timori ),  Dioctophyme  spp. (e.g.  Dioctophyme renale ),  Dracunculus  spp. (e.g.  Dracunculus medinensis ),  Enterobius  spp. (e.g.  Enterobius vermicularis, Enterobius gregorii ),  Gnathostoma  spp. (e.g.  Gnathostoma spinigerum, Gnathostoma hispidum ),  Halicephalobus  spp. (e.g.  Halicephalobus gingivalis ),  Loa loa  spp. (e.g.  Loa loa  filaria),  Mansonella  spp. (e.g.  Mansonella streptocerca ),  Onchocerca  spp. (e.g.  Onchocerca volvulus ),  Strongyloides  spp. (e.g.  Strongyloides stercoralis ),  Thelazia  spp. (e.g.  Thelazia californiensis, Thelazia callipaeda ),  Toxocara  spp. (e.g.  Toxocara canis, Toxocara cati , Toxascaris leonine),  Trichinella  spp. (e.g.  Trichinella spiralis, Trichinella britovi, Trichinella nelsoni, Trichinella nativa ),  Trichuris  spp. (e.g.  Trichuris trichiura, Trichuris vulpis ),  Wuchereria  spp. (e.g.  Wuchereria bancrofti ),  Dermatobia  spp. (e.g.  Dermatobia hominis ),  Tunga  spp. (e.g.  Tunga penetrans ),  Cochliomyia  spp. (e.g.  Cochliomyia hominivorax ),  Linguatula  spp. (e.g.  Linguatula serrata ), Archiacanthocephala sp.,  Moniliformis  sp. (e.g.  Moniliformis moniliformis ),  Pediculus  spp. (e.g.  Pediculus humanus  capitis,  Pediculus humanus humanus ),  Pthirus  spp. (e.g.  Pthirus pubis ),  Arachnida  spp. (e.g. Trombiculidae, Ixodidae, Argaside),  Siphonaptera  spp (e.g.  Siphonaptera: Pulicinae ),  Cimicidae  spp. (e.g.  Cimex lectularius  and  Cimex hemipterus ),  Diptera  spp.,  Demodex  spp. (e.g.  Demodex folliculorum/brevis/canis ),  Sarcoptes  spp. (e.g.  Sarcoptes scabiei ),  Dermanyssus  spp. (e.g.  Dermanyssus gallinae ),  Ornithonyssus  spp. (e.g.  Ornithonyssus sylviarum, Ornithonyssus bursa, Ornithonyssus bacoti ),  Laelaps  spp. (e.g.  Laelaps echidnina ),  Liponyssoides  spp. (e.g.  Liponyssoides sanguineus ). 
     In some embodiments the gene targets can be any of those as set forth in Table 1 of Strich and Chertow. 2019. J. Clin. Microbio. 57:4 e01307-18, which is incorporated herein as if expressed in its entirety herein. 
     In some embodiments, the method can include delivering a CRISPR-Cas system and/or component thereof to a pathogenic organism described herein, allowing the CRISPR-Cas system and/or component thereof to specifically bind and modify one or more targets in the pathogenic organism, whereby the modification kills, inhibits, reduces the pathogenicity of the pathogenic organism, or otherwise renders the pathogenic organism non-pathogenic. In some embodiments, delivery of the CRISPR-Cas system occurs in vivo (i.e. in the subject being treated). In some embodiments occurs by an intermediary, such as microorganism or phage that is non-pathogenic to the subject but is capable of transferring polynucleotides and/or infecting the pathogenic microorganism. In some embodiments, the intermediary microorganism can be an engineered bacteria, virus, or phage that contains the CRISPR-Cas system(s) and/or component(s) thereof and/or CRISPR-Cas vectors and/or vector systems. The method can include administering an intermediary microorganism containing the CRISPR-Cas system(s) and/or component(s) thereof and/or CRISPR-Cas vectors and/or vector systems to the subject to be treated. The intermediary microorganism can then produce the CRISPR-system and/or component thereof or transfer a CRISPR-Cas system polynucleotide to the pathogenic organism. In embodiments, where the CRISPR-system and/or component thereof, vector, or vector system is transferred to the pathogenic microorganism, the CRISPR-Cas system or component thereof is then produced in the pathogenic microorganism and modifies the pathogenic microorganism such that it is less virulent, killed, inhibited, or is otherwise rendered incapable of causing disease and/or infecting and/or replicating in a host or cell thereof. 
     In some embodiments, where the pathogenic microorganism inserts its genetic material into the host cell&#39;s genome (e.g. a virus), the CRISPR-Cas system can be designed such that it modifies the host cell&#39;s genome such that the viral DNA or cDNA cannot be replicated by the host cell&#39;s machinery into a functional virus. In some embodiments, where the pathogenic microorganism inserts its genetic material into the host cell&#39;s genome (e.g. a virus), the CRISPR-Cas system can be designed such that it modifies the host cell&#39;s genome such that the viral DNA or cDNA is deleted from the host cell&#39;s genome. 
     It will be appreciated that inhibiting or killing the pathogenic microorganism, the disease and/or condition that its infection causes in the subject can be treated or prevented. Thus, also provided herein are methods of treating and/or preventing one or more diseases or symptoms thereof caused by any one or more pathogenic microorganisms, such as any of those described herein. 
     Mitochondrial Diseases 
     Some of the most challenging mitochondrial disorders arise from mutations in mitochondrial DNA (mtDNA), a high copy number genome that is maternally inherited. In some embodiments, mtDNA mutations can be modified using a CRISPR-Cas system described herein. In some embodiments, the mitochondrial disease that can be diagnosed, prognosed, treated, and/or prevented can be MELAS (mitochondrial myopathy encephalopathy, and lactic acidosis and stroke-like episodes), CPEO/PEO (chronic progressive external ophthalmoplegia syndrome/progressive external ophthalmoplegia), KSS (Kearns-Sayre syndrome), MIDD (maternally inherited diabetes and deafness), MERRF (myoclonic epilepsy associated with ragged red fibers), NIDDM (noninsulin-dependent diabetes mellitus), LHON (Leber hereditary optic neuropathy), LS (Leigh Syndrome) an aminoglycoside induced hearing disorder, NARP (neuropathy, ataxia, and pigmentary retinopathy), Extrapyramidal disorder with akinesia-rigidity, psychosis and SNHL, Nonsyndromic hearing loss a cardiomyopathy, an encephalomyopathy, Pearson&#39;s syndrome, or a combination thereof. 
     In some embodiments, the mtDNA of a subject can be modified in vivo or ex vivo. In some embodiments, where the mtDNA is modified ex vivo, after modification the cells containing the modified mitochondria can be administered back to the subject. In some embodiments, the CRISPR-Cas system or component thereof can be capable of correcting an mtDNA mutation. 
     In some embodiments, at least one of the one or more mtDNA mutations is selected from the group consisting of: A3243G, C3256T, T3271C, G1019A, A1304T, A15533G, C1494T, C4467A, T1658C, G12315A, A3421G, A8344G, T8356C, G8363A, A13042T, T3200C, G3242A, A3252G, T3264C, G3316A, T3394C, T14577C, A4833G, G3460A, G9804A, G11778A, G14459A, A14484G, G15257A, T8993C, T8993G, G10197A, G13513A, T1095C, C1494T, A1555G, G1541A, C1634T, A3260G, A4269G, T7587C, A8296G, A8348G, G8363A, T9957C, T9997C, G12192A, C12297T, A14484G, G15059A, duplication of CCCCCTCCCC-tandem repeats at positions 305-314 and/or 956-965, deletion at positions from 8,469-13,447, 4,308-14,874, and/or 4,398-14,822, 961ins/delC, the mitochondrial common deletion (e.g. mtDNA 4,977 bp deletion), and combinations thereof. 
     In some embodiments, the mitochondrial mutation can be any mutation as set forth in or as identified by use of one or more bioinformatic tools available at Mitomap available at mitomap.org. Such tools include, but are not limited to, “Variant Search, aka Market Finder”, Find Sequences for Any Haplogroup, aka “Sequence Finder”, “Variant Info”, “POLG Pathogenicity Prediction Server”, “MITOMASTER”, “Allele Search”, “Sequence and Variant Downloads”, “Data Downloads”. MitoMap contains reports of mutations in mtDNA that can be associated with disease and maintains a database of reported mitochondrial DNA Base Substitution Diseases: rRNA/tRNA mutations. 
     In some embodiments, the method includes delivering a CRISPR-Cas system and/or a component thereof to a cell, and more specifically one or more mitochondria in a cell, allowing the CRISPR-Cas system and/or component thereof to modify one or more target polynucleotides in the cell, and more specifically one or more mitochondria in the cell. The target polynucleotides can correspond to a mutation in the mtDNA, such as any one or more of those described herein. In some embodiments, the modification can alter a function of the mitochondria such that the mitochondria functions normally or at least is/are less dysfunctional as compared to an unmodified mitochondria. Modification can occur in vivo or ex vivo. Where modification is performed ex vivo, cells containing modified mitochondria can be administered to a subject in need thereof in an autologous or allogenic manner. 
     Microbiome Modification 
     Microbiomes play important roles in health and disease. For example, the gut microbiome can play a role in health by controlling digestion, preventing growth of pathogenic microorganisms and have been suggested to influence mood and emotion. Imbalanced microbiomes can promote disease and are suggested to contribute to weight gain, unregulated blood sugar, high cholesterol, cancer, and other disorders. A healthy microbiome has a series of joint characteristics that can be distinguished from non-healthy individuals, thus detection and identification of the disease-associated microbiome can be used to diagnose and detect disease in an individual. The CRISPR-Cas systems and components thereof can be used to screen the microbiome cell population and be used to identify a disease associated microbiome. Cell screening methods utilizing CRISPR-Cas systems and components thereof are described elsewhere herein and can be applied to screening a microbiome, such as a gut, skin, vagina, and/or oral microbiome, of a subject. 
     In some embodiments, the microbe population of a microbiome in a subject can be modified using a CRISPR-Cas system and/or component thereof described herein. In some embodiments, the CRISPR-Cas system and/or component thereof can be used to identify and select one or more cell types in the microbiome and remove them from the microbiome population. Exemplary methods of selecting cells using a CRISPR-Cas system and/or component thereof are described elsewhere herein. In this way the make-up or microorganism profile of the microbiome can be altered. In some embodiments, the alteration causes a change from a diseased microbiome composition to a healthy microbiome composition. In this way the ratio of one type or species of microorganism to another can be modified, such as going from a diseased ratio to a healthy ratio. In some embodiments, the cells selected are pathogenic microorganisms. 
     In some embodiments, the CRISPR-Cas systems described herein can be used to modify a polynucleotide in a microorganism of a microbiome in a subject. In some embodiments, the microorganism is a pathogenic microorganism. In some embodiments, the microorganism is a commensal and non-pathogenic microorganism. Methods of modifying polynucleotides in a cell in the subject are described elsewhere herein and can be applied to these embodiments. 
     Adoptive Therapy 
     The CRISPR-Cas systems and components thereof described herein can be used to modify cells for an adoptive cell therapy. 
     Aspects of the invention accordingly involve the adoptive transfer of immune system cells, such as T cells, specific for selected antigens, such as tumor associated antigens (see Maus et al., 2014, Adoptive Immunotherapy for Cancer or Viruses, Annual Review of Immunology, Vol. 32: 189-225; Rosenberg and Restifo, 2015, Adoptive cell transfer as personalized immunotherapy for human cancer, Science Vol. 348 no. 6230 pp. 62-68; and, Restifo et al., 2015, Adoptive immunotherapy for cancer: harnessing the T cell response. Nat. Rev. Immunol. 12(4): 269-281; and Jenson and Riddell, 2014, Design and implementation of adoptive therapy with chimeric antigen receptor-modified T cells. Immunol Rev. 257(1): 127-144). Various strategies may for example be employed to genetically modify T cells by altering the specificity of the T cell receptor (TCR) for example by introducing new TCR α and β chains with selected peptide specificity (see U.S. Pat. No. 8,697,854; PCT Patent Publications: WO2003020763, WO2004033685, WO2004044004, WO2005114215, WO2006000830, WO2008038002, WO2008039818, WO2004074322, WO2005113595, WO2006125962, WO2013166321, WO2013039889, WO2014018863, WO2014083173; U.S. Pat. No. 8,088,379). 
     As an alternative to, or addition to, TCR modifications, chimeric antigen receptors (CARs) may be used in order to generate immunoresponsive cells, such as T cells, specific for selected targets, such as malignant cells, with a wide variety of receptor chimera constructs having been described (see U.S. Pat. Nos. 5,843,728; 5,851,828; 5,912,170; 6,004,811; 6,284,240; 6,392,013; 6,410,014; 6,753,162; 8,211,422; and, PCT Publication WO9215322). Alternative CAR constructs may be characterized as belonging to successive generations. First-generation CARs typically consist of a single-chain variable fragment of an antibody specific for an antigen, for example comprising a VL linked to a VH of a specific antibody, linked by a flexible linker, for example by a CD8a hinge domain and a CD8α transmembrane domain, to the transmembrane and intracellular signaling domains of either CD3ζ or FcRγ (scFv-CD3ζ or scFv-FcRγ; see U.S. Pat. Nos. 7,741,465; 5,912,172; 5,906,936). Second-generation CARs incorporate the intracellular domains of one or more costimulatory molecules, such as CD28, OX40 (CD134), or 4-1BB (CD137) within the endodomain (for example scFv-CD28/0X40/4-1BB-CD3ζ; see U.S. Pat. Nos. 8,911,993; 8,916,381; 8,975,071; 9,101,584; 9,102,760; 9,102,761). Third-generation CARs include a combination of costimulatory endodomains, such a CD3ζ-chain, CD97, GDI1a-CD18, CD2, ICOS, CD27, CD154, CDS, OX40, 4-1BB, or CD28 signaling domains (for example scFv-CD28-4-1BB-CD3ζ or scFv-CD28-OX40-CD3ζ; see U.S. Pat. Nos. 8,906,682; 8,399,645; 5,686,281; PCT Publication No. WO2014134165; PCT Publication No. WO2012079000). Alternatively, costimulation may be orchestrated by expressing CARs in antigen-specific T cells, chosen so as to be activated and expanded following engagement of their native αβTCR, for example by antigen on professional antigen-presenting cells, with attendant costimulation. In addition, additional engineered receptors may be provided on the immunoresponsive cells, for example to improve targeting of a T-cell attack and/or minimize side effects. 
     Alternative techniques may be used to transform target immunoresponsive cells, such as protoplast fusion, lipofection, transfection or electroporation. A wide variety of vectors may be used, such as retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, plasmids or transposons, such as a Sleeping Beauty transposon (see U.S. Pat. Nos. 6,489,458; 7,148,203; 7,160,682; 7,985,739; 8,227,432), may be used to introduce CARs, for example using 2nd generation antigen-specific CARs signaling through CD3ζ and either CD28 or CD137. Viral vectors may for example include vectors based on HIV, SV40, EBV, HSV or BPV. 
     Cells that are targeted for transformation may for example include T cells, Natural Killer (NK) cells, cytotoxic T lymphocytes (CTL), regulatory T cells, human embryonic stem cells, tumor-infiltrating lymphocytes (TIL) or a pluripotent stem cell from which lymphoid cells may be differentiated. T cells expressing a desired CAR may for example be selected through co-culture with γ-irradiated activating and propagating cells (AaPC), which co-express the cancer antigen and co-stimulatory molecules. The engineered CAR T-cells may be expanded, for example by co-culture on AaPC in presence of soluble factors, such as IL-2 and IL-21. This expansion may for example be carried out so as to provide memory CAR+ T cells (which may for example be assayed by non-enzymatic digital array and/or multi-panel flow cytometry). In this way, CAR T cells may be provided that have specific cytotoxic activity against antigen-bearing tumors (optionally in conjunction with production of desired chemokines such as interferon-γ). CART cells of this kind may for example be used in animal models, for example to threat tumor xenografts. 
     Approaches such as the foregoing may be adapted to provide methods of treating and/or increasing survival of a subject having a disease, such as a neoplasia, for example by administering an effective amount of an immunoresponsive cell comprising an antigen recognizing receptor that binds a selected antigen, wherein the binding activates the immunoresponsive cell, thereby treating or preventing the disease (such as a neoplasia, a pathogen infection, an autoimmune disorder, or an allogeneic transplant reaction). Dosing in CAR T cell therapies may for example involve administration of from 106 to 109 cells/kg, with or without a course of lymphodepletion, for example with cyclophosphamide. 
     In one embodiment, the treatment can be administrated into patients undergoing an immunosuppressive treatment. The cells or population of cells, may be made resistant to at least one immunosuppressive agent due to the inactivation of a gene encoding a receptor for such immunosuppressive agent. Not being bound by a theory, the immunosuppressive treatment should help the selection and expansion of the immunoresponsive or T cells according to the invention within the patient. 
     The administration of the cells or population of cells according to the present invention may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The cells or population of cells may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous or intralymphatic injection, or intraperitoneally. In one embodiment, the cell compositions of the present invention are preferably administered by intravenous injection. 
     The administration of the cells or population of cells can consist of the administration of 104-109 cells per kg body weight, preferably 105 to 106 cells/kg body weight including all integer values of cell numbers within those ranges. Dosing in CAR T cell therapies may for example involve administration of from 106 to 109 cells/kg, with or without a course of lymphodepletion, for example with cyclophosphamide. The cells or population of cells can be administrated in one or more doses. In another embodiment, the effective amount of cells are administrated as a single dose. In another embodiment, the effective amount of cells are administrated as more than one dose over a period time. Timing of administration is within the judgment of managing physician and depends on the clinical condition of the patient. The cells or population of cells may be obtained from any source, such as a blood bank or a donor. While individual needs vary, determination of optimal ranges of effective amounts of a given cell type for a particular disease or conditions are within the skill of one in the art. An effective amount means an amount which provides a therapeutic or prophylactic benefit. The dosage administrated will be dependent upon the age, health and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment and the nature of the effect desired. 
     In another embodiment, the effective amount of cells or composition comprising those cells are administrated parenterally. The administration can be an intravenous administration. The administration can be directly done by injection within a tumor. 
     To guard against possible adverse reactions, engineered immunoresponsive cells may be equipped with a transgenic safety switch, in the form of a transgene that renders the cells vulnerable to exposure to a specific signal. For example, the herpes simplex viral thymidine kinase (TK) gene may be used in this way, for example by introduction into allogeneic T lymphocytes used as donor lymphocyte infusions following stem cell transplantation (Greco, et al., Improving the safety of cell therapy with the TK-suicide gene. Front. Pharmacol. 2015; 6: 95). In such cells, administration of a nucleoside prodrug such as ganciclovir or acyclovir causes cell death. Alternative safety switch constructs include inducible caspase 9, for example triggered by administration of a small-molecule dimerizer that brings together two nonfunctional icasp9 molecules to form the active enzyme. A wide variety of alternative approaches to implementing cellular proliferation controls have been described (see U.S. Patent Publication No. 20130071414; PCT Patent Publication WO2011146862; PCT Patent Publication WO2014011987; PCT Patent Publication WO2013040371; Zhou et al. BLOOD, 2014, 123/25:3895-3905; Di Stasi et al., The New England Journal of Medicine 2011; 365:1673-1683; Sadelain M, The New England Journal of Medicine 2011; 365:1735-173; Ramos et al., Stem Cells 28(6):1107-15 (2010)). 
     In a further refinement of adoptive therapies, genome editing with a CRISPR-Cas system as described herein may be used to tailor immunoresponsive cells to alternative implementations, for example providing edited CAR T cells (see Poirot et al., 2015, Multiplex genome edited T-cell manufacturing platform for “off-the-shelf” adoptive T-cell immunotherapies, Cancer Res 75 (18): 3853). For example, immunoresponsive cells may be edited to delete expression of some or all of the class of HLA type II and/or type I molecules, or to knockout selected genes that may inhibit the desired immune response, such as the PD1 gene. 
     Cells may be edited using any CRISPR system and method of use thereof as described herein. CRISPR systems may be delivered to an immune cell by any method described herein. In preferred embodiments, cells are edited ex vivo and transferred to a subject in need thereof. Immunoresponsive cells, CAR T cells or any cells used for adoptive cell transfer may be edited. Editing may be performed to eliminate potential alloreactive T-cell receptors (TCR), disrupt the target of a chemotherapeutic agent, block an immune checkpoint, activate a T cell, and/or increase the differentiation and/or proliferation of functionally exhausted or dysfunctional CD8+ T-cells (see PCT Patent Publications: WO2013176915, WO2014059173, WO2014172606, WO2014184744, and WO2014191128). Editing may result in inactivation of a gene. 
     T cell receptors (TCR) are cell surface receptors that participate in the activation of T cells in response to the presentation of antigen. The TCR is generally made from two chains, a and β, which assemble to form a heterodimer and associates with the CD3-transducing subunits to form the T cell receptor complex present on the cell surface. Each α and β chain of the TCR consists of an immunoglobulin-like N-terminal variable (V) and constant (C) region, a hydrophobic transmembrane domain, and a short cytoplasmic region. As for immunoglobulin molecules, the variable region of the α and β chains are generated by V(D)J recombination, creating a large diversity of antigen specificities within the population of T cells. However, in contrast to immunoglobulins that recognize intact antigen, T cells are activated by processed peptide fragments in association with an MHC molecule, introducing an extra dimension to antigen recognition by T cells, known as MHC restriction. Recognition of MHC disparities between the donor and recipient through the T cell receptor leads to T cell proliferation and the potential development of graft versus host disease (GVHD). The inactivation of TCRα or TCRβ can result in the elimination of the TCR from the surface of T cells preventing recognition of alloantigen and thus GVHD. However, TCR disruption generally results in the elimination of the CD3 signaling component and alters the means of further T cell expansion. 
     Allogeneic cells are rapidly rejected by the host immune system. It has been demonstrated that, allogeneic leukocytes present in non-irradiated blood products will persist for no more than 5 to 6 days (Boni, Muranski et al. 2008 Blood 1; 112(12):4746-54). Thus, to prevent rejection of allogeneic cells, the host&#39;s immune system usually has to be suppressed to some extent. However, in the case of adoptive cell transfer the use of immunosuppressive drugs also have a detrimental effect on the introduced therapeutic T cells. Therefore, to effectively use an adoptive immunotherapy approach in these conditions, the introduced cells would need to be resistant to the immunosuppressive treatment. Thus, in a particular embodiment, the present invention further comprises a step of modifying T cells to make them resistant to an immunosuppressive agent, preferably by inactivating at least one gene encoding a target for an immunosuppressive agent. An immunosuppressive agent is an agent that suppresses immune function by one of several mechanisms of action. An immunosuppressive agent can be, but is not limited to a calcineurin inhibitor, a target of rapamycin, an interleukin-2 receptor α-chain blocker, an inhibitor of inosine monophosphate dehydrogenase, an inhibitor of dihydrofolic acid reductase, a corticosteroid or an immunosuppressive antimetabolite. The present invention allows conferring immunosuppressive resistance to T cells for immunotherapy by inactivating the target of the immunosuppressive agent in T cells. As non-limiting examples, targets for an immunosuppressive agent can be a receptor for an immunosuppressive agent such as: CD52, glucocorticoid receptor (GR), a FKBP family gene member and a cyclophilin family gene member. 
     Immune checkpoints are inhibitory pathways that slow down or stop immune reactions and prevent excessive tissue damage from uncontrolled activity of immune cells. In certain embodiments, the immune checkpoint targeted is the programmed death-1 (PD-1 or CD279) gene (PDCD1). In other embodiments, the immune checkpoint targeted is cytotoxic T-lymphocyte-associated antigen (CTLA-4). In additional embodiments, the immune checkpoint targeted is another member of the CD28 and CTLA4 Ig superfamily such as BTLA, LAG3, ICOS, PDL1 or KIR. In further additional embodiments, the immune checkpoint targeted is a member of the TNFR superfamily such as CD40, OX40, CD137, GITR, CD27 or TIM-3. 
     Additional immune checkpoints include Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP-1) (Watson H A, et al., SHP-1: the next checkpoint target for cancer immunotherapy? Biochem Soc Trans. 2016 Apr. 15; 44(2):356-62). SHP-1 is a widely expressed inhibitory protein tyrosine phosphatase (PTP). In T-cells, it is a negative regulator of antigen-dependent activation and proliferation. It is a cytosolic protein, and therefore not amenable to antibody-mediated therapies, but its role in activation and proliferation makes it an attractive target for genetic manipulation in adoptive transfer strategies, such as chimeric antigen receptor (CAR) T cells. Immune checkpoints may also include T cell immunoreceptor with Ig and ITIM domains (TIGIT/Vstm3/WUCAM/VSIG9) and VISTA (Le Mercier I, et al., (2015) Beyond CTLA-4 and PD-1, the generation Z of negative checkpoint regulators. Front. Immunol. 6:418). 
     WO2014172606 relates to the use of MT1 and/or MT1 inhibitors to increase proliferation and/or activity of exhausted CD8+ T-cells and to decrease CD8+ T-cell exhaustion (e.g., decrease functionally exhausted or unresponsive CD8+ immune cells). In certain embodiments, metallothioneins are targeted by gene editing in adoptively transferred T cells. 
     In certain embodiments, targets of gene editing may be at least one targeted locus involved in the expression of an immune checkpoint protein. Such targets may include, but are not limited to CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, ICOS (CD278), PDL1, KIR, LAG3, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244 (2B4), TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, VISTA, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, MT1, MT2, CD40, OX40, CD137, GITR, CD27, SHP-1 or TIM-3. In preferred embodiments, the gene locus involved in the expression of PD-1 or CTLA-4 genes is targeted. In other preferred embodiments, combinations of genes are targeted, such as but not limited to PD-1 and TIGIT. 
     In other embodiments, at least two genes are edited. Pairs of genes may include, but are not limited to PD1 and TCRα, PD1 and TCRβ, CTLA-4 and TCRα, CTLA-4 and TCRβ, LAG3 and TCRα, LAG3 and TCRβ, Tim3 and TCRα, Tim3 and TCRβ, BTLA and TCRα, BTLA and TCRβ, BY55 and TCRα, BY55 and TCRβ, TIGIT and TCRα, TIGIT and TCRβ, B7H5 and TCRα, B7H5 and TCRβ, LAIR1 and TCRα, LAIR1 and TCRβ, SIGLEC10 and TCRα, SIGLEC10 and TCRβ, 2B4 and TCRα, 2B4 and TCRβ. 
     Whether prior to or after genetic modification of the T cells, the T cells can be activated and expanded generally using methods as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and 7,572,631. T cells can be expanded in vitro or in vivo. 
     The practice of the present invention employs, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which are within the skill of the art. See MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989) (Sambrook, Fritsch and Maniatis); MOLECULAR CLONING: A LABORATORY MANUAL, 4th edition (2012) (Green and Sambrook); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (1987) (F. M. Ausubel, et al. eds.); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.); PCR 2: A PRACTICAL APPROACH (1995) (M. J. MacPherson, B. D. Hames and G. R. Taylor eds.); ANTIBODIES, A LABORATORY MANUAL (1988) (Harlow and Lane, eds.); ANTIBODIES A LABORATORY MANUAL, 2nd edition (2013) (E. A. Greenfield ed.); and ANIMAL CELL CULTURE (1987) (R. I. Freshney, ed.). 
     The practice of the present invention employs, unless otherwise indicated, conventional techniques for generation of genetically modified mice. See Marten H. Hofker and Jan van Deursen, TRANSGENIC MOUSE METHODS AND PROTOCOLS, 2nd edition (2011). 
     In some embodiments, the invention described herein relates to a method for adoptive immunotherapy, in which T cells are edited ex vivo by CRISPR to modulate at least one gene and subsequently administered to a patient in need thereof. In some embodiments, the CRISPR editing comprising knocking-out or knocking-down the expression of a target gene in the edited T cells. In some embodiments, in addition to modulating the target gene, the T cells are also edited ex vivo by CRISPR to (1) knock-in an exogenous gene encoding a chimeric antigen receptor (CAR) or a T-cell receptor (TCR), (2) knock-out or knock-down expression of an immune checkpoint receptor, (3) knock-out or knock-down expression of an endogenous TCR, (4) knock-out or knock-down expression of a human leukocyte antigen class I (HLA-I) proteins, and/or (5) knock-out or knock-down expression of an endogenous gene encoding an antigen targeted by an exogenous CAR or TCR. 
     In some embodiments, the T cells are contacted ex vivo with an adeno-associated virus (AAV) vector encoding a CRISPR effector protein, and a guide molecule comprising a guide sequence hybridizable to a target sequence, a tracr mate sequence, and a tracr sequence hybridizable to the tracr mate sequence. In some embodiments, the T cells are contacted ex vivo (e.g., by electroporation) with a ribonucleoprotein (RNP) comprising a CRISPR effector protein complexed with a guide molecule, wherein the guide molecule comprising a guide sequence hybridizable to a target sequence, a tracr mate sequence, and a tracr sequence hybridizable to the tracr mate sequence. See Rupp et al., Scientific Reports 7:737 (2017); Liu et al., Cell Research 27:154-157 (2017). In some embodiments, the T cells are contacted ex vivo (e.g., by electroporation) with an mRNA encoding a CRISPR effector protein, and a guide molecule comprising a guide sequence hybridizable to a target sequence, a tracr mate sequence, and a tracr sequence hybridizable to the tracr mate sequence. See Eyquem et al., Nature 543:113-117 (2017). In some embodiments, the T cells are not contacted ex vivo with a lentivirus or retrovirus vector. 
     In some embodiments, the method comprises editing T cells ex vivo by CRISPR to knock-in an exogenous gene encoding a CAR, thereby allowing the edited T cells to recognize cancer cells based on the expression of specific proteins located on the cell surface. In some embodiments, T cells are edited ex vivo by CRISPR to knock-in an exogenous gene encoding a TCR, thereby allowing the edited T cells to recognize proteins derived from either the surface or inside of the cancer cells. In some embodiments, the method comprising providing an exogenous CAR-encoding or TCR-encoding sequence as a donor sequence, which can be integrated by homology-directed repair (HDR) into a genomic locus targeted by a CRISPR guide sequence. In some embodiments, targeting the exogenous CAR or TCR to an endogenous TCR α constant (TRAC) locus can reduce tonic CAR signaling and facilitate effective internalization and re-expression of the CAR following single or repeated exposure to antigen, thereby delaying effector T-cell differentiation and exhaustion. See Eyquem et al., Nature 543:113-117 (2017). 
     In some embodiments, the method comprises editing T cells ex vivo by CRISPR to block one or more immune checkpoint receptors to reduce immunosuppression by cancer cells. In some embodiments, T cells are edited ex vivo by CRISPR to knock-out or knock-down an endogenous gene involved in the programmed death-1 (PD-1) signaling pathway, such as PD-1 and PD-L1. In some embodiments, T cells are edited ex vivo by CRISPR to mutate the Pdcd1 locus or the CD274 locus. In some embodiments, T cells are edited ex vivo by CRISPR using one or more guide sequences targeting the first exon of PD-1. See Rupp et al., Scientific Reports 7:737 (2017); Liu et al., Cell Research 27:154-157 (2017). 
     In some embodiments, the method comprises editing T cells ex vivo by CRISPR to eliminate potential alloreactive TCRs to allow allogeneic adoptive transfer. In some embodiments, T cells are edited ex vivo by CRISPR to knock-out or knock-down an endogenous gene encoding a TCR (e.g., an αβ TCR) to avoid graft-versus-host-disease (GVHD). In some embodiments, T cells are edited ex vivo by CRISPR to mutate the TRAC locus. In some embodiments, T cells are edited ex vivo by CRISPR using one or more guide sequences targeting the first exon of TRAC. See Liu et al., Cell Research 27:154-157 (2017). In some embodiments, the method comprises use of CRISPR to knock-in an exogenous gene encoding a CAR or a TCR into the TRAC locus, while simultaneously knocking-out the endogenous TCR (e.g., with a donor sequence encoding a self-cleaving P2A peptide following the CAR cDNA). See Eyquem et al., Nature 543:113-117 (2017). In some embodiments, the exogenous gene comprises a promoter-less CAR-encoding or TCR-encoding sequence which is inserted operably downstream of an endogenous TCR promoter. 
     In some embodiments, the method comprises editing T cells ex vivo by CRISPR to knock-out or knock-down an endogenous gene encoding an HLA-I protein to minimize immunogenicity of the edited T cells. In some embodiments, T cells are edited ex vivo by CRISPR to mutate the beta-2 microglobulin (B2M) locus. In some embodiments, T cells are edited ex vivo by CRISPR using one or more guide sequences targeting the first exon of B2M. See Liu et al., Cell Research 27:154-157 (2017). In some embodiments, the method comprises use of CRISPR to knock-in an exogenous gene encoding a CAR or a TCR into the B2M locus, while simultaneously knocking-out the endogenous B2M (e.g., with a donor sequence encoding a self-cleaving P2A peptide following the CAR cDNA). See Eyquem et al., Nature 543:113-117 (2017). In some embodiments, the exogenous gene comprises a promoter-less CAR-encoding or TCR-encoding sequence which is inserted operably downstream of an endogenous B2M promoter. 
     In some embodiments, the method comprises editing T cells ex vivo by CRISPR to knock-out or knock-down an endogenous gene encoding an antigen targeted by an exogenous CAR or TCR. In some embodiments, the T cells are edited ex vivo by CRISPR to knock-out or knock-down the expression of a tumor antigen selected from human telomerase reverse transcriptase (hTERT), survivin, mouse double minute 2 homolog (MDM2), cytochrome P450 1B 1 (CYP1B), HER2/neu, Wilms&#39; tumor gene 1 (WT1), livin, alphafetoprotein (AFP), carcinoembryonic antigen (CEA), mucin 16 (MUC16), MUC1, prostate-specific membrane antigen (PSMA), p53 or cyclin (DI) (see WO2016/011210). In some embodiments, the T cells are edited ex vivo by CRISPR to knock-out or knock-down the expression of an antigen selected from B cell maturation antigen (BCMA), transmembrane activator and CAML Interactor (TACT), or B-cell activating factor receptor (BAFF-R), CD38, CD138, CS-1, CD33, CD26, CD30, CD53, CD92, CD100, CD148, CD150, CD200, CD261, CD262, or CD362 (see WO2017/011804). 
     Gene Drives 
     In some embodiments, the CRISPR-Cas systems described herein can be used to provide RNA-guided gene drives, for example in systems analogous to gene drives described in PCT Patent Publication WO 2015/105928. Systems of this kind may for example provide methods for altering eukaryotic germline cells, by introducing into the germline cell a nucleic acid sequence encoding an RNA-guided DNA nuclease and one or more guide RNAs. The guide RNAs may be designed to be complementary to one or more target locations on genomic DNA of the germline cell. The nucleic acid sequence encoding the RNA guided DNA nuclease and the nucleic acid sequence encoding the guide RNAs may be provided on constructs between flanking sequences, with promoters arranged such that the germline cell may express the RNA guided DNA nuclease and the guide RNAs, together with any desired cargo-encoding sequences that are also situated between the flanking sequences. The flanking sequences will typically include a sequence which is identical to a corresponding sequence on a selected target chromosome, so that the flanking sequences work with the components encoded by the construct to facilitate insertion of the foreign nucleic acid construct sequences into genomic DNA at a target cut site by mechanisms such as homologous recombination, to render the germline cell homozygous for the foreign nucleic acid sequence. In this way, gene-drive systems are capable of introgressing desired cargo genes throughout a breeding population (Gantz et al., 2015, Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito  Anopheles stephensi , PNAS 2015, published ahead of print Nov. 23, 2015, doi:10.1073/pnas.1521077112; Esvelt et al., 2014, Concerning RNA-guided gene drives for the alteration of wild populations eLife 2014; 3:e03401). In select embodiments, target sequences may be selected which have few potential off-target sites in a genome. Targeting multiple sites within a target locus, using multiple guide RNAs, may increase the cutting frequency and hinder the evolution of drive resistant alleles. Truncated guide RNAs may reduce off-target cutting. Paired nickases may be used instead of a single nuclease, to further increase specificity. Gene drive constructs may include cargo sequences encoding transcriptional regulators, for example to activate homologous recombination genes and/or repress non-homologous end-joining. Target sites may be chosen within an essential gene, so that non-homologous end-joining events may cause lethality rather than creating a drive-resistant allele. The gene drive constructs can be engineered to function in a range of hosts at a range of temperatures (Cho et al. 2013, Rapid and Tunable Control of Protein Stability in  Caenorhabditis elegans  Using a Small Molecule, PLoS ONE 8(8): e72393. doi:10.1371/journal.pone.0072393). 
     Xenotransplantation 
     In some embodiments, the CRISPR-Cas systems described herein can be used to provide RNA-guided DNA nucleases adapted to be used to provide modified tissues for transplantation. For example, RNA-guided DNA nucleases may be used to knockout, knockdown or disrupt selected genes in an animal, such as a transgenic pig (such as the human heme oxygenase-1 transgenic pig line), for example by disrupting expression of genes that encode epitopes recognized by the human immune system, i.e. xenoantigen genes. Candidate porcine genes for disruption may for example include α(1,3)-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase genes (see PCT Patent Publication WO 2014/066505). In addition, genes encoding endogenous retroviruses may be disrupted, for example the genes encoding all porcine endogenous retroviruses (see Yang et al., 2015, Genome-wide inactivation of porcine endogenous retroviruses (PERVs), Science 27 Nov. 2015: Vol. 350 no. 6264 pp. 1101-1104). In addition, RNA-guided DNA nucleases may be used to target a site for integration of additional genes in xenotransplant donor animals, such as a human CD55 gene to improve protection against hyperacute rejection. 
     Treating and Preventing Diseases Using RNA Editing 
     In some embodiments, the disease, disorder, and/condition or symptom thereof can be treated or prevented using an RNA editing system described herein. In some embodiments, the CRISPR-Cas system described herein is an RNA editing system. In some embodiments, treatment or prevention using a CRISPR-Cas RNA editing system described herein can have the advantage of less immunogenicity than a DNA editing CRISPR-Cas system and is not as hindered by limitations on viral vector packaging size. Further, as the effect is transient, the effect can be better controlled over time and can potentially be reversible. Thus, they pose less risk of causing permeant detrimental effects than DNA editing-based preventatives and treatments. 
     In some of these embodiments, the CRISPR-Cas system contains an ADAR enzyme or effector domain thereof. Such systems are described elsewhere herein. In some embodiments, the CIRSPR-Cas system includes a Cas13 or Cas13d effector. 
     Any disease involving a dysfunctional RNA molecule, where the dysfunction is the result of a mutation in the RNA sequence can be treated or prevented by modifying its sequence using a CRISPR-Cas system capable of RNA modification described elsewhere herein. In some embodiments, the disease that can be treated or prevented using a CRISPR-Cas system capable of RNA modification can be one or more of those listed in Tables 12-13 or a combination thereof. In some embodiments, the coding sequence for the gene involved in the disease is greater than the packaging capacity of a viral vector system, particularly an AAV vector system. 
     The potential for RNA editing has now been demonstrated in vitro and in vivo for pathogenic mutations in genes related to cystic fibrosis, Duchenne&#39;s muscular dystrophy, Hurler&#39;s syndrome, and Ornithine transcarbamylase (OTC) deficiency, among others. See e.g. Katrekar et al. Nat. Methods. 2019. 16:239-242; Montieel-Gonzalez et al. 2013. PNAS USA. 110: 18285-18290; Sinnamon et al. PNAS USA 2017; Wettengel et al. Curr. Gene Ther. 2018, 18:31-39; Qu et al. BioRxiv. 2019, 605972; and Fry et al. 2020. Int. J. Mol. Sci. 12:777, which are incorporated by reference as if expressed in their entirety here and the teachings of which can be adapted in view of the description herein to the CRISPR-Cas Systems described herein. 
     In some embodiments, the disease is an inherited retinal degeneration disease. In some embodiments, gene whose transcript can be modified using a CRISPR-Cas system described herein capable of RNA modification that is associated with inherited retinal degeneration and whose coding sequence is too large for packaging in a single AAV can be ABC4, USH2A, CEP290, MYO7A, EYS, and CDH23. 
     Models of Diseases and Conditions 
     In an aspect, the invention provides a method of modeling a disease associated with a genomic locus in a eukaryotic organism or a non-human organism comprising manipulation of a target sequence within a coding, non-coding or regulatory element of said genomic locus comprising delivering a non-naturally occurring or engineered composition comprising a viral vector system comprising one or more viral vectors operably encoding a composition for expression thereof, wherein the composition comprises particle delivery system or the delivery system or the virus particle of any one of the above embodiments or the cell of any one of the above embodiment. 
     In one aspect, the invention provides a method of generating a model eukaryotic cell that can include one or more a mutated disease genes and/or infectious microorganisms. In some embodiments, a disease gene is any gene associated an increase in the risk of having or developing a disease. In some embodiments, the method includes (a) introducing one or more vectors into a eukaryotic cell, wherein the one or more vectors comprise a CRISPR-Cas system and/or component thereof and/or a CRISPR-Cas vector or vector system that is capable of driving expression of a CRISPR-Cas system and/or component thereof including, but not limited to: a guide sequence optionally linked to a tracr mate sequence, a tracr sequence, one or more Cas effectors, and combinations thereof and (b) allowing a CRISPR-Cas complex to bind to one or more target polynucleotides, e.g., to effect cleavage, nicking, or other modification of the target polynucleotide within said disease gene, wherein the CRISPR-Cas complex is composed of one or more CRISPR-Cas effectors complexed with (1) one or more guide sequences that is/are hybridized to the target sequence(s) within the target polynucleotide(s), and optionally (2) the tracr mate sequence(s) that is/are hybridized to the tracr sequence(s), thereby generating a model eukaryotic cell comprising one or more mutated disease gene(s). Thus, in some embodiments the CRISPR-Cas system contains nucleic acid molecules for and drives expression of one or more of: a Cas effector, a guide sequence linked to a tracr mate sequence, and a tracr sequence and/or a Homologous Recombination template and/or a stabilizing ligand if the Cas effector has a destabilization domain. In some embodiments, said cleavage comprises cleaving one or two strands at the location of the target sequence by the Cas effector(s). In some embodiments, nicking comprises nicking one or two strands at the location of the target sequence by the Cas effector(s). In some embodiments, said cleavage or nicking results in modified transcription of a target polynucleotide. In some embodiments, modification results in decreased transcription of the target polynucleotide. In some embodiments, the method further comprises repairing said cleaved or nicked target polynucleotide by homologous recombination with an exogenous template polynucleotide, wherein said repair results in a mutation comprising an insertion, deletion, or substitution of one or more nucleotides of said target polynucleotide. In some embodiments, said mutation results in one or more amino acid changes in a protein expression from a gene comprising the target sequence. 
     The disease modeled can be any disease with a genetic or epigenetic component. In some embodiments, the disease modeled can be any as discussed elsewhere herein, including but not limited to any as set forth in Tables 12-13 herein and combinations thereof. 
     In Situ Disease Detection 
     The CRISPR-Cas systems and/or components thereof can be used for diagnostic methods of detection such as in CASFISH (see e.g. Deng et al. 2015. PNAS USA 112(38): 11870-11875), CRISPR-Live FISH (see e.g. Wang et al. 2020. Science; 365(6459):1301-1305), sm-FISH (Lee and Jefcoate. 2017. Front. Endocrinol. doi.org/10.3389/fendo.2017.00289), sequential FISH CRISPRainbow (Ma et al. Nat Biotechnol, 34 (2016), pp. 528-530), CRISPR-Sirius (Nat Methods, 15 (2018), pp. 928-931), Casilio (Cheng et al. Cell Res, 26 (2016), pp. 254-257), Halo-Tag based genomic loci visualization techniques (e.g. Deng et al. 2015. PNAS USA 112(38): 11870-11875; Knight et al., Science, 350 (2015), pp. 823-826), RNA-aptamer based methods (e.g. Ma et al., J Cell Biol, 214 (2016), pp. 529-537), molecular beacon-based methods (e.g. Zhao et al. Biomaterials, 100 (2016), pp. 172-183; Wu et al. Nucleic Acids Res (2018)), Quantum Dot-based systems (e.g. Ma et al. Anal Chem, 89 (2017), pp. 12896-12901), multiplexed methods (e.g. Ma et al., Proc Natl Acad Sci USA, 112 (2015), pp. 3002-3007; Fu et al. Nat Commun, 7 (2016), p. 11707; Ma et al. Nat Biotechnol, 34 (2016), pp. 528-530; Shao et al. Nucleic Acids Res, 44 (2016), Article e86); Wang et al. Sci Rep, 6 (2016), p. 26857), ç, and other in situ CRISPR-hybridization based methods (e.g. Chen et al. Cell, 155 (2013), pp. 1479-1491; Gu et al. Science, 359 (2018), pp. 1050-1055; Tanebaum et al. Cell, 159 (2014), pp. 635-646; Ye et al. Protein Cell, 8 (2017), pp. 853-855; Chen et al. Nat Commun, 9 (2018), p. 5065; Shao et al. ACS Synth Biol (2017); Fu et al. Nat Commun, 7 (2016), p. 11707; Shao et al. Nucleic Acids Res, 44 (2016), Article e86; Wang et al., Sci Rep, 6 (2016), p. 26857), all of which are incorporated by reference herein as if expressed in their entirety and whose teachings can be adapted to the CRISPR-Cas systems and components thereof described herein in view of the description herein. 
     In some embodiments, the CRISPR-Cas system or component thereof can be used in a detection method, such as an in situ detection method described herein. In some embodiments, the CRISPR-Cas system or component thereof can include a catalytically inactivate Cas effector described herein, preferably an inactivated Cas9 (dCas9) and/or inactivated Cas12 (dCas12) protein(s) and use this system in detection methods such as fluorescence in situ hybridization (FISH) or any other described herein. In some embodiments, the inactivated Cas effector, which lacks the ability to produce DNA double-strand breaks may be fused with a marker, such as fluorescent protein, such as the enhanced green fluorescent protein (eEGFP) and co-expressed with small guide RNAs to target pericentric, centric and telomeric repeats in vivo. The dCas effector or system thereof can be used to visualize both repetitive sequences and individual genes in the human genome. Such new applications of labelled dCas effector and CRISPR-Cas systems thereof can be important in imaging cells and studying the functional nuclear architecture, especially in cases with a small nucleus volume or complex 3-D structures. 
     Cell Selection 
     In some embodiments, the CRISPR-Cas systems and/or components thereof described herein can be used in a method to screen and/or select cells. In some embodiments, CRISPR-Cas system-based screening/selection method can be used to identify diseased cells in a cell population. In some embodiments, selection of the cells results in a modification in the cells such that the selected cells die. In this way, diseased cells can be identified, and removed from the healthy cell population. In some embodiments, the diseased cells can be a cancer cell, pre-cancerous cell, a virus or other pathogenic organism infected cells, or otherwise abnormal cell. In some embodiments, the modification can impart another detectable change in the cells to be selected (e.g. a functional change and/or genomic barcode) that facilitates selection of the desired cells. In some embodiments a negative selection scheme can be used to obtain a desired cell population. In these embodiments, the cells to be selected against are modified, thus can be removed from the cell population based on their death or identification or sorting based the detectable change imparted on the cells. Thus, in these embodiments, the remaining cells after selection are the desired cell population. 
     In some embodiments, a method of selecting one or more cell(s) containing a polynucleotide modification can include: introducing one or more CRISPR-Cas system(s) and/or components thereof, and/or CRISPR-Cas vectors or vector systems into the cell(s), wherein the CRISPR-Cas system(s) and/or components thereof, and/or CRISPR-Cas vectors or vector systems contains and/or is capable of expressing one or more of: a Cas effector, a guide sequence optionally linked to a tracr mate sequence, a tracr sequence, and an editing template; wherein, for example that which is being expressed is within and expressed in vivo by the CRISPR-Cas system vector or vector system and/or the editing template comprises the one or more mutations that abolish Cas effector cleavage; allowing homologous recombination of the editing template with the target polynucleotide in the cell(s) to be selected; allowing a CRISPR complex to bind to a target polynucleotide to effect cleavage of the target polynucleotide within said gene, wherein the AAV-CRISPR complex comprises the Cas effector complexed with (1) the guide sequence that is hybridized to the target sequence within the target polynucleotide, and (2) the tracr mate sequence that is hybridized to the tracr sequence, wherein binding of the CRISPR complex to the target polynucleotide induces cell death or imparts some other detectable change to the cell, thereby allowing one or more cell(s) in which one or more mutations have been introduced to be selected. In a preferred embodiment, the Cas effector is a Cas 9 or Cas12. In some embodiments, the cell to be selected may be a eukaryotic cell. In some embodiments, the cell to be selected may be a prokaryotic cell. Selection of specific cells via the methods herein can be performed without requiring a selection marker or a two-step process that may include a counter-selection system. 
     Therapeutic Agent Development 
     The CRISPR-Cas systems and components thereof described herein can be used to develop CRISPR-Cas-based and non-CRISPR-Cas-based biologically active agents, such as small molecule therapeutics. Thus, described herein are methods for developing a biologically active agent that modulates a cell function and/or signaling event associated with a disease and/or disease gene. As used herein, “active agent” or “active ingredient” refers to a substance, compound, or molecule, which is biologically active or otherwise, induces a biological or physiological effect on a subject to which it is administered to. In other words, “active agent” or “active ingredient” refers to a component or components of a composition to which the whole or part of the effect of the composition is attributed. As used herein, “agent” refers to any substance, compound, molecule, and the like, which can be biologically active or otherwise can induce a biological and/or physiological effect on a subject to which it is administered to. An agent can be a primary active agent, or in other words, the component(s) of a composition to which the whole or part of the effect of the composition is attributed. An agent can be a secondary agent, or in other words, the component(s) of a composition to which an additional part and/or other effect of the composition is attributed. In some embodiments, the method comprises (a) contacting a test compound with a diseased cell and/or a cell containing a disease gene cell; and (b) detecting a change in a readout that is indicative of a reduction or an augmentation of a cell signaling event or other cell functionality associated with said disease or disease gene, thereby developing said biologically active agent that modulates said cell signaling event or other functionality associated with said disease gene. In some embodiments, the diseased cell is a model cell described elsewhere herein. In some embodiments, the diseased cell is a diseased cell isolated from a subject in need of treatment. In some embodiments, the test compound is a small molecule agent. In some embodiments, test compound is a small molecule agent. In some embodiments, the test compound is a biologic molecule agent. 
     In some embodiments, the method involves developing a therapeutic based on the CRISPR-Cas system described herein. In particular embodiments, the therapeutic comprises a Cas effector and/or a guide RNA capable of hybridizing to a target sequence of interest. In particular embodiments, the therapeutic is a CRISPR-Cas vector or vector system that can contain a) a first regulatory element operably linked to a nucleotide sequence encoding the Cas effector protein(s); and b) a second regulatory element operably linked to one or more nucleotide sequences encoding one or more nucleic acid molecules comprising a guide RNA comprising a guide sequence, a direct repeat sequence; wherein components (a) and (b) are located on same or different vectors. In particular embodiments, the biologically active agent is a composition comprising a delivery system operably configured to deliver CRISPR-Cas system or components thereof, and/or or one or more polynucleotide sequences, vectors, or vector systems containing or encoding said components into a cell and capable of forming a CRISPR-Cas complex, and wherein said CRISPR-Cas complex is operable in the cell. In some embodiments, the CRISPR-Cas complex can include the Cas effector protein(s) as described herein, guide RNA comprising the guide sequence, and a direct repeat sequence. In any such compositions, the delivery system can be a yeast system, a lipofection system, a microinjection system, a biolistic system, virosomes, liposomes, immunoliposomes, polycations, lipid:nucleic acid conjugates or artificial virions, or any other system as described herein. In particular embodiments, the delivery is via a particle, a nanoparticle, a lipid or a cell penetrating peptide (CPP). 
     Also described herein are methods for developing or designing a CRISPR-Cas system, optionally a CRISPR-Cas system based therapy or therapeutic, comprising (a) selecting for a (therapeutic) locus of interest gRNA target sites, wherein said target sites have minimal sequence variation across a population, and from said selected target sites subselecting target sites, wherein a gRNA directed against said target sites recognizes a minimal number of off-target sites across said population, or (b) selecting for a (therapeutic) locus of interest gRNA target sites, wherein said target sites have minimal sequence variation across a population, or selecting for a (therapeutic) locus of interest gRNA target sites, wherein a gRNA directed against said target sites recognizes a minimal number of off-target sites across said population, and optionally estimating the number of (sub)selected target sites needed to treat or otherwise modulate or manipulate a population, and optionally validating one or more of the (sub)selected target sites for an individual subject, optionally designing one or more gRNA recognizing one or more of said (sub)selected target sites. 
     In some embodiments, the method for developing or designing a gRNA for use in a CRISPR-Cas system, optionally a CRISPR-Cas system based therapy or therapeutic, can include (a) selecting for a (therapeutic) locus of interest gRNA target sites, wherein said target sites have minimal sequence variation across a population, and from said selected target sites subselecting target sites, wherein a gRNA directed against said target sites recognizes a minimal number of off-target sites across said population, or (b) selecting for a (therapeutic) locus of interest gRNA target sites, wherein said target sites have minimal sequence variation across a population, or selecting for a (therapeutic) locus of interest gRNA target sites, wherein a gRNA directed against said target sites recognizes a minimal number of off-target sites across said population, and optionally estimating the number of (sub)selected target sites needed to treat or otherwise modulate or manipulate a population, optionally validating one or more of the (sub)selected target sites for an individual subject, optionally designing one or more gRNA recognizing one or more of said (sub)selected target sites. 
     In some embodiments, the method for developing or designing a CRISPR-Cas system, optionally a CRISPR-Cas system based therapy or therapeutic in a population, can include (a) selecting for a (therapeutic) locus of interest gRNA target sites, wherein said target sites have minimal sequence variation across a population, and from said selected target sites subselecting target sites, wherein a gRNA directed against said target sites recognizes a minimal number of off-target sites across said population, or (b) selecting for a (therapeutic) locus of interest gRNA target sites, wherein said target sites have minimal sequence variation across a population, or selecting for a (therapeutic) locus of interest gRNA target sites, wherein a gRNA directed against said target sites recognizes a minimal number of off-target sites across said population, and optionally estimating the number of (sub)selected target sites needed to treat or otherwise modulate or manipulate a population, optionally validating one or more of the (sub)selected target sites for an individual subject, optionally designing one or more gRNA recognizing one or more of said (sub)selected target sites. 
     In some embodiments the method for developing or designing a gRNA for use in a CRISPR-Cas system, optionally a CRISPR-Cas system based therapy or therapeutic in a population, can include (a) selecting for a (therapeutic) locus of interest gRNA target sites, wherein said target sites have minimal sequence variation across a population, and from said selected target sites subselecting target sites, wherein a gRNA directed against said target sites recognizes a minimal number of off-target sites across said population, or (b) selecting for a (therapeutic) locus of interest gRNA target sites, wherein said target sites have minimal sequence variation across a population, or selecting for a (therapeutic) locus of interest gRNA target sites, wherein a gRNA directed against said target sites recognizes a minimal number of off-target sites across said population, and optionally estimating the number of (sub)selected target sites needed to treat or otherwise modulate or manipulate a population, optionally validating one or more of the (sub)selected target sites for an individual subject, optionally designing one or more gRNA recognizing one or more of said (sub)selected target sites. 
     In some embodiments, the method for developing or designing a CRISPR-Cas system, such as a CRISPR-Cas system based therapy or therapeutic, optionally in a population; or for developing or designing a gRNA for use in a CRISPR-Cas system, optionally a CRISPR-Cas system based therapy or therapeutic, optionally in a population, can include: selecting a set of target sequences for one or more loci in a target population, wherein the target sequences do not contain variants occurring above a threshold allele frequency in the target population (i.e. platinum target sequences); removing from said selected (platinum) target sequences any target sequences having high frequency off-target candidates (relative to other (platinum) targets in the set) to define a final target sequence set; preparing one or more, such as a set of CRISPR-Cas systems based on the final target sequence set, optionally wherein a number of CRISP-Cas systems prepared is based (at least in part) on the size of a target population. 
     In certain embodiments, off-target candidates/off-targets, PAM restrictiveness, target cleavage efficiency, or effector protein specificity is identified or determined using a sequencing-based double-strand break (DSB) detection assay, such as described herein elsewhere. In certain embodiments, off-target candidates/off-targets are identified or determined using a sequencing-based double-strand break (DSB) detection assay, such as described herein elsewhere. In certain embodiments, off-targets, or off target candidates have at least 1, preferably 1-3, mismatches or (distal) PAM mismatches, such as 1 or more, such as 1, 2, 3, or more (distal) PAM mismatches. In certain embodiments, sequencing-based DSB detection assay comprises labeling a site of a DSB with an adapter comprising a primer binding site, labeling a site of a DSB with a barcode or unique molecular identifier, or combination thereof, as described herein elsewhere. 
     It will be understood that the guide sequence of the gRNA is 100% complementary to the target site, i.e. does not comprise any mismatch with the target site. It will be further understood that “recognition” of an (off-)target site by a gRNA presupposes CRISPR-Cas system functionality, i.e. an (off-)target site is only recognized by a gRNA if binding of the gRNA to the (off-)target site leads to CRISPR-Cas system activity (such as induction of single or double strand DNA cleavage, transcriptional modulation, etc.). 
     In certain embodiments, the target sites having minimal sequence variation across a population are characterized by absence of sequence variation in at least 99%, preferably at least 99.9%, more preferably at least 99.99% of the population. In certain embodiments, optimizing target location comprises selecting target sequences or loci having an absence of sequence variation in at least 99%, %, preferably at least 99.9%, more preferably at least 99.99% of a population. These targets are referred to herein elsewhere also as “platinum targets”. In certain embodiments, said population comprises at least 1000 individuals, such as at least 5000 individuals, such as at least 10000 individuals, such as at least 50000 individuals. 
     In certain embodiments, the off-target sites are characterized by at least one mismatch between the off-target site and the gRNA. In certain embodiments, the off-target sites are characterized by at most five, preferably at most four, more preferably at most three mismatches between the off-target site and the gRNA. In certain embodiments, the off-target sites are characterized by at least one mismatch between the off-target site and the gRNA and by at most five, preferably at most four, more preferably at most three mismatches between the off-target site and the gRNA. 
     In certain embodiments, said minimal number of off-target sites across said population is determined for high-frequency haplotypes in said population. In certain embodiments, said minimal number of off-target sites across said population is determined for high-frequency haplotypes of the off-target site locus in said population. In certain embodiments, said minimal number of off-target sites across said population is determined for high-frequency haplotypes of the target site locus in said population. In certain embodiments, the high-frequency haplotypes are characterized by occurrence in at least 0.1% of the population. 
     In certain embodiments, the number of (sub)selected target sites needed to treat a population is estimated based on based low frequency sequence variation, such as low frequency sequence variation captured in large scale sequencing datasets. In certain embodiments, the number of (sub)selected target sites needed to treat a population of a given size is estimated. In certain embodiments, the method further comprises obtaining genome sequencing data of a subject to be treated; and treating the subject with a CRISPR-Cas system selected from the set of CRISPR-Cas systems, wherein the CRISPR-Cas system selected is based (at least in part) on the genome sequencing data of the individual. In certain embodiments, the ((sub)selected) target is validated by genome sequencing, preferably whole genome sequencing. 
     In certain embodiments, target sequences or loci as described herein are (further) selected based on optimization of one or more parameters, such as PAM type (natural or modified), PAM nucleotide content, PAM length, target sequence length, PAM restrictiveness, target cleavage efficiency, and target sequence position within a gene, a locus or other genomic region. Methods of optimization are discussed in greater detail elsewhere herein. 
     In certain embodiments, target sequences or loci as described herein are (further) selected based on optimization of one or more of target loci location, target length, target specificity, and PAM characteristics. As used herein, PAM characteristics may comprise for instance PAM sequence, PAM length, and/or PAM GC contents. In certain embodiments, optimizing PAM characteristics comprises optimizing nucleotide content of a PAM. In certain embodiments, optimizing nucleotide content of PAM is selecting a PAM with a motif that maximizes abundance in the one or more target loci, minimizes mutation frequency, or both. Minimizing mutation frequency can for instance be achieved by selecting PAM sequences devoid of or having low or minimal CpG. 
     In certain embodiments, the effector protein for each CRISPR-Cas system in the set of CRISPR-Cas systems is selected based on optimization of one or more parameters selected from the group consisting of; effector protein size, ability of effector protein to access regions of high chromatin accessibility, degree of uniform enzyme activity across genomic targets, epigenetic tolerance, mismatch/budge tolerance, effector protein specificity, effector protein stability or half-life, effector protein immunogenicity or toxicity. Methods of optimization are discussed in greater detail elsewhere herein. 
     Optimization of CRISPR-Cas Systems 
     The methods of the present invention can involve optimization of selected parameters or variables associated with the CRISPR-Cas system and/or its functionality, as described herein further elsewhere. Optimization of selected parameters or variables in the methods as described herein may result in optimized or improved CRISPR-Cas system, such as CRISPR-Cas system-based therapy or therapeutic, specificity, efficacy, and/or safety. Optimization of the CRISPR-Cas system in the methods as described herein may depend on the target(s), such as the therapeutic target or therapeutic targets, the mode or type of CRISPR-Cas system modulation, such as CRISPR-Cas system based therapeutic target(s) modulation, modification, or manipulation, as well as the delivery of the CRISPR-Cas system components. One or more targets may be selected, depending on the genotypic and/or phenotypic outcome. For instance, one or more therapeutic targets may be selected, depending on (genetic) disease etiology or the desired therapeutic outcome. The (therapeutic) target(s) may be a single gene, locus, or other genomic site, or may be multiple genes, loci or other genomic sites. As is known in the art, a single gene, locus, or other genomic site may be targeted more than once, such as by use of multiple gRNAs. 
     CRISPR-Cas system activity, such as CRISPR-Cas system-based therapy or therapeutics may involve target disruption, such as target mutation, such as leading to gene knockout. CRISPR-Cas system activity, such as CRISPR-Cas system-based therapy or therapeutics may involve replacement of particular target sites, such as leading to target correction. CRISPR-Cas system-based therapy or therapeutics may involve removal of particular target sites, such as leading to target deletion. CRISPR-Cas system activity, such as CRISPR-Cas system-based therapy or therapeutics may involve modulation of target site functionality, such as target site activity or accessibility, leading for instance to (transcriptional and/or epigenetic) gene or genomic region activation or gene or genomic region silencing. The skilled person will understand that modulation of target site functionality may involve CRISPR effector mutation (such as for instance generation of a catalytically inactive CRISPR effector) and/or functionalization (such as for instance fusion of the CRISPR effector with a heterologous functional domain, such as a transcriptional activator or repressor), as described herein elsewhere. 
     Accordingly, in an aspect, the invention relates to a method as described herein, comprising selection of one or more (therapeutic) target, selecting one or more CRISPR-Cas system functionality, and optimization of selected parameters or variables associated with the CRISPR-Cas system and/or its functionality. In a related aspect, the invention relates to a method as described herein, comprising (a) selecting one or more (therapeutic) target loci, (b) selecting one or more CRISPR-Cas system functionalities, (c) optionally selecting one or more modes of delivery, and preparing, developing, or designing a CRISPR-Cas system selected based on steps (a)-(c). 
     In certain embodiments, CRISPR-Cas system functionality comprises genomic mutation. In certain embodiments, CRISPR-Cas system functionality comprises single genomic mutation. In certain embodiments, CRISPR-Cas system functionality comprises multiple genomic mutation. In certain embodiments, CRISPR-Cas system functionality comprises gene knockout. In certain embodiments, CRISPR-Cas system functionality comprises single gene knockout. In certain embodiments, CRISPR-Cas system functionality comprises multiple gene knockout. In certain embodiments, CRISPR-Cas system functionality comprises gene correction. In certain embodiments, CRISPR-Cas system functionality comprises single gene correction. In certain embodiments, CRISPR-Cas system functionality comprises multiple gene correction. In certain embodiments, CRISPR-Cas system functionality comprises genomic region correction. In certain embodiments, CRISPR-Cas system functionality comprises single genomic region correction. In certain embodiments, CRISPR-Cas system functionality comprises multiple genomic region correction. In certain embodiments, CRISPR-Cas system functionality comprises gene deletion. In certain embodiments, CRISPR-Cas system functionality comprises single gene deletion. In certain embodiments, CRISPR-Cas system functionality comprises multiple gene deletion. In certain embodiments, CRISPR-Cas system functionality comprises genomic region deletion. In certain embodiments, CRISPR-Cas system functionality comprises single genomic region deletion. In certain embodiments, CRISPR-Cas system functionality comprises multiple genomic region deletion. In certain embodiments, CRISPR-Cas system functionality comprises modulation of gene or genomic region functionality. In certain embodiments, CRISPR-Cas system functionality comprises modulation of single gene or genomic region functionality. In certain embodiments, CRISPR-Cas system functionality comprises modulation of multiple gene or genomic region functionality. In certain embodiments, CRISPR-Cas system functionality comprises gene or genomic region functionality, such as gene or genomic region activity. In certain embodiments, CRISPR-Cas system functionality comprises single gene or genomic region functionality, such as gene or genomic region activity. In certain embodiments, CRISPR-Cas system functionality comprises multiple gene or genomic region functionality, such as gene or genomic region activity. In certain embodiments, CRISPR-Cas system functionality comprises modulation gene activity or accessibility optionally leading to transcriptional and/or epigenetic gene or genomic region activation or gene or genomic region silencing. In certain embodiments, CRISPR-Cas system functionality comprises modulation single gene activity or accessibility optionally leading to transcriptional and/or epigenetic gene or genomic region activation or gene or genomic region silencing. In certain embodiments, CRISPR-Cas system functionality comprises modulation multiple gene activity or accessibility optionally leading to transcriptional and/or epigenetic gene or genomic region activation or gene or genomic region silencing. 
     Optimization of selected parameters or variables in the methods as described herein may result in optimized or improved CRISPR-Cas system, such as CRISPR-Cas system-based therapy or therapeutic, specificity, efficacy, and/or safety. In certain embodiments, one or more of the following parameters or variables are taken into account, are selected, or are optimized in the methods of the invention as described herein: Cas protein allosteric interactions, Cas protein functional domains and functional domain interactions, CRISPR effector specificity, gRNA specificity, CRISPR-Cas complex specificity, PAM restrictiveness, PAM type (natural or modified), PAM nucleotide content, PAM length, CRISPR effector activity, gRNA activity, CRISPR-Cas complex activity, target cleavage efficiency, target site selection, target sequence length, ability of effector protein to access regions of high chromatin accessibility, degree of uniform enzyme activity across genomic targets, epigenetic tolerance, mismatch/budge tolerance, CRISPR effector stability, CRISPR effector mRNA stability, gRNA stability, CRISPR-Cas complex stability, CRISPR effector protein or mRNA immunogenicity or toxicity, gRNA immunogenicity or toxicity, CRISPR-Cas complex immunogenicity or toxicity, CRISPR effector protein or mRNA dose or titer, gRNA dose or titer, CRISPR-Cas complex dose or titer, CRISPR effector protein size, CRISPR effector expression level, gRNA expression level, CRISPR-Cas complex expression level, CRISPR effector spatiotemporal expression, gRNA spatiotemporal expression, CRISPR-Cas complex spatiotemporal expression. 
     By means of example, and without limitation, parameter or variable optimization may be achieved as follows. CRISPR effector specificity may be optimized by selecting the most specific CRISPR effector. This may be achieved for instance by selecting the most specific CRISPR effector orthologue or by specific CRISPR effector mutations which increase specificity. gRNA specificity may be optimized by selecting the most specific gRNA. This can be achieved for instance by selecting gRNA having low homology, i.e. at least one or preferably more, such as at least 2, or preferably at least 3, mismatches to off-target sites. CRISPR-Cas complex specificity may be optimized by increasing CRISPR effector specificity and/or gRNA specificity as above. PAM restrictiveness may be optimized by selecting a CRISPR effector having to most restrictive PAM recognition. This can be achieved for instance by selecting a CRISPR effector orthologue having more restrictive PAM recognition or by specific CRISPR effector mutations which increase or alter PAM restrictiveness. PAM type may be optimized for instance by selecting the appropriate CRISPR effector, such as the appropriate CRISPR effector recognizing a desired PAM type. The CRISPR effector or PAM type may be naturally occurring or may for instance be optimized based on CRISPR effector mutants having an altered PAM recognition, or PAM recognition repertoire. PAM nucleotide content may for instance be optimized by selecting the appropriate CRISPR effector, such as the appropriate CRISPR effector recognizing a desired PAM nucleotide content. The CRISPR effector or PAM type may be naturally occurring or may for instance be optimized based on CRISPR effector mutants having an altered PAM recognition, or PAM recognition repertoire. PAM length may for instance be optimized by selecting the appropriate CRISPR effector, such as the appropriate CRISPR effector recognizing a desired PAM nucleotide length. The CRISPR effector or PAM type may be naturally occurring or may for instance be optimized based on CRISPR effector mutants having an altered PAM recognition, or PAM recognition repertoire. 
     Target length or target sequence length may for instance be optimized by selecting the appropriate CRISPR effector, such as the appropriate CRISPR effector recognizing a desired target or target sequence nucleotide length. Alternatively, or in addition, the target (sequence) length may be optimized by providing a target having a length deviating from the target (sequence) length typically associated with the CRISPR effector, such as the naturally occurring CRISPR effector. The CRISPR effector or target (sequence) length may be naturally occurring or may for instance be optimized based on CRISPR effector mutants having an altered target (sequence) length recognition, or target (sequence) length recognition repertoire. For instance, increasing or decreasing target (sequence) length may influence target recognition and/or off-target recognition. CRISPR effector activity may be optimized by selecting the most active CRISPR effector. This may be achieved for instance by selecting the most active CRISPR effector orthologue or by specific CRISPR effector mutations which increase activity. The ability of the CRISPR effector protein to access regions of high chromatin accessibility, may be optimized by selecting the appropriate CRISPR effector or mutant thereof, and can consider the size of the CRISPR effector, charge, or other dimensional variables etc. The degree of uniform CRISPR effector activity may be optimized by selecting the appropriate CRISPR effector or mutant thereof, and can consider CRISPR effector specificity and/or activity, PAM specificity, target length, mismatch tolerance, epigenetic tolerance, CRISPR effector and/or gRNA stability and/or half-life, CRISPR effector and/or gRNA immunogenicity and/or toxicity, etc. gRNA activity may be optimized by selecting the most active gRNA. In some embodiments, this can be achieved by increasing gRNA stability through RNA modification. CRISPR-Cas complex activity may be optimized by increasing CRISPR effector activity and/or gRNA activity as above. 
     The target site selection may be optimized by selecting the optimal position of the target site within a gene, locus or other genomic region. The target site selection may be optimized by optimizing target location comprises selecting a target sequence with a gene, locus, or other genomic region having low variability. This may be achieved for instance by selecting a target site in an early and/or conserved exon or domain (i.e. having low variability, such as polymorphisms, within a population). 
     In certain embodiments, optimizing target (sequence) length comprises selecting a target sequence within one or more target loci between 5 and 25 nucleotides. In certain embodiments, a target sequence is 20 nucleotides. 
     In certain embodiments, optimizing target specificity comprises selecting targets loci that minimize off-target candidates. 
     In some embodiments, the target site may be selected by minimization of off-target effects (e.g. off-targets qualified as having 1-5, 1-4, or preferably 1-3 mismatches compared to target and/or having one or more PAM mismatches, such as distal PAM mismatches), preferably also considering variability within a population. CRISPR effector stability may be optimized by selecting CRISPR effector having appropriate half-life, such as preferably a short half-life while still capable of maintaining sufficient activity. In some embodiments, this can be achieved by selecting an appropriate CRISPR effector orthologue having a specific half-life or by specific CRISPR effector mutations or modifications which affect half-life or stability, such as inclusion (e.g. fusion) of stabilizing or destabilizing domains or sequences. CRISPR effector mRNA stability may be optimized by increasing or decreasing CRISPR effector mRNA stability. In some embodiments, this can be achieved by increasing or decreasing CRISPR effector mRNA stability through mRNA modification. gRNA stability may be optimized by increasing or decreasing gRNA stability. In some embodiments, this can be achieved by increasing or decreasing gRNA stability through RNA modification. CRISPR-Cas complex stability may be optimized by increasing or decreasing CRISPR effector stability and/or gRNA stability as above. CRISPR effector protein or mRNA immunogenicity or toxicity may be optimized by decreasing CRISPR effector protein or mRNA immunogenicity or toxicity. In some embodiments, this can be achieved by mRNA or protein modifications. Similarly, in case of DNA based expression systems, DNA immunogenicity or toxicity may be decreased. gRNA immunogenicity or toxicity may be optimized by decreasing gRNA immunogenicity or toxicity. In some embodiments, this can be achieved by gRNA modifications. Similarly, in case of DNA based expression systems, DNA immunogenicity or toxicity may be decreased. CRISPR-Cas complex immunogenicity or toxicity may be optimized by decreasing CRISPR effector immunogenicity or toxicity and/or gRNA immunogenicity or toxicity as above, or by selecting the least immunogenic or toxic CRISPR effector/gRNA combination. Similarly, in case of DNA based expression systems, DNA immunogenicity or toxicity may be decreased. CRISPR effector protein or mRNA dose or titer may be optimized by selecting dosage or titer to minimize toxicity and/or maximize specificity and/or efficacy. gRNA dose or titer may be optimized by selecting dosage or titer to minimize toxicity and/or maximize specificity and/or efficacy. CRISPR-Cas complex dose or titer may be optimized by selecting dosage or titer to minimize toxicity and/or maximize specificity and/or efficacy. CRISPR effector protein size may be optimized by selecting minimal protein size to increase efficiency of delivery, in particular for virus mediated delivery. CRISPR effector, gRNA, or CRISPR-Cas complex expression level may be optimized by limiting (or extending) the duration of expression and/or limiting (or increasing) expression level. This may be achieved for instance by using self-inactivating CRISPR-Cas systems, such as including a self-targeting (e.g. CRISPR effector targeting) gRNA, by using viral vectors having limited expression duration, by using appropriate promoters for low (or high) expression levels, by combining different delivery methods for individual CRISP-Cas system components, such as virus mediated delivery of CRISPR-effector encoding nucleic acid combined with non-virus mediated delivery of gRNA, or virus mediated delivery of gRNA combined with non-virus mediated delivery of CRISPR effector protein or mRNA. CRISPR effector, gRNA, or CRISPR-Cas complex spatiotemporal expression may be optimized by appropriate choice of conditional and/or inducible expression systems, including controllable CRISPR effector activity optionally a destabilized CRISPR effector and/or a split CRISPR effector, and/or cell- or tissue-specific expression systems. 
     In an aspect, the invention relates to a method as described herein, comprising selection of one or more (therapeutic) target, selecting CRISPR-Cas system functionality, selecting CRISPR-Cas system mode of delivery, selecting CRISPR-Cas system delivery vehicle or expression system, and optimization of selected parameters or variables associated with the CRISPR-Cas system and/or its functionality, optionally wherein the parameters or variables are one or more selected from CRISPR effector specificity, gRNA specificity, CRISPR-Cas complex specificity, PAM restrictiveness, PAM type (natural or modified), PAM nucleotide content, PAM length, CRISPR effector activity, gRNA activity, CRISPR-Cas complex activity, target cleavage efficiency, target site selection, target sequence length, ability of effector protein to access regions of high chromatin accessibility, degree of uniform enzyme activity across genomic targets, epigenetic tolerance, mismatch/budge tolerance, CRISPR effector stability, CRISPR effector mRNA stability, gRNA stability, CRISPR-Cas complex stability, CRISPR effector protein or mRNA immunogenicity or toxicity, gRNA immunogenicity or toxicity, CRISPR-Cas complex immunogenicity or toxicity, CRISPR effector protein or mRNA dose or titer, gRNA dose or titer, CRISPR-Cas complex dose or titer, CRISPR effector protein size, CRISPR effector expression level, gRNA expression level, CRISPR-Cas complex expression level, CRISPR effector spatiotemporal expression, gRNA spatiotemporal expression, CRISPR-Cas complex spatiotemporal expression. 
     In an aspect, the invention relates to a method as described herein, comprising selecting one or more (therapeutic) target, selecting one or more CRISPR-Cas system functionality, selecting one or more CRISPR-Cas system mode of delivery, selecting one or more CRISPR-Cas system delivery vehicle or expression system, and optimization of selected parameters or variables associated with the CRISPR-Cas system and/or its functionality, wherein specificity, efficacy, and/or safety are optimized, and optionally wherein optimization of specificity comprises optimizing one or more parameters or variables selected from CRISPR effector specificity, gRNA specificity, CRISPR-Cas complex specificity, PAM restrictiveness, PAM type (natural or modified), PAM nucleotide content, PAM length, wherein optimization of efficacy comprises optimizing one or more parameters or variables selected from CRISPR effector activity, gRNA activity, CRISPR-Cas complex activity, target cleavage efficiency, target site selection, target sequence length, CRISPR effector protein size, ability of effector protein to access regions of high chromatin accessibility, degree of uniform enzyme activity across genomic targets, epigenetic tolerance, mismatch/budge tolerance, and wherein optimization of safety comprises optimizing one or more parameters or variables selected from CRISPR effector stability, CRISPR effector mRNA stability, gRNA stability, CRISPR-Cas complex stability, CRISPR effector protein or mRNA immunogenicity or toxicity, gRNA immunogenicity or toxicity, CRISPR-Cas complex immunogenicity or toxicity, CRISPR effector protein or mRNA dose or titer, gRNA dose or titer, CRISPR-Cas complex dose or titer, CRISPR effector expression level, gRNA expression level, CRISPR-Cas complex expression level, CRISPR effector spatiotemporal expression, gRNA spatiotemporal expression, CRISPR-Cas complex spatiotemporal expression. 
     In an aspect, the invention relates to a method as described herein, comprising optionally selecting one or more (therapeutic) target, optionally selecting one or more CRISPR-Cas system functionality, optionally selecting one or more CRISPR-Cas system mode of delivery, optionally selecting one or more CRISPR-Cas system delivery vehicle or expression system, and optimization of selected parameters or variables associated with the CRISPR-Cas system and/or its functionality, wherein specificity, efficacy, and/or safety are optimized, and optionally wherein optimization of specificity comprises optimizing one or more parameters or variables selected from CRISPR effector specificity, gRNA specificity, CRISPR-Cas complex specificity, PAM restrictiveness, PAM type (natural or modified), PAM nucleotide content, PAM length, wherein optimization of efficacy comprises optimizing one or more parameters or variables selected from CRISPR effector activity, gRNA activity, CRISPR-Cas complex activity, target cleavage efficiency, target site selection, target sequence length, CRISPR effector protein size, ability of effector protein to access regions of high chromatin accessibility, degree of uniform enzyme activity across genomic targets, epigenetic tolerance, mismatch/budge tolerance, and wherein optimization of safety comprises optimizing one or more parameters or variables selected from CRISPR effector stability, CRISPR effector mRNA stability, gRNA stability, CRISPR-Cas complex stability, CRISPR effector protein or mRNA immunogenicity or toxicity, gRNA immunogenicity or toxicity, CRISPR-Cas complex immunogenicity or toxicity, CRISPR effector protein or mRNA dose or titer, gRNA dose or titer, CRISPR-Cas complex dose or titer, CRISPR effector expression level, gRNA expression level, CRISPR-Cas complex expression level, CRISPR effector spatiotemporal expression, gRNA spatiotemporal expression, CRISPR-Cas complex spatiotemporal expression. 
     In an aspect, the invention relates to a method as described herein, comprising optimization of selected parameters or variables associated with the CRISPR-Cas system and/or its functionality, wherein specificity, efficacy, and/or safety are optimized, and optionally wherein optimization of specificity comprises optimizing one or more parameters or variables selected from CRISPR effector specificity, gRNA specificity, CRISPR-Cas complex specificity, PAM restrictiveness, PAM type (natural or modified), PAM nucleotide content, PAM length, wherein optimization of efficacy comprises optimizing one or more parameters or variables selected from CRISPR effector activity, gRNA activity, CRISPR-Cas complex activity, target cleavage efficiency, target site selection, target sequence length, CRISPR effector protein size, ability of effector protein to access regions of high chromatin accessibility, degree of uniform enzyme activity across genomic targets, epigenetic tolerance, mismatch/budge tolerance, and wherein optimization of safety comprises optimizing one or more parameters or variables selected from CRISPR effector stability, CRISPR effector mRNA stability, gRNA stability, CRISPR-Cas complex stability, CRISPR effector protein or mRNA immunogenicity or toxicity, gRNA immunogenicity or toxicity, CRISPR-Cas complex immunogenicity or toxicity, CRISPR effector protein or mRNA dose or titer, gRNA dose or titer, CRISPR-Cas complex dose or titer, CRISPR effector expression level, gRNA expression level, CRISPR-Cas complex expression level, CRISPR effector spatiotemporal expression, gRNA spatiotemporal expression, CRISPR-Cas complex spatiotemporal expression. 
     It will be understood that the parameters or variables to be optimized as well as the nature of optimization may depend on the (therapeutic) target, the CRISPR-Cas system functionality, the CRISPR-Cas system mode of delivery, and/or the CRISPR-Cas system delivery vehicle or expression system. 
     In an aspect, the invention relates to a method as described herein, comprising optimization of gRNA specificity at the population level. Preferably, said optimization of gRNA specificity comprises minimizing gRNA target site sequence variation across a population and/or minimizing gRNA off-target incidence across a population. 
     In some embodiments, optimization can result in selection of a CRISPR-Cas effector that is naturally occurring or is modified. In some embodiments, optimization can result in selection of a CRISPR-Cas effector that has nuclease, nickase, deaminase, transposase, and/or has one or more effector functionalities deactivated or eliminated. In some embodiments, optimizing a PAM specificity can include selecting a CRISPR-Cas effector with a modified PAM specificity. In some embodiments, optimizing can include selecting a CRISPR-Cas effector having a minimal size. In certain embodiments, optimizing effector protein stability comprises selecting an effector protein having a short half-life while maintaining sufficient activity, such as by selecting an appropriate CRISPR effector orthologue having a specific half-life or stability. In certain embodiments, optimizing immunogenicity or toxicity comprises minimizing effector protein immunogenicity or toxicity by protein modifications. In certain embodiments, optimizing functional specific comprises selecting a protein effector with reduced tolerance of mismatches and/or bulges between the guide RNA and one or more target loci. 
     In certain embodiments, optimizing efficacy comprises optimizing overall efficiency, epigenetic tolerance, or both. In certain embodiments, maximizing overall efficiency comprises selecting an effector protein with uniform enzyme activity across target loci with varying chromatin complexity, selecting an effector protein with enzyme activity limited to areas of open chromatin accessibility. In certain embodiments, chromatin accessibility is measured using one or more of ATAC-seq, or a DNA-proximity ligation assay. In certain embodiments, optimizing epigenetic tolerance comprises optimizing methylation tolerance, epigenetic mark competition, or both. In certain embodiments, optimizing methylation tolerance comprises selecting an effector protein that modify methylated DNA. In certain embodiments, optimizing epigenetic tolerance comprises selecting an effector protein unable to modify silenced regions of a chromosome, selecting an effector protein able to modify silenced regions of a chromosome, or selecting target loci not enriched for epigenetic markers. 
     In certain embodiments, selecting an optimized guide RNA comprises optimizing gRNA stability, gRNA immunogenicity, or both, or other gRNA associated parameters or variables as described herein elsewhere. 
     In certain embodiments, optimizing gRNA stability and/or gRNA immunogenicity comprises RNA modification, or other gRNA associated parameters or variables as described herein elsewhere. In certain embodiments, the modification comprises removing 1-3 nucleotides form the 3′ end of a target complementarity region of the gRNA. In certain embodiments, modification comprises an extended gRNA and/or trans RNA/DNA element that create stable structures in the gRNA that compete with gRNA base pairing at a target of off-target loci, or extended complimentary nucleotides between the gRNA and target sequence, or both. 
     In certain embodiments, the mode of delivery comprises delivering gRNA and/or CRISPR effector protein, delivering gRNA and/or CRISPR effector mRNA, or delivery gRNA and/or CRISPR effector as a DNA based expression system. In certain embodiments, the mode of delivery further comprises selecting a delivery vehicle and/or expression systems from the group consisting of liposomes, lipid particles, nanoparticles, biolistics, or viral-based expression/delivery systems. In certain embodiments, expression is spatiotemporal expression is optimized by choice of conditional and/or inducible expression systems, including controllable CRISPR effector activity optionally a destabilized CRISPR effector and/or a split CRISPR effector, and/or cell- or tissue-specific expression system. 
     The methods as described herein may further involve selection of the CRISPR-Cas system mode of delivery. In certain embodiments, gRNA (and tracr, if and where needed, optionally provided as a sgRNA) and/or CRISPR effector protein are or are to be delivered. In certain embodiments, gRNA (and tracr, if and where needed, optionally provided as a sgRNA) and/or CRISPR effector mRNA are or are to be delivered. In certain embodiments, gRNA (and tracr, if and where needed, optionally provided as a sgRNA) and/or CRISPR effector provided in a DNA-based expression system are or are to be delivered. In certain embodiments, delivery of the individual CRISPR-Cas system components comprises a combination of the above modes of delivery. In certain embodiments, delivery comprises delivering gRNA and/or CRISPR effector protein, delivering gRNA and/or CRISPR effector mRNA, or delivering gRNA and/or CRISPR effector as a DNA based expression system. 
     The methods as described herein may further involve selection of the CRISPR-Cas system delivery vehicle and/or expression system. Delivery vehicles and expression systems are described herein elsewhere. By means of example, delivery vehicles of nucleic acids and/or proteins include nanoparticles, liposomes, etc. Delivery vehicles for DNA, such as DNA-based expression systems include for instance biolistics, viral based vector systems (e.g. adenoviral, AAV, lentiviral), etc. the skilled person will understand that selection of the mode of delivery, as well as delivery vehicle or expression system may depend on for instance the cell or tissues to be targeted. In certain embodiments, the delivery vehicle and/or expression system for delivering the CRISPR-Cas systems or components thereof comprises liposomes, lipid particles, nanoparticles, biolistics, or viral-based expression/delivery systems. 
     Considerations for Therapeutic Applications 
     A consideration in genome editing therapy is the choice of sequence-specific nuclease, such as a variant of a Cas (e.g. Cas9 and/or Cas12) nuclease. Each nuclease variant may possess its own unique set of strengths and weaknesses, many of which must be balanced in the context of treatment to maximize therapeutic benefit. For a specific editing therapy to be efficacious, a sufficiently high level of modification must be achieved in target cell populations to reverse disease symptoms. This therapeutic modification ‘threshold’ is determined by the fitness of edited cells following treatment and the amount of gene product necessary to reverse symptoms. With regard to fitness, editing creates three potential outcomes for treated cells relative to their unedited counterparts: increased, neutral, or decreased fitness. In the case of increased fitness, corrected cells may be able and expand relative to their diseased counterparts to mediate therapy. In this case, where edited cells possess a selective advantage, even low numbers of edited cells can be amplified through expansion, providing a therapeutic benefit to the patient. Where the edited cells possess no change in fitness, an increase the therapeutic modification threshold can be warranted. As such, significantly greater levels of editing may be needed to treat diseases, where editing creates a neutral fitness advantage, relative to diseases where editing creates increased fitness for target cells. If editing imposes a fitness disadvantage, as would be the case for restoring function to a tumor suppressor gene in cancer cells, modified cells would be outcompeted by their diseased counterparts, causing the benefit of treatment to be low relative to editing rates. This may be overcome with supplemental therapies to increase the potency and/or fitness of the edited cells relative to the diseased counterparts. 
     In addition to cell fitness, the amount of gene product necessary to treat disease can also influence the minimal level of therapeutic genome editing that can treat or prevent a disease or a symptom thereof. In cases where a small change in the gene product levels can result in significant changes in clinical outcome, the minimal level of therapeutic genome editing is less relative to cases where a larger change in the gene product levels are needed to gain a clinically relevant response. In some embodiments, the minimal level of therapeutic genome editing can range from 0.1 to 1%, 1-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%. 45-50%, or 50-55%. Thus, where a small change in gene product levels can influence clinical outcomes and diseases where there is a fitness advantage for edited cells, are ideal targets for genome editing therapy, as the therapeutic modification threshold is low enough to permit a high chance of success. 
     The activity of NHEJ and HDR DSB repair can vary by cell type and cell state. NHEJ is not highly regulated by the cell cycle and is efficient across cell types, allowing for high levels of gene disruption in accessible target cell populations. In contrast, HDR acts primarily during S/G2 phase, and is therefore restricted to cells that are actively dividing, limiting treatments that require precise genome modifications to mitotic cells [Ciccia, A. &amp; Elledge, S. J. Molecular cell 40, 179-204 (2010); Chapman, J. R., et al. Molecular cell 47, 497-510 (2012)]. 
     The efficiency of correction via HDR may be controlled by the epigenetic state or sequence of the targeted locus, or the specific repair template configuration (single vs. double stranded, long vs. short homology arms) used [Hacein-Bey-Abina, S., et al. The New England journal of medicine 346, 1185-1193 (2002); Gaspar, H. B., et al. Lancet 364, 2181-2187 (2004); Beumer, K. J., et al. G3 (2013)]. The relative activity of NHEJ and HDR machineries in target cells may also affect gene correction efficiency, as these pathways may compete to resolve DSBs [Beumer, K. J., et al. Proceedings of the National Academy of Sciences of the United States of America 105, 19821-19826 (2008)]. HDR also imposes a delivery challenge not seen with NHEJ strategies, as it uses the concurrent delivery of nucleases and repair templates. Thus, these differences can be kept in mind when designing, optimizing, and/or selecting a CRISPR-Cas based therapeutic as described in greater detail elsewhere herein. 
     CRISPR-Cas-based polynucleotide modification application can include combinations of proteins, small RNA molecules, and/or repair templates, and can make, in some embodiments, delivery of these multiple parts substantially more challenging than, for example, traditional small molecule therapeutics. Two main strategies for delivery of CRISPR-Cas systems and components thereof have been developed: ex vivo and in vivo. In some embodiments of ex vivo treatments, diseased cells are removed from a subject, edited and then transplanted back into the patient. In other embodiments, cells from a healthy allogeneic donor are collected, modified using a CRISPR-Cas system or component thereof, to impart various functionalities and/or reduce immunogenicity, and administered to an allogeneic recipient in need of treatment. Ex vivo editing has the advantage of allowing the target cell population to be well defined and the specific dosage of therapeutic molecules delivered to cells to be specified. The latter consideration may be particularly important when off-target modifications are a concern, as titrating the amount of nuclease may decrease such mutations (Hsu et al., 2013). Another advantage of ex vivo approaches is the typically high editing rates that can be achieved, due to the development of efficient delivery systems for proteins and nucleic acids into cells in culture for research and gene therapy applications. 
     In vivo polynucleotide modification via CRISPR-Cas systems and/or components thereof involves direct delivery of the CRISPR-Cas systems and/or components thereof to cell types in their native tissues. In vivo polynucleotide modification via CRISPR-Cas systems and/or components thereof allows diseases in which the affected cell population is not amenable to ex vivo manipulation to be treated. Furthermore, delivering CRISPR-Cas systems and/or components thereof to cells in situ allows for the treatment of multiple tissue and cell types. 
     In some embodiments, such as those where viral vector systems are used to generate viral particles to deliver the CRISPR-Cas system and/or component thereof to a cell, the total cargo size of the CRISPR-Cas system and/or component thereof should be considered as vector systems can have limits on the size of a polynucleotide that can be expressed therefrom and/or packaged into cargo inside of a viral particle. In some embodiments, the tropism of a vector system, such as a viral vector system, should be considered as it can impact the cell type to which the CRISPR-Cas system or component thereof can be efficiently and/or effectively delivered. 
     When delivering a CRISPR-Cas system or component thereof via a viral-based system, it can be important to consider the amount of viral particles that will be needed to achieve a therapeutic effect so as to account for the potential immune response that can be elicited by the viral particles when delivered to a subject or cell(s). When delivering a CRISPR-Cas system or component thereof via a viral based system, it can be important to consider mechanisms of controlling the distribution and/or dosage of the CRISRP-Cas system in vivo. Generally, to reduce the potential for off-target effects, it is optimal but not necessarily required, that the amount of the CRISPR-Cas system be as close to the minimum or least effective dose. In practice this can be challenging to do. 
     In some embodiments, it can be important to considered the immunogenicity of the CRISPR-Cas system or component thereof. In embodiments, where the immunogenicity of the CRISPR-Cas system or component thereof is of concern, the immunogenicity CRISPR-Cas system or component thereof can be reduced. By way of example only, the immunogenicity of the CRISPR-Cas system or component thereof can be reduced using the approach set out in Tangri et al. Accordingly, directed evolution or rational design may be used to reduce the immunogenicity of the CRISPR enzyme (for instance a Cas (e.g. Cas9 and/or Cas12)) in the host species (human or other species). 
     Screening 
     In some aspects, the non-class I CRISPR-Cas systems, components thereof, polynucleotides thereof, and vectors thereof described here in can used in a screening assay within or outside of a cell. In some embodiments, the screen can include delivery of one or more CRISPR-Cas systems described herein to a cell (e.g. in vitro or ex vivo) and optionally obtaining data or results from an output or change in the cell induced by delivery of and/or activity of the CRISPR-Cas system(s) on the cell, and optionally transmitting the data and/or results. 
     The CRISPR-Cas systems can be used in gain of function screens. In some embodiments, cells which are artificially forced to overexpress a gene are be able to down regulate the gene over time (re-establishing equilibrium) e.g. by negative feedback loops. By the time the screen starts the unregulated gene might be reduced again. 
     In an aspect, the invention provides a cell from or of an in vitro method of delivery, wherein the method comprises contacting the delivery system with a cell, optionally a eukaryotic cell, whereby there is delivery into the cell of constituents of the delivery system, and optionally obtaining data or results from the contacting, and transmitting the data or results; and wherein the cell product is altered compared to the cell not contacted with the delivery system, for example altered from that which would have been wild type of the cell but for the contacting. Delivery methods and vehicles are described in greater detail elsewhere herein. 
     In embodiments of a screen described herein the cell is a eukaryotic cell. In embodiments of a screen described herein the cell is a prokaryotic cell. In embodiments of a screen described herein the cell is a non-human animal cell. In embodiments of a screen described herein the cell is a non-human primate cell. In embodiments of a screen described herein the cell is a human cell. In embodiments of a screen described herein the cell is a plant, fungal, or microorganism cell. 
     In some embodiments, the CRISPR-Cas systems and components thereof described herein can be used to screen endogenous plant genes to identify genes of value encoding enzymes involved in the production of a component of added nutritional value or generally genes affecting agronomic traits of interest, across species, phyla, and plant kingdom. By selectively targeting e.g. genes encoding enzymes of metabolic pathways in plants using the CRISPR-Cas system as described herein, the genes responsible for certain nutritional aspects of a plant can be identified. Similarly, by selectively targeting genes which may affect a desirable agronomic trait, the relevant genes can be identified. Accordingly, the present invention encompasses screening methods for genes encoding enzymes involved in the production of compounds with a particular nutritional value and/or agronomic traits. 
     Sequencing 
     The CRISPR-Cas compositions, systems, and methods described herein can be used in a sequencing method or technique. For example, some sequencing techniques utilize capture or identification, separation, and or isolation of polynucleotides to be sequenced. Thus, the CRISPR-Cas systems and/or components thereof described herein can be used in any sequencing method where it is necessary and/or advantageous to specifically identify polynucleotides to be sequenced and/or otherwise facilitate polynucleotide sequencing. 
     In some embodiments, the CRISPR-Cas system described herein can be used in a single cell sequencing technique. Such single-cell sequencing techniques include, but are not limited to Drop-Seq (see e.g., International Patent Publication No. WO 2016/040476), RASIN-Seq (see e.g., International Patent Publication No. WO 2020/077236), Seq-Well (US Patent Publication No. 20190144936; International Patent Publication No. WO 2017/124101), Single cell ATAC-seq (see e.g., Cusanovich, D. A., Daza, R., Adey, A., Pliner, H., Christiansen, L., Gunderson, K. L., Steemers, F. J., Trapnell, C. &amp; Shendure, J. Multiplex single-cell profiling of chromatin accessibility by combinatorial cellular indexing. Science. 2015 May 22; 348(6237):910-4. doi: 10.1126/science.aab1601. Epub 2015 May 7; US20160208323A1; US20160060691A1; and WO2017156336A1) and the like. 
     In some embodiments, the CRISPR-Cas system described herein can be used in a single cell sequencing technique adapted from e.g., Kalisky, T., Blainey, P. &amp; Quake, S. R. Genomic Analysis at the Single-Cell Level. Annual review of genetics 45, 431-445, (2011); Kalisky, T. &amp; Quake, S. R. Single-cell genomics. Nature Methods 8, 311-314 (2011); Islam, S. et al. Characterization of the single-cell transcriptional landscape by highly multiplex RNA-seq. Genome Research, (2011); Tang, F. et al. RNA-Seq analysis to capture the transcriptome landscape of a single cell. Nature Protocols 5, 516-535, (2010); Tang, F. et al. mRNA-Seq whole-transcriptome analysis of a single cell. Nature Methods 6, 377-382, (2009); Ramskold, D. et al. Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells. Nature Biotechnology 30, 777-782, (2012); and Hashimshony, T., Wagner, F., Sher, N. &amp; Yanai, I. CEL-Seq: Single-Cell RNA-Seq by Multiplexed Linear Amplification. Cell Reports, Cell Reports, Volume 2, Issue 3, p 666-6′73, 2012. 
     In some embodiments, the single cell sequencing method can be high-throughput single-cell sequencing (see e.g., Macosko et al., 2015, “Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets” Cell 161, 1202-1214; International patent application number PCT/US2015/049178, published as WO2016/040476 on Mar. 17, 2016; Klein et al., 2015, “Droplet Barcoding for Single-Cell Transcriptomics Applied to Embryonic Stem Cells” Cell 161, 1187-1201; International patent application number PCT/US2016/027734, published as WO2016168584A1 on Oct. 20, 2016; Zheng, et al., 2016, “Haplotyping germline and cancer genomes with high-throughput linked-read sequencing” Nature Biotechnology 34, 303-311; Zheng, et al., 2017, “Massively parallel digital transcriptional profiling of single cells” Nat. Commun. 8, 14049 doi: 10.1038/ncomms14049; International patent publication number WO2014210353A2; Zilionis, et al., 2017, “Single-cell barcoding and sequencing using droplet microfluidics” Nat Protoc. January; 12(1):44-73; Cao et al., 2017, “Comprehensive single cell transcriptional profiling of a multicellular organism by combinatorial indexing” bioRxiv preprint first posted online Feb. 2, 2017, doi: dx.doi.org/10.1101/104844; Rosenberg et al., 2017, “Scaling single cell transcriptomics through split pool barcoding” bioRxiv preprint first posted online Feb. 2, 2017, doi: dx.doi.org/10.1101/105163; Rosenberg et al., “Single-cell profiling of the developing mouse brain and spinal cord with split-pool barcoding” Science 15 Mar. 2018; Vitak, et al., “Sequencing thousands of single-cell genomes with combinatorial indexing” Nature Methods, 14(3):302-308, 2017; Cao, et al., Comprehensive single-cell transcriptional profiling of a multicellular organism. Science, 357(6352):661-667, 2017; Gierahn et al., “Seq-Well: portable, low-cost RNA sequencing of single cells at high throughput” Nature Methods 14, 395-398 (2017); and Hughes, et al., “Highly Efficient, Massively-Parallel Single-Cell RNA-Seq Reveals Cellular States and Molecular Features of Human Skin Pathology” bioRxiv 689273; doi: doi.org/10.1101/689273, all the contents and disclosure of each of which are herein incorporated by reference in their entirety). 
     In some embodiments, the CRISPR-Cas systems and/or components thereof can be used in a single nucleus sequencing method or technique (see e.g., Swiech et al., 2014, “In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9” Nature Biotechnology Vol. 33, pp. 102-106; Habib et al., 2016, “Div-Seq: Single-nucleus RNA-Seq reveals dynamics of rare adult newborn neurons” Science, Vol. 353, Issue 6302, pp. 925-928; Habib et al., 2017, “Massively parallel single-nucleus RNA-seq with DroNc-seq” Nat Methods. 2017 October; 14(10):955-958; International patent application number PCT/US2016/059239, published as WO2017164936 on Sep. 28, 2017; International patent application number PCT/US2018/060860, published as WO/2019/094984 on May 16, 2019; International patent application number PCT/US2019/055894, published as WO/2020/077236 on Apr. 16, 2020; and Drokhlyansky, et al., “The enteric nervous system of the human and mouse colon at a single-cell resolution,” bioRxiv 746743; doi: doi.org/10.1101/746743, which are herein incorporated by reference in their entirety). 
     In some embodiments, the CRISPR-Cas systems and/or components thereof can be used in a Assay for Transposase Accessible Chromatin using sequencing (ATAC-seq) method or technique (see e.g., Buenrostro, et al., Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nature methods 2013; 10 (12): 1213-1218; Buenrostro et al., Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-490 (2015); Cusanovich, D. A., Daza, R., Adey, A., Pliner, H., Christiansen, L., Gunderson, K. L., Steemers, F. J., Trapnell, C. &amp; Shendure, J. Multiplex single-cell profiling of chromatin accessibility by combinatorial cellular indexing.  Science.  2015 May 22; 348(6237):910-4. doi: 10.1126/science.aab1601. Epub 2015 May 7; U.S. Patent Publication Nos. US20160208323A1 and US20160060691A1; and International Patent Publication No. W02017156336A1). 
     Uses in Non-Animal Agriculture 
     The CRISPR-Cas compositions, systems, and methods described herein can be used to perform gene or genome interrogation or editing or manipulation in plants and fungi. For example, the applications include investigation and/or selection and/or interrogations and/or comparison and/or manipulations and/or transformation of plant genes or genomes; e.g., to create, identify, develop, optimize, or confer trait(s) or characteristic(s) to plant(s) or to transform a plant or fungus genome. There can accordingly be improved production of plants, new plants with new combinations of traits or characteristics or new plants with enhanced traits. The compositions, systems, and methods can be used with regard to plants in Site-Directed Integration (SDI) or Gene Editing (GE) or any Near Reverse Breeding (NRB) or Reverse Breeding (RB) techniques. 
     The compositions, systems, and methods herein may be used to confer desired traits (e.g., enhanced nutritional quality, increased resistance to diseases and resistance to biotic and abiotic stress, and increased production of commercially valuable plant products or heterologous compounds) on essentially any plants and fungi, and their cells and tissues. The compositions, systems, and methods may be used to modify endogenous genes or to modify their expression without the permanent introduction into the genome of any foreign gene. 
     In some embodiments, compositions, systems, and methods may be used in genome editing in plants or where RNAi or similar genome editing techniques have been used previously; see, e.g., Nekrasov, “Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR-Cas system,” Plant Methods 2013, 9:39 (doi:10.1186/1746-4811-9-39); Brooks, “Efficient gene editing in tomato in the first generation using the CRISPR-Cas9 system,” Plant Physiology September 2014 pp 114.247577; Shan, “Targeted genome modification of crop plants using a CRISPR-Cas system,” Nature Biotechnology 31, 686-688 (2013); Feng, “Efficient genome editing in plants using a CRISPR/Cas system,” Cell Research (2013) 23:1229-1232. doi:10.1038/cr.2013.114; published online 20 Aug. 2013; Xie, “RNA-guided genome editing in plants using a CRISPR-Cas system,” Mol Plant. 2013 November; 6(6):1975-83. doi: 10.1093/mp/sst119. Epub 2013 August 17; Xu, “Gene targeting using the  Agrobacterium tumefaciens -mediated CRISPR-Cas system in rice,” Rice 2014, 7:5 (2014), Zhou et al., “Exploiting SNPs for biallelic CRISPR mutations in the outcrossing woody perennial  Populus  reveals 4-coumarate: CoA ligase specificity and Redundancy,” New Phytologist (2015) (Forum) 1-4 (available online only at www.newphytologist.com); Caliando et al, “Targeted DNA degradation using a CRISPR device stably carried in the host genome, NATURE COMMUNICATIONS 6:6989, DOI: 10.1038/ncomms7989, www.nature.com/naturecommunications DOI: 10.1038/ncomms7989; U.S. Pat. No. 6,603,061— Agrobacterium -Mediated Plant Transformation Method; U.S. Pat. No. 7,868,149—Plant Genome Sequences and Uses Thereof and US 2009/0100536—Transgenic Plants with Enhanced Agronomic Traits, Morrell et al “Crop genomics: advances and applications,” Nat Rev Genet. 2011 Dec. 29; 13(2):85-96, all the contents and disclosure of each of which are herein incorporated by reference in their entirety. Aspects of utilizing the compositions, systems, and methods may be analogous to the use of the CRISPR-Cas (e.g. CRISPR-Cas9) system in plants, and mention is made of the University of Arizona website “CRISPR-PLANT” (www.genome.arizona.edu/crispr/) (supported by Penn State and AGI). 
     The compositions, systems, and methods may also be used on protoplasts. A “protoplast” refers to a plant cell that has had its protective cell wall completely or partially removed using, for example, mechanical or enzymatic means resulting in an intact biochemical competent unit of living plant that can reform their cell wall, proliferate and regenerate grow into a whole plant under proper growing conditions. 
     The compositions, systems, and methods may be used for screening genes (e.g., endogenous, mutations) of interest. In some examples, genes of interest include those encoding enzymes involved in the production of a component of added nutritional value or generally genes affecting agronomic traits of interest, across species, phyla, and plant kingdom. By selectively targeting e.g. genes encoding enzymes of metabolic pathways, the genes responsible for certain nutritional aspects of a plant can be identified. Similarly, by selectively targeting genes which may affect a desirable agronomic trait, the relevant genes can be identified. Accordingly, the present invention encompasses screening methods for genes encoding enzymes involved in the production of compounds with a particular nutritional value and/or agronomic traits. 
     It is also understood that reference herein to animal cells may also apply, mutatis mutandis, to plant or fungal cells unless otherwise apparent; and, the enzymes herein having reduced off-target effects and systems employing such enzymes can be used in plant applications, including those mentioned herein. 
     In some cases, nucleic acids introduced to plants and fungi may be codon optimized for expression in the plants and fungi. Methods of codon optimization include those described in Kwon K C, et al., Codon Optimization to Enhance Expression Yields Insights into Chloroplast Translation, Plant Physiol. 2016 September; 172(1):62-77. 
     The components (e.g., Cas proteins) in the compositions and systems may further comprise one or more functional domains described herein. In some examples, the functional domains may be an exonuclease. Such exonuclease may increase the efficiency of the Cas proteins&#39; function, e.g., mutagenesis efficiency. An example of the functional domain is Trex2, as described in Weiss T et al., www.biorxiv.org/content/10.1101/2020.04.11.037572v1, doi: https://doi.org/10.1101/2020.04.11.037572. 
     Examples of Plants 
     The compositions, systems, and methods herein can be used to confer desired traits on essentially any plant. A wide variety of plants and plant cell systems may be engineered for the desired physiological and agronomic characteristics. In general, the term “plant” relates to any various photosynthetic, eukaryotic, unicellular or multicellular organism of the kingdom Plantae characteristically growing by cell division, containing chloroplasts, and having cell walls comprised of cellulose. The term plant encompasses monocotyledonous and dicotyledonous plants. 
     The compositions, systems, and methods may be used over a broad range of plants, such as for example with dicotyledonous plants belonging to the orders Magniolales, Illiciales, Laurales, Piperales, Aristochiales, Nymphaeales, Ranunculales, Papeverales, Sarraceniaceae, Trochodendrales, Hamamelidales, Eucomiales, Leitneriales, Myricales, Fagales, Casuarinales, Caryophyllales, Batales, Polygonales, Plumbaginales, Dilleniales, Theales, Malvales, Urticales, Lecythidales, Violales, Salicales, Capparales, Ericales, Diapensales, Ebenales, Primulales, Rosales, Fabales, Podostemales, Haloragales, Myrtales, Cornales, Proteales, San tales, Raffiesiales, Celastrales, Euphorbiales, Rhamnales, Sapindales, Juglandales, Geraniales, Polygalales, Umbellales, Gentianales, Polemoniales, Lamiales, Plantaginales, Scrophulariales, Campanulales, Rubiales, Dipsacales, and Asterales; monocotyledonous plants such as those belonging to the orders Alismatales, Hydrocharitales, Najadales, Triuridales, Commelinales, Eriocaulales, Restionales, Poales, Juncales, Cyperales, Typhales, Bromeliales, Zingiberales, Arecales, Cyclanthales, Pandanales, Arales, Lilliales, and Orchid ales, or with plants belonging to Gymnospermae, e.g. those belonging to the orders Pinales, Ginkgoales, Cycadales, Araucariales, Cupressales and Gnetales. 
     The compositions, systems, and methods herein can be used over a broad range of plant species, included in the non-limitative list of dicot, monocot or gymnosperm genera hereunder:  Atropa, Alseodaphne, Anacardium, Arachis, Beilschmiedia, Brassica, Carthamus, Cocculus, Croton, Cucumis, Citrus, Citrullus, Capsicum, Catharanthus, Cocos, Coffea, Cucurbita, Daucus, Duguetia, Eschscholzia, Ficus, Fragaria, Glaucium, Glycine, Gossypium, Helianthus, Hevea, Hyoscyamus, Lactuca, Landolphia, Linum, Litsea, Lycopersicon, Lupinus, Manihot, Majorana, Malus, Medicago, Nicotiana, Olea, Parthenium, Papaver, Persea, Phaseolus, Pistacia, Pisum, Pyrus, Prunus, Raphanus, Ricinus, Senecio, Sinomenium, Stephania, Sinapis, Solanum, Theobroma, Trifolium, Trigonella, Vicia, Vinca, Vilis , and  Vigna ; and the genera  Allium, Andropogon, Aragrostis, Asparagus, Avena, Cynodon, Elaeis, Festuca, Festulolium, Heterocallis, Hordeum, Lemna, Lolium, Musa, Oryza, Panicum, Pannesetum, Phleum, Poa, Secale, Sorghum, Triticum, Zea, Abies, Cunninghamia, Ephedra, Picea, Pinus , and  Pseudotsuga.    
     In some embodiments, target plants and plant cells for engineering include those monocotyledonous and dicotyledonous plants, such as crops including grain crops (e.g., wheat, maize, rice, millet, barley), fruit crops (e.g., tomato, apple, pear, strawberry, orange), forage crops (e.g., alfalfa), root vegetable crops (e.g., carrot, potato, sugar beets, yam), leafy vegetable crops (e.g., lettuce, spinach); flowering plants (e.g., petunia, rose, chrysanthemum), conifers and pine trees (e.g., pine fir, spruce); plants used in phytoremediation (e.g., heavy metal accumulating plants); oil crops (e.g., sunflower, rape seed) and plants used for experimental purposes (e.g.,  Arabidopsis ). Specifically, the plants are intended to comprise without limitation angiosperm and gymnosperm plants such as acacia, alfalfa, amaranth, apple, apricot, artichoke, ash tree, asparagus, avocado, banana, barley, beans, beet, birch, beech, blackberry, blueberry, broccoli, Brussel&#39;s sprouts, cabbage, canola, cantaloupe, carrot, cassava, cauliflower, cedar, a cereal, celery, chestnut, cherry, Chinese cabbage, citrus, clementine, clover, coffee, corn, cotton, cowpea, cucumber, cypress, eggplant, elm, endive, eucalyptus, fennel, figs, fir, geranium, grape, grapefruit, groundnuts, ground cherry, gum hemlock, hickory, kale, kiwifruit, kohlrabi, larch, lettuce, leek, lemon, lime, locust, pine, maidenhair, maize, mango, maple, melon, millet, mushroom, mustard, nuts, oak, oats, oil palm, okra, onion, orange, an ornamental plant or flower or tree, papaya, palm, parsley, parsnip, pea, peach, peanut, pear, peat, pepper, persimmon, pigeon pea, pine, pineapple, plantain, plum, pomegranate, potato, pumpkin, radicchio, radish, rapeseed, raspberry, rice, rye, sorghum, safflower, sallow, soybean, spinach, spruce, squash, strawberry, sugar beet, sugarcane, sunflower, sweet potato, sweet corn, tangerine, tea, tobacco, tomato, trees, triticale, turf grasses, turnips, vine, walnut, watercress, watermelon, wheat, yams, yew, and zucchini. 
     The term plant also encompasses Algae, which are mainly photoautotrophs unified primarily by their lack of roots, leaves and other organs that characterize higher plants. The compositions, systems, and methods can be used over a broad range of “algae” or “algae cells.” Examples of algae include eukaryotic phyla, including the Rhodophyta (red algae), Chlorophyta (green algae), Phaeophyta (brown algae), Bacillariophyta (diatoms), Eustigmatophyta and dinoflagellates as well as the prokaryotic phylum Cyanobacteria (blue-green algae). Examples of algae species include those of  Amphora, Anabaena, Anikstrodesmis, Botryococcus, Chaetoceros, Chlamydomonas, Chlorella, Chlorococcum, Cyclotella, Cylindrotheca, Dunaliella, Emiliana, Euglena, Hematococcus, Isochrysis, Monochrysis, Monoraphidium, Nannochloris, Nannnochloropsis, Navicula, Nephrochloris, Nephroselmis, Nitzschia, Nodularia, Nostoc, Oochromonas, Oocystis, Oscillartoria, Pavlova, Phaeodactylum, Playtmonas, Pleurochrysis, Porhyra, Pseudoanabaena, Pyramimonas, Stichococcus, Synechococcus, Synechocystis, Tetraselmis, Thalassiosira , and  Trichodesmium.    
     Plant Promoters 
     In order to ensure appropriate expression in a plant cell, the components of the components and systems herein may be placed under control of a plant promoter. A plant promoter is a promoter operable in plant cells. A plant promoter is capable of initiating transcription in plant cells, whether or not its origin is a plant cell. The use of different types of promoters is envisaged. 
     In some examples, the plant promoter is a constitutive plant promoter, which is a promoter that is able to express the open reading frame (ORF) that it controls in all or nearly all of the plant tissues during all or nearly all developmental stages of the plant (referred to as “constitutive expression”). One example of a constitutive promoter is the cauliflower mosaic virus 35S promoter. In some examples, the plant promoter is a regulated promoter, which directs gene expression not constitutively, but in a temporally- and/or spatially-regulated manner, and includes tissue-specific, tissue-preferred and inducible promoters. Different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. In some examples, the plant promoter is a tissue-preferred promoters, which can be utilized to target enhanced expression in certain cell types within a particular plant tissue, for instance vascular cells in leaves or roots or in specific cells of the seed. 
     Exemplary plant promoters include those obtained from plants, plant viruses, and bacteria such as  Agrobacterium  or  Rhizobium  which comprise genes expressed in plant cells. Additional examples of promoters include those described in Kawamata et al., (1997) Plant Cell Physiol 38:792-803; Yamamoto et al., (1997) Plant J 12:255-65; Hire et al, (1992) Plant Mol Biol 20:207-18, Kuster et al, (1995) Plant Mol Biol 29:759-72, and Capana et al., (1994) Plant Mol Biol 25:681-91. 
     In some examples, a plant promoter may be an inducible promoter, which is inducible and allows for spatiotemporal control of gene editing or gene expression may use a form of energy. The form of energy may include sound energy, electromagnetic radiation, chemical energy and/or thermal energy. Examples of inducible systems include tetracycline inducible promoters (Tet-On or Tet-Off), small molecule two-hybrid transcription activations systems (FKBP, ABA, etc.), or light inducible systems (Phytochrome, LOV domains, or cryptochrome), such as a Light Inducible Transcriptional Effector (LITE) that direct changes in transcriptional activity in a sequence-specific manner. In a particular example, of the components of a light inducible system include a Cas protein, a light-responsive cytochrome heterodimer (e.g. from  Arabidopsis thaliana ), and a transcriptional activation/repression domain. 
     In some examples, the promoter may be a chemical-regulated promotor (where the application of an exogenous chemical induces gene expression) or a chemical-repressible promoter (where application of the chemical represses gene expression). Examples of chemical-inducible promoters include maize 1n2-2 promoter (activated by benzene sulfonamide herbicide safeners), the maize GST promoter (activated by hydrophobic electrophilic compounds used as pre-emergent herbicides), the tobacco PR-1 a promoter (activated by salicylic acid), promoters regulated by antibiotics (such as tetracycline-inducible and tetracycline-repressible promoters). 
     Stable Integration in the Genome of Plants 
     In some embodiments, polynucleotides encoding the components of the compositions and systems may be introduced for stable integration into the genome of a plant cell. In some cases, vectors or expression systems may be used for such integration. The design of the vector or the expression system can be adjusted depending on for when, where and under what conditions the guide RNA and/or the Cas gene are expressed. In some cases, the polynucleotides may be integrated into an organelle of a plant, such as a plastid, mitochondrion or a chloroplast. The elements of the expression system may be on one or more expression constructs which are either circular such as a plasmid or transformation vector, or non-circular such as linear double stranded DNA. 
     In some embodiments, the method of integration generally comprises the steps of selecting a suitable host cell or host tissue, introducing the construct(s) into the host cell or host tissue, and regenerating plant cells or plants therefrom. In some examples, the expression system for stable integration into the genome of a plant cell may contain one or more of the following elements: a promoter element that can be used to express the RNA and/or Cas enzyme in a plant cell; a 5′ untranslated region to enhance expression; an intron element to further enhance expression in certain cells, such as monocot cells; a multiple-cloning site to provide convenient restriction sites for inserting the guide RNA and/or the Cas gene sequences and other desired elements; and a 3′ untranslated region to provide for efficient termination of the expressed transcript. 
     Transient Expression in Plants 
     In some embodiments, the components of the compositions and systems may be transiently expressed in the plant cell. In some examples, the compositions and systems may modify a target nucleic acid only when both the guide RNA and the Cas protein are present in a cell, such that genomic modification can further be controlled. As the expression of the Cas protein is transient, plants regenerated from such plant cells typically contain no foreign DNA. In certain examples, the Cas protein is stably expressed and the guide sequence is transiently expressed. 
     DNA and/or RNA (e.g., mRNA) may be introduced to plant cells for transient expression. In such cases, the introduced nucleic acid may be provided in sufficient quantity to modify the cell but do not persist after a contemplated period of time has passed or after one or more cell divisions. 
     The transient expression may be achieved using suitable vectors. Exemplary vectors that may be used for transient expression include a pEAQ vector (may be tailored for  Agrobacterium -mediated transient expression) and Cabbage Leaf Curl virus (CaLCuV), and vectors described in Sainsbury F. et al., Plant Biotechnol J. 2009 September; 7(7):682-93; and Yin K et al., Scientific Reports volume 5, Article number: 14926 (2015). 
     Combinations of the different methods described above are also envisaged. 
     Translocation to and/or Expression in Specific Plant Organelles 
     The compositions and systems herein may comprise elements for translocation to and/or expression in a specific plant organelle. 
     Chloroplast Targeting 
     In some embodiments, it is envisaged that the compositions and systems are used to specifically modify chloroplast genes or to ensure expression in the chloroplast. The compositions and systems (e.g., Cas proteins, guide molecules, or their encoding polynucleotides) may be transformed, compartmentalized, and/or targeted to the chloroplast. In an example, the introduction of genetic modifications in the plastid genome can reduce biosafety issues such as gene flow through pollen. 
     Examples of methods of chloroplast transformation include Particle bombardment, PEG treatment, and microinjection, and the translocation of transformation cassettes from the nuclear genome to the plastid. In some examples, targeting of chloroplasts may be achieved by incorporating in chloroplast localization sequence, and/or the expression construct a sequence encoding a chloroplast transit peptide (CTP) or plastid transit peptide, operably linked to the 5′ region of the sequence encoding the components of the compositions and systems. Additional examples of transforming, targeting and localization of chloroplasts include those described in WO2010061186, Protein Transport into Chloroplasts, 2010, Annual Review of Plant Biology, Vol. 61: 157-180, and US 20040142476, which are incorporated by reference herein in their entireties. 
     Exemplary Applications in Plants 
     The compositions, systems, and methods may be used to generate genetic variation(s) in a plant (e.g., crop) of interest. One or more, e.g., a library of, guide molecules targeting one or more locations in a genome may be provided and introduced into plant cells together with the Cas effector protein. For example, a collection of genome-scale point mutations and gene knock-outs can be generated. In some examples, the compositions, systems, and methods may be used to generate a plant part or plant from the cells so obtained and screening the cells for a trait of interest. The target genes may include both coding and non-coding regions. In some cases, the trait is stress tolerance and the method is a method for the generation of stress-tolerant crop varieties. 
     In some embodiments, the compositions, systems, and methods are used to modify endogenous genes or to modify their expression. The expression of the components may induce targeted modification of the genome, either by direct activity of the Cas nuclease and optionally introduction of template DNA, or by modification of genes targeted. The different strategies described herein above allow Cas-mediated targeted genome editing without requiring the introduction of the components into the plant genome. 
     In some cases, the modification may be performed without the permanent introduction into the genome of the plant of any foreign gene, including those encoding CRISPR components, so as to avoid the presence of foreign DNA in the genome of the plant. This can be of interest as the regulatory requirements for non-transgenic plants are less rigorous. Components which are transiently introduced into the plant cell are typically removed upon crossing. 
     For example, the modification may be performed by transient expression of the components of the compositions and systems. The transient expression may be performed by delivering the components of the compositions and systems with viral vectors, delivery into protoplasts, with the aid of particulate molecules such as nanoparticles or CPPs. 
     Generation of Plants with Desired Traits 
     The compositions, systems, and methods herein may be used to introduce desired traits to plants. The approaches include introduction of one or more foreign genes to confer a trait of interest, editing or modulating endogenous genes to confer a trait of interest. 
     Agronomic Traits 
     In some embodiments, crop plants can be improved by influencing specific plant traits. Examples of the traits include improved agronomic traits such as herbicide resistance, disease resistance, abiotic stress tolerance, high yield, and superior quality, pesticide-resistance, disease resistance, insect and nematode resistance, resistance against parasitic weeds, drought tolerance, nutritional value, stress tolerance, self-pollination voidance, forage digestibility biomass, and grain yield. 
     In some embodiments, genes that confer resistance to pests or diseases may be introduced to plants. In cases there are endogenous genes that confer such resistance in a plants, their expression and function may be enhanced (e.g., by introducing extra copies, modifications that enhance expression and/or activity). 
     Examples of genes that confer resistance include plant disease resistance genes (e.g., Cf-9, Pto, RSP2, SIDMR6-1), genes conferring resistance to a pest (e.g., those described in WO96/30517),  Bacillus thuringiensis  proteins, lectins, Vitamin-binding proteins (e.g., avidin), enzyme inhibitors (e.g., protease or proteinase inhibitors or amylase inhibitors), insect-specific hormones or pheromones (e.g., ecdysteroid or a juvenile hormone, variant thereof, a mimetic based thereon, or an antagonist or agonist thereof) or genes involved in the production and regulation of such hormone and pheromones, insect-specific peptides or neuropeptide, Insect-specific venom (e.g., produced by a snake, a wasp, etc., or analog thereof), Enzymes responsible for a hyperaccumulation of a monoterpene, a sesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivative or another nonprotein molecule with insecticidal activity, Enzymes involved in the modification of biologically active molecule (e.g., a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme, a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, a phosphatase, a kinase, a phosphorylase, a polymerase, an elastase, a chitinase and a glucanase, whether natural or synthetic), molecules that stimulates signal transduction, Viral-invasive proteins or a complex toxin derived therefrom, Developmental-arrestive proteins produced in nature by a pathogen or a parasite, a developmental-arrestive protein produced in nature by a plant, or any combination thereof. 
     The compositions, systems, and methods may be used to identify, screen, introduce or remove mutations or sequences lead to genetic variability that give rise to susceptibility to certain pathogens, e.g., host specific pathogens. Such approach may generate plants that are non-host resistance, e.g., the host and pathogen are incompatible or there can be partial resistance against all races of a pathogen, typically controlled by many genes and/or also complete resistance to some races of a pathogen but not to other races. 
     In some embodiments, compositions, systems, and methods may be used to modify genes involved in plant diseases. Such genes may be removed, inactivated, or otherwise regulated or modified. Examples of plant diseases include those described in [0045]-[0080] of US20140213619A1, which is incorporated by reference herein in its entirety. 
     In some embodiments, genes that confer resistance to herbicides may be introduced to plants. Examples of genes that confer resistance to herbicides include genes conferring resistance to herbicides that inhibit the growing point or meristem, such as an imidazolinone or a sulfonylurea, genes conferring glyphosate tolerance (e.g., resistance conferred by, e.g., mutant 5-enolpyruvylshikimate-3-phosphate synthase genes, aroA genes and glyphosate acetyl transferase (GAT) genes, respectively), or resistance to other phosphono compounds such as by glufosinate (phosphinothricin acetyl transferase (PAT) genes from  Streptomyces  species, including  Streptomyces hygroscopicus  and  Streptomyces viridichromogenes ), and to pyridinoxy or phenoxy proprionic acids and cyclohexones by ACCase inhibitor-encoding genes), genes conferring resistance to herbicides that inhibit photosynthesis (such as a triazine (psbA and gs+ genes) or a benzonitrile (nitrilase gene), and glutathione S-transferase), genes encoding enzymes detoxifying the herbicide or a mutant glutamine synthase enzyme that is resistant to inhibition, genes encoding a detoxifying enzyme is an enzyme encoding a phosphinothricin acetyltransferase (such as the bar or pat protein from  Streptomyces  species), genes encoding hydroxyphenylpyruvatedioxygenases (HPPD) inhibitors, e.g., naturally occurring HPPD resistant enzymes, and genes encoding a mutated or chimeric HPPD enzyme. 
     In some embodiments, genes involved in Abiotic stress tolerance may be introduced to plants. Examples of genes include those capable of reducing the expression and/or the activity of poly(ADP-ribose) polymerase (PARP) gene, transgenes capable of reducing the expression and/or the activity of the PARG encoding genes, genes coding for a plant-functional enzyme of the nicotineamide adenine dinucleotide salvage synthesis pathway including nicotinamidase, nicotinate phosphoribosyltransferase, nicotinic acid mononucleotide adenyl transferase, nicotinamide adenine dinucleotide synthetase or nicotine amide phosphorybosyltransferase, enzymes involved in carbohydrate biosynthesis, enzymes involved in the production of polyfructose (e.g., the inulin and levan-type), the production of alpha-1,6 branched alpha-1,4-glucans, the production of alternan, the production of hyaluronan. 
     In some embodiments, genes that improve drought resistance may be introduced to plants. Examples of genes Ubiquitin Protein Ligase protein (UPL) protein (UPL3), DR02, DR03, ABC transporter, and DREB1A. 
     Nutritionally Improved Plants 
     In some embodiments, the compositions, systems, and methods may be used to produce nutritionally improved plants. In some examples, such plants may provide functional foods, e.g., a modified food or food ingredient that may provide a health benefit beyond the traditional nutrients it contains. In certain examples, such plants may provide nutraceuticals foods, e.g., substances that may be considered a food or part of a food and provides health benefits, including the prevention and treatment of disease. The nutraceutical foods may be useful in the prevention and/or treatment of diseases in animals and humans, e.g., cancers, diabetes, cardiovascular disease, and hypertension. 
     An improved plant may naturally produce one or more desired compounds and the modification may enhance the level or activity or quality of the compounds. In some cases, the improved plant may not naturally produce the compound(s), while the modification enables the plant to produce such compound(s). In some cases, the compositions, systems, and methods used to modify the endogenous synthesis of these compounds indirectly, e.g. by modifying one or more transcription factors that controls the metabolism of this compound. 
     Examples of nutritionally improved plants include plants comprising modified protein quality, content and/or amino acid composition, essential amino acid contents, oils and fatty acids, carbohydrates, vitamins and carotenoids, functional secondary metabolites, and minerals. In some examples, the improved plants may comprise or produce compounds with health benefits. Examples of nutritionally improved plants include those described in Newell-McGloughlin, Plant Physiology, July 2008, Vol. 147, pp. 939-953. 
     Examples of compounds that can be produced include carotenoids (e.g., α-Carotene or β-Carotene), lutein, lycopene, Zeaxanthin, Dietary fiber (e.g., insoluble fibers, β-Glucan, soluble fibers, fatty acids (e.g., ω-3 fatty acids, Conjugated linoleic acid, GLA), Flavonoids (e.g., Hydroxycinnamates, flavonols, catechins and tannins), Glucosinolates, indoles, isothiocyanates (e.g., Sulforaphane), Phenolics (e.g., stilbenes, caffeic acid and ferulic acid, epicatechin), Plant stanols/sterols, Fructans, inulins, fructo-oligosaccharides, Saponins, Soybean proteins, Phytoestrogens (e.g., isoflavones, lignans), Sulfides and thiols such as diallyl sulphide, Allyl methyl trisulfide, dithiolthiones, Tannins, such as proanthocyanidins, or any combination thereof. 
     The compositions, systems, and methods may also be used to modify protein/starch functionality, shelf life, taste/aesthetics, fiber quality, and allergen, antinutrient, and toxin reduction traits. 
     Examples of genes and nucleic acids that can be modified to introduce the traits include stearyl-ACP desaturase, DNA associated with the single allele which may be responsible for maize mutants characterized by low levels of phytic acid, Tf RAP2.2 and its interacting partner SINAT2, TfDof1, and DOF Tf AtDof1.1 (OBP2). 
     Modification of Polyploid Plants 
     The compositions, systems, and methods may be used to modify polyploid plants. Polyploid plants carry duplicate copies of their genomes (e.g. as many as six, such as in wheat). In some cases, the compositions, systems, and methods may be can be multiplexed to affect all copies of a gene, or to target dozens of genes at once. For instance, the compositions, systems, and methods may be used to simultaneously ensure a loss of function mutation in different genes responsible for suppressing defenses against a disease. The modification may be simultaneous suppression the expression of the TaMLO-A1, TaMLO-B1 and TaMLO-D1 nucleic acid sequence in a wheat plant cell and regenerating a wheat plant therefrom, in order to ensure that the wheat plant is resistant to powdery mildew (e.g., as described in WO2015109752). 
     Regulation of Fruit-Ripening 
     The compositions, systems, and methods may be used to regulate ripening of fruits. Ripening is a normal phase in the maturation process of fruits and vegetables. Only a few days after it starts it may render a fruit or vegetable inedible, which can bring significant losses to both farmers and consumers. 
     In some embodiments, the compositions, systems, and methods are used to reduce ethylene production. In some examples, the compositions, systems, and methods may be used to suppress the expression and/or activity of ACC synthase, insert a ACC deaminase gene or a functional fragment thereof, insert a SAM hydrolase gene or functional fragment thereof, suppress ACC oxidase gene expression 
     Alternatively or additionally, the compositions, systems, and methods may be used to modify ethylene receptors (e.g., suppressing ETR1) and/or Polygalacturonase (PG). Suppression of a gene may be achieved by introducing a mutation, an antisense sequence, and/or a truncated copy of the gene to the genome. 
     Increasing Storage Life of Plants 
     In some embodiments, the compositions, systems, and methods are used to modify genes involved in the production of compounds which affect storage life of the plant or plant part. The modification may be in a gene that prevents the accumulation of reducing sugars in potato tubers. Upon high-temperature processing, these reducing sugars react with free amino acids, resulting in brown, bitter-tasting products and elevated levels of acrylamide, which is a potential carcinogen. In particular embodiments, the methods provided herein are used to reduce or inhibit expression of the vacuolar invertase gene (VInv), which encodes a protein that breaks down sucrose to glucose and fructose. 
     Reducing Allergens in Plants 
     In some embodiments, the compositions, systems, and methods are used to generate plants with a reduced level of allergens, making them safer for consumers. To this end, the compositions, systems, and methods may be used to identify and modify (e.g., suppress) one or more genes responsible for the production of plant allergens. Examples of such genes include Lol p5, as well as those in peanuts, soybeans, lentils, peas, lupin, green beans, mung beans, such as those described in Nicolaou et al., Current Opinion in Allergy and Clinical Immunology 2011; 11(3):222), which is incorporated by reference herein in its entirety. 
     Generation of Male Sterile Plants 
     The compositions, systems, and methods may be used to generate male sterile plants. Hybrid plants typically have advantageous agronomic traits compared to inbred plants. However, for self-pollinating plants, the generation of hybrids can be challenging. In different plant types (e.g., maize and rice), genes have been identified which are important for plant fertility, more particularly male fertility. Plants that are as such genetically altered can be used in hybrid breeding programs. 
     The compositions, systems, and methods may be used to modify genes involved male fertility, e.g., inactivating (such as by introducing mutations to) genes required for male fertility. Examples of the genes involved in male fertility include cytochrome P450-like gene (MS26) or the meganuclease gene (MS45), and those described in Wan X et al., Mol Plant. 2019 Mar. 4; 12(3):321-342; and Kim Y J, et al., Trends Plant Sci. 2018 January; 23(1):53-65. 
     Increasing the Fertility Stage in Plants 
     In some embodiments, the compositions, systems, and methods may be used to prolong the fertility stage of a plant such as of a rice. For instance, a rice fertility stage gene such as Ehd3 can be targeted in order to generate a mutation in the gene and plantlets can be selected for a prolonged regeneration plant fertility stage. 
     Production of Early Yield of Products 
     In some embodiments, the compositions, systems, and methods may be used to produce early yield of the product. For example, flowering process may be modulated, e.g., by mutating flowering repressor gene such as SP5G. Examples of such approaches include those described in Soyk S, et al., Nat Genet. 2017 January; 49(1):162-168. 
     Oil and Biofuel Production 
     The compositions, systems, and methods may be used to generate plants for oil and biofuel production. Biofuels include fuels made from plant and plant-derived resources. Biofuels may be extracted from organic matter whose energy has been obtained through a process of carbon fixation or are made through the use or conversion of biomass. This biomass can be used directly for biofuels or can be converted to convenient energy containing substances by thermal conversion, chemical conversion, and biochemical conversion. This biomass conversion can result in fuel in solid, liquid, or gas form. Biofuels include bioethanol and biodiesel. Bioethanol can be produced by the sugar fermentation process of cellulose (starch), which may be derived from maize and sugar cane. Biodiesel can be produced from oil crops such as rapeseed, palm, and soybean. Biofuels can be used for transportation. 
     Generation of Plants for Production of Vegetable Oils and Biofuels 
     The compositions, systems, and methods may be used to generate algae (e.g., diatom) and other plants (e.g., grapes) that express or overexpress high levels of oil or biofuels. 
     In some cases, the compositions, systems, and methods may be used to modify genes involved in the modification of the quantity of lipids and/or the quality of the lipids. Examples of such genes include those involved in the pathways of fatty acid synthesis, e.g., acetyl-CoA carboxylase, fatty acid synthase, 3-ketoacyl acyl-carrier protein synthase III, glycerol-3-phospate dehydrogenase (G3PDH), Enoyl-acyl carrier protein reductase (Enoyl-ACP-reductase), glycerol-3-phosphate acyltransferase, lysophosphatidic acyl transferase or diacylglycerol acyltransferase, phospholipid: diacylglycerol acyltransferase, phoshatidate phosphatase, fatty acid thioesterase such as palmitoyi protein thioesterase, or malic enzyme activities. 
     In further embodiments it is envisaged to generate diatoms that have increased lipid accumulation. This can be achieved by targeting genes that decrease lipid catabolization. Examples of genes include those involved in the activation of triacylglycerol and free fatty acids, β-oxidation of fatty acids, such as genes of acyl-CoA synthetase, 3-ketoacyl-CoA thiolase, acyl-CoA oxidase activity and phosphoglucomutase. 
     In some examples, algae may be modified for production of oil and biofuels, including fatty acids (e.g., fatty esters such as acid methyl esters (FAME) and fatty acid ethyl esters (FAEE)). Examples of methods of modifying microalgae include those described in Stovicek et al. Metab. Eng. Comm., 2015; 2:1; U.S. Pat. No. 8,945,839; and WO 2015086795. 
     In some examples, one or more genes may be introduced (e.g., overexpressed) to the plants (e.g., algae) to produce oils and biofuels (e.g., fatty acids) from a carbon source (e.g., alcohol). Examples of the genes include genes encoding acyl-CoA synthases, ester synthases, thioesterases (e.g., tesA, ‘tesA, tesB, fatB, fatB2, fatB3, fatA1, or fatA), acyl-CoA synthases (e.g., fadD, JadK, BH3103, pfl-4354, EAV15023, fadD1, fadD2, RPC_4074, fadDD35, fadDD22, faa39), ester synthases (e.g., synthase/acyl-CoA:diacylglycerl acyltransferase from  Simmondsia chinensis, Acinetobacter  sp. ADP,  Alcanivorax borkumensis, Pseudomonas aeruginosa, Fundibacter jadensis, Arabidopsis thaliana , or  Alkaligenes eutrophus , or variants thereof). 
     Additionally or alternatively, one or more genes in the plants (e.g., algae) may be inactivated (e.g., expression of the genes is decreased). For examples, one or more mutations may be introduced to the genes. Examples of such genes include genes encoding acyl-CoA dehydrogenases (e.g., fade), outer membrane protein receptors, and transcriptional regulator (e.g., repressor) of fatty acid biosynthesis (e.g., fabR), pyruvate formate lyases (e.g., pflB), lactate dehydrogenases (e.g., IdhA). 
     Organic Acid Production 
     In some embodiments, plants may be modified to produce organic acids such as lactic acid. The plants may produce organic acids using sugars, pentose or hexose sugars. To this end, one or more genes may be introduced (e.g., and overexpressed) in the plants. An example of such genes include LDH gene. 
     In some examples, one or more genes may be inactivated (e.g., expression of the genes is decreased). For examples, one or more mutations may be introduced to the genes. The genes may include those encoding proteins involved an endogenous metabolic pathway which produces a metabolite other than the organic acid of interest and/or wherein the endogenous metabolic pathway consumes the organic acid. 
     Examples of genes that can be modified or introduced include those encoding pyruvate decarboxylases (pdc), fumarate reductases, alcohol dehydrogenases (adh), acetaldehyde dehydrogenases, phosphoenolpyruvate carboxylases (ppc), D-lactate dehydrogenases (d-ldh), L-lactate dehydrogenases (l-ldh), lactate 2-monooxygenases, lactate dehydrogenase, cytochrome-dependent lactate dehydrogenases (e.g., cytochrome B2-dependent L-lactate dehydrogenases). 
     Enhancing Plant Properties for Biofuel Production 
     In some embodiments, the compositions, systems, and methods are used to alter the properties of the cell wall of plants to facilitate access by key hydrolyzing agents for a more efficient release of sugars for fermentation. By reducing the proportion of lignin in a plant the proportion of cellulose can be increased. In particular embodiments, lignin biosynthesis may be downregulated in the plant so as to increase fermentable carbohydrates. 
     In some examples, one or more lignin biosynthesis genes may be down regulated. Examples of such genes include 4-coumarate 3-hydroxylases (C3H), phenylalanine ammonia-lyases (PAL), cinnamate 4-hydroxylases (C4H), hydroxycinnamoyl transferases (HCT), caffeic acid O-methyltransferases (COMT), caffeoyl CoA 3-O-methyltransferases (CCoAOMT), ferulate 5-hydroxylases (F5H), cinnamyl alcohol dehydrogenases (CAD), cinnamoyl CoA-reductases (CCR), 4-coumarate-CoA ligases (4CL), monolignol-lignin-specific glycosyltransferases, and aldehyde dehydrogenases (ALDH), and those described in WO 2008064289. 
     In some examples, plant mass that produces lower level of acetic acid during fermentation may be reduced. To this end, genes involved in polysaccharide acetylation (e.g., Cas1L and those described in WO 2010096488) may be inactivated. 
     Other Microorganisms for Oils and Biofuel Production 
     In some embodiments, microorganisms other than plants may be used for production of oils and biofuels using the compositions, systems, and methods herein. Examples of the microorganisms include those of the genus of  Escherichia, Bacillus, Lactobacillus, Rhodococcus, Synechococcus, Synechoystis, Pseudomonas, Aspergillus, Trichoderma, Neurospora, Fusarium, Humicola, Rhizomucor, Kluyveromyces, Pichia, Mucor, Myceliophtora, Penicillium, Phanerochaete, Pleurotus, Trametes, Chrysosporium, Saccharomyces, Stenotrophamonas, Schizosaccharomyces, Yarrowia , or  Streptomyces.    
     Plant Cultures and Regeneration 
     In some embodiments, the modified plants or plant cells may be cultured to regenerate a whole plant which possesses the transformed or modified genotype and thus the desired phenotype. Examples of regeneration techniques include those relying on manipulation of certain phytohormones in a tissue culture growth medium, relying on a biocide and/or herbicide marker which has been introduced together with the desired nucleotide sequences, obtaining from cultured protoplasts, plant callus, explants, organs, pollens, embryos or parts thereof. 
     Detecting Modifications in the Plant Genome-Selectable Markers 
     When the compositions, systems, and methods are used to modify a plant, suitable methods may be used to confirm and detect the modification made in the plant. In some examples, when a variety of modifications are made, one or more desired modifications or traits resulting from the modifications may be selected and detected. The detection and confirmation may be performed by biochemical and molecular biology techniques such as Southern analysis, PCR, Northern blot, Si RNase protection, primer-extension or reverse transcriptase-PCR, enzymatic assays, ribozyme activity, gel electrophoresis, Western blot, immunoprecipitation, enzyme-linked immunoassays, in situ hybridization, enzyme staining, and immunostaining. 
     In some cases, one or more markers, such as selectable and detectable markers, may be introduced to the plants. Such markers may be used for selecting, monitoring, isolating cells and plants with desired modifications and traits. A selectable marker can confer positive or negative selection and is conditional or non-conditional on the presence of external substrates. Examples of such markers include genes and proteins that confer resistance to antibiotics, such as hygromycin (hpt) and kanamycin (nptII), and genes that confer resistance to herbicides, such as phosphinothricin (bar) and chlorosulfuron (als), enzyme capable of producing or processing a colored substances (e.g., the β-glucuronidase, luciferase, B or Cl genes). 
     Applications in Fungi 
     The compositions, systems, and methods described herein can be used to perform efficient and cost effective gene or genome interrogation or editing or manipulation in fungi or fungal cells, such as yeast. The approaches and applications in plants may be applied to fungi as well. 
     A fungal cell may be any type of eukaryotic cell within the kingdom of fungi, such as phyla of Ascomycota, Basidiomycota, Blastocladiomycota, Chytridiomycota, Glomeromycota, Microsporidia, and Neocallimastigomycota. Examples of fungi or fungal cells in include yeasts, molds, and filamentous fungi. 
     In some embodiments, the fungal cell is a yeast cell. A yeast cell refers to any fungal cell within the phyla Ascomycota and Basidiomycota. Examples of yeasts include budding yeast, fission yeast, and mold,  S. cerevisiae, Kluyveromyces marxianus, Issatchenkia orientalis, Candida  spp. (e.g.,  Candida albicans ),  Yarrowia  spp. (e.g.,  Yarrowia lipolytica ),  Pichia  spp. (e.g.,  Pichia pastoris ),  Kluyveromyces  spp. (e.g.,  Kluyveromyces lactis  and  Kluyveromyces marxianus ),  Neurospora  spp. (e.g.,  Neurospora crassa ),  Fusarium  spp. (e.g.,  Fusarium oxysporum ), and  Issatchenkia  spp. (e.g.,  Issatchenkia orientalis, Pichia kudriavzevii  and  Candida acidothermophilum ). 
     In some embodiments, the fungal cell is a filamentous fungal cell, which grow in filaments, e.g., hyphae or mycelia. Examples of filamentous fungal cells include  Aspergillus  spp. (e.g.,  Aspergillus niger ),  Trichoderma  spp. (e.g.,  Trichoderma reesei ),  Rhizopus  spp. (e.g.,  Rhizopus oryzae ), and  Mortierella  spp. (e.g.,  Mortierella isabellina ). 
     In some embodiments, the fungal cell is of an industrial strain. Industrial strains include any strain of fungal cell used in or isolated from an industrial process, e.g., production of a product on a commercial or industrial scale. Industrial strain may refer to a fungal species that is typically used in an industrial process, or it may refer to an isolate of a fungal species that may be also used for non-industrial purposes (e.g., laboratory research). Examples of industrial processes include fermentation (e.g., in production of food or beverage products), distillation, biofuel production, production of a compound, and production of a polypeptide. Examples of industrial strains include, without limitation, JAY270 and ATCC4124. 
     In some embodiments, the fungal cell is a polyploid cell whose genome is present in more than one copy. Polyploid cells include cells naturally found in a polyploid state, and cells that has been induced to exist in a polyploid state (e.g., through specific regulation, alteration, inactivation, activation, or modification of meiosis, cytokinesis, or DNA replication). A polyploid cell may be a cell whose entire genome is polyploid, or a cell that is polyploid in a particular genomic locus of interest. In some examples, the abundance of guide RNA may more often be a rate-limiting component in genome engineering of polyploid cells than in haploid cells, and thus the methods using the CRISPR system described herein may take advantage of using certain fungal cell types. 
     In some embodiments, the fungal cell is a diploid cell, whose genome is present in two copies. Diploid cells include cells naturally found in a diploid state, and cells that have been induced to exist in a diploid state (e.g., through specific regulation, alteration, inactivation, activation, or modification of meiosis, cytokinesis, or DNA replication). A diploid cell may refer to a cell whose entire genome is diploid, or it may refer to a cell that is diploid in a particular genomic locus of interest. 
     In some embodiments, the fungal cell is a haploid cell, whose genome is present in one copy. Haploid cells include cells naturally found in a haploid state, or cells that have been induced to exist in a haploid state (e.g., through specific regulation, alteration, inactivation, activation, or modification of meiosis, cytokinesis, or DNA replication). A haploid cell may refer to a cell whose entire genome is haploid, or it may refer to a cell that is haploid in a particular genomic locus of interest. 
     The compositions and systems, and nucleic acid encoding thereof may be introduced to fungi cells using the delivery systems and methods herein. Examples of delivery systems include lithium acetate treatment, bombardment, electroporation, and those described in Kawai et al., 2010, Bioeng Bugs. 2010 November-December; 1(6): 395-403. 
     In some examples, a yeast expression vector (e.g., those with one or more regulatory elements) may be used. Examples of such vectors include a centromeric (CEN) sequence, an autonomous replication sequence (ARS), a promoter, such as an RNA Polymerase III promoter, operably linked to a sequence or gene of interest, a terminator such as an RNA polymerase III terminator, an origin of replication, and a marker gene (e.g., auxotrophic, antibiotic, or other selectable markers). Examples of expression vectors for use in yeast may include plasmids, yeast artificial chromosomes, 2μ plasmids, yeast integrative plasmids, yeast replicative plasmids, shuttle vectors, and episomal plasmids. 
     Biofuel and Materials Production by Fungi 
     In some embodiments, the compositions, systems, and methods may be used for generating modified fungi for biofuel and material productions. For instance, the modified fungi for production of biofuel or biopolymers from fermentable sugars and optionally to be able to degrade plant-derived lignocellulose derived from agricultural waste as a source of fermentable sugars. Foreign genes required for biofuel production and synthesis may be introduced in to fungi In some examples, the genes may encode enzymes involved in the conversion of pyruvate to ethanol or another product of interest, degrade cellulose (e.g., cellulase), endogenous metabolic pathways which compete with the biofuel production pathway. 
     In some examples, the compositions, systems, and methods may be used for generating and/or selecting yeast strains with improved xylose or cellobiose utilization, isoprenoid biosynthesis, and/or lactic acid production. One or more genes involved in the metabolism and synthesis of these compounds may be modified and/or introduced to yeast cells. Examples of the methods and genes include lactate dehydrogenase, PDC1 and PDC5, and those described in Ha, S. J., et al. (2011) Proc. Natl. Acad. Sci. USA 108(2):504-9 and Galazka, J. M., et al. (2010) Science 330(6000):84-6; Jakočiūnas T et al., Metab Eng. 2015 March; 28:213-222; Stovicek V, et al., FEMS Yeast Res. 2017 Aug. 1; 17(5). 
     Improved Plants and Yeast Cells 
     The present disclosure further provides improved plants and fungi. The improved and fungi may comprise one or more genes introduced, and/or one or more genes modified by the compositions, systems, and methods herein. The improved plants and fungi may have increased food or feed production (e.g., higher protein, carbohydrate, nutrient or vitamin levels), oil and biofuel production (e.g., methanol, ethanol), tolerance to pests, herbicides, drought, low or high temperatures, excessive water, etc. 
     The plants or fungi may have one or more parts that are improved, e.g., leaves, stems, roots, tubers, seeds, endosperm, ovule, and pollen. The parts may be viable, nonviable, regeneratable, and/or non-regeneratable. 
     The improved plants and fungi may include gametes, seeds, embryos, either zygotic or somatic, progeny and/or hybrids of improved plants and fungi. The progeny may be a clone of the produced plant or fungi, or may result from sexual reproduction by crossing with other individuals of the same species to introgress further desirable traits into their offspring. The cell may be in vivo or ex vivo in the cases of multicellular organisms, particularly plants. 
     Further Applications of the CRISPR-Cas System in Plants 
     Further applications of the compositions, systems, and methods on plants and fungi include visualization of genetic element dynamics (e.g., as described in Chen B, et al., Cell. 2013 Dec. 19; 155(7):1479-91), targeted gene disruption positive-selection in vitro and in vivo (as described in Malina A et al., Genes Dev. 2013 Dec. 1; 27(23):2602-14), epigenetic modification such as using fusion of Cas and histone-modifying enzymes (e.g., as described in Rusk N, Nat Methods. 2014 January; 11(1):28), identifying transcription regulators (e.g., as described in Waldrip Z J, Epigenetics. 2014 September; 9(9):1207-11), anti-virus treatment for both RNA and DNA viruses (e.g., as described in Price A A, et al., Proc Natl Acad Sci USA. 2015 May 12; 112(19):6164-9; Ramanan V et al., Sci Rep. 2015 Jun. 2; 5:10833), alteration of genome complexity such as chromosome numbers (e.g., as described in Karimi-Ashtiyani R et al., Proc Natl Acad Sci USA. 2015 Sep. 8; 112(36):11211-6; Anton T, et al., Nucleus. 2014 March-April; 5(2):163-72), self-cleavage of the CRISPR system for controlled inactivation/activation (e.g., as described Sugano S S et al., Plant Cell Physiol. 2014 March; 55(3):475-81), multiplexed gene editing (as described in Kabadi A M et al., Nucleic Acids Res. 2014 Oct. 29; 42(19):e147), development of kits for multiplex genome editing (as described in Xing H L et al., BMC Plant Biol. 2014 Nov. 29; 14:327), starch production (as described in Hebelstrup K H et al., Front Plant Sci. 2015 Apr. 23; 6:247), targeting multiple genes in a family or pathway (e.g., as described in Ma X et al., Mol Plant. 2015 August; 8(8):1274-84), regulation of non-coding genes and sequences (e.g., as described in Lowder L G, et al., Plant Physiol. 2015 October; 169(2):971-85), editing genes in trees (e.g., as described in Belhaj K et al., Plant Methods. 2013 Oct. 11; 9(1):39; Harrison M M, et al., Genes Dev. 2014 Sep. 1; 28(17):1859-72; Zhou X et al., New Phytol. 2015 October; 208(2):298-301), introduction of mutations for resistance to host-specific pathogens and pests. 
     Additional examples of modifications of plants and fungi that may be performed using the compositions, systems, and methods include those described in International Patent Publication Nos. WO2016/099887, WO2016/025131, WO2016/073433, WO2017/066175, WO2017/100158, WO 2017/105991, WO2017/106414, WO2016/100272, WO2016/100571, WO 2016/100568, WO 2016/100562, and WO 2017/019867. 
     Applications in Non-Human Animals 
     The compositions, systems, and methods may be used to study and modify non-human animals, e.g., introducing desirable traits and disease resilience, treating diseases, facilitating breeding, etc. In some embodiments, the compositions, systems, and methods may be used to improve breeding and introducing desired traits, e.g., increasing the frequency of trait-associated alleles, introgression of alleles from other breeds/species without linkage drag, and creation of de novo favorable alleles. Genes and other genetic elements that can be targeted may be screened and identified. Examples of application and approaches include those described in Tait-Burkard C, et al., Livestock 2.0—genome editing for fitter, healthier, and more productive farmed animals. Genome Biol. 2018 Nov. 26; 19(1):204; Lillico S, Agricultural applications of genome editing in farmed animals. Transgenic Res. 2019 August; 28(Suppl 2):57-60; Houston R D, et al., Harnessing genomics to fast-track genetic improvement in aquaculture. Nat Rev Genet. 2020 Apr. 16. doi: 10.1038/s41576-020-0227-y, which are incorporated herein by reference in their entireties. Applications described in other sections such as therapeutic, diagnostic, etc. can also be used on the animals herein. 
     The compositions, systems, and methods may be used on animals such as fish, amphibians, reptiles, mammals, and birds. The animals may be farm and agriculture animals, or pets. Examples of farm and agriculture animals include horses, goats, sheep, swine, cattle, llamas, alpacas, and birds, e.g., chickens, turkeys, ducks, and geese. The animals may be a non-human primate, e.g., baboons, capuchin monkeys, chimpanzees, lemurs, macaques, marmosets, tamarins, spider monkeys, squirrel monkeys, and vervet monkeys. Examples of pets include dogs, cats horses, wolfs, rabbits, ferrets, gerbils, hamsters, chinchillas, fancy rats, guinea pigs, canaries, parakeets, and parrots. 
     In some embodiments, one or more genes may be introduced (e.g., overexpressed) in the animals to obtain or enhance one or more desired traits. Growth hormones, insulin-like growth factors (IGF-1) may be introduced to increase the growth of the animals, e.g., pigs or salmon (such as described in Pursel V G et al., J Reprod Fertil Suppl. 1990; 40:235-45; Waltz E, Nature. 2017; 548:148). Fat-1 gene (e.g., from  C elegans ) may be introduced for production of larger ratio of n-3 to n-6 fatty acids may be induced, e.g. in pigs (such as described in Li M, et al., Genetics. 2018; 8:1747-54). Phytase (e.g., from  E coli ) xylanase (e.g., from  Aspergillus niger ), beta-glucanase (e.g., from  Bacillus licheniformis ) may be introduced to reduce the environmental impact through phosphorous and nitrogen release reduction, e.g. in pigs (such as described in Golovan S P, et al., Nat Biotechnol. 2001; 19:741-5; Zhang X et al., elife. 2018). shRNA decoy may be introduced to induce avian influenza resilience e.g. in chicken (such as described in Lyall et al., Science. 2011; 331:223-6). Lysozyme or lysostaphin may be introduced to induce mastitis resilience e.g., in goat and cow (such as described in Maga E A et al., Foodborne Pathog Dis. 2006; 3:384-92; Wall R J, et al., Nat Biotechnol. 2005; 23:445-51). Histone deacetylase such as HDAC6 may be introduced to induce PRRSV resilience, e.g., in pig (such as described in Lu T., et al., PLoS One. 2017; 12:e0169317). CD163 may be modified (e.g., inactivated or removed) to introduce PRRSV resilience in pigs (such as described in Prather R S et al., Sci Rep. 2017 Oct. 17; 7(1):13371). Similar approaches may be used to inhibit or remove viruses and bacteria (e.g., Swine Influenza Virus (SIV) strains which include influenza C and the subtypes of influenza A known as H1N1, H1N2, H2N1, H3N1, H3N2, and H2N3, as well as pneumonia, meningitis and oedema) that may be transmitted from animals to humans. 
     In some embodiments, one or more genes may be modified or edited for disease resistance and production traits. Myostatin (e.g., GDF8) may be modified to increase muscle growth, e.g., in cow, sheep, goat, catfish, and pig (such as described in Crispo M et al., PLoS One. 2015; 10:e0136690; Wang X, et al., Anim Genet. 2018; 49:43-51; Khalil K, et al., Sci Rep. 2017; 7:7301; Kang J-D, et al., RSC Adv. 2017; 7:12541-9). Pc POLLED may be modified to induce horlessness, e.g., in cow (such as described in Carlson D F et al., Nat Biotechnol. 2016; 34:479-81). KISS1R may be modified to induce boretaint (hormone release during sexual maturity leading to undesired meat taste), e.g., in pigs. Dead end protein (dnd) may be modified to induce sterility, e.g., in salmon (such as described in Wargelius A, et al., Sci Rep. 2016; 6:21284). Nano2 and DDX may be modified to induce sterility (e.g., in surrogate hosts), e.g., in pigs and chicken (such as described Park K-E, et al., Sci Rep. 2017; 7:40176; Taylor L et al., Development. 2017; 144:928-34). CD163 may be modified to induce PRRSV resistance, e.g., in pigs (such as described in Whitworth K M, et al., Nat Biotechnol. 2015; 34:20-2) RELA may be modified to induce ASFV resilience, e.g., in pigs (such as described in Lillico S G, et al., Sci Rep. 2016; 6:21645). CD18 may be modified to induce Mannheimia ( Pasteurella )  haemolytica  resilience, e.g., in cows (such as described in Shanthalingam S, et al., roc Natl Acad Sci USA. 2016; 113:13186-90). NRAMP1 may be modified to induce tuberculosis resilience, e.g., in cows (such as described in Gao Y et al., Genome Biol. 2017; 18:13). Endogenous retrovirus genes may be modified or removed for xenotransplantation such as described in Yang L, et al. Science. 2015; 350:1101-4; Niu D et al., Science. 2017; 357:1303-7). Negative regulators of muscle mass (e.g., Myostatin) may be modified (e.g., inactivated) to increase muscle mass, e.g., in dogs (as described in Zou Q et al., J Mol Cell Biol. 2015 December; 7(6):580-3). 
     Animals such as pigs with severe combined immunodeficiency (SCID) may generated (e.g., by modifying RAG2) to provide useful models for regenerative medicine, xenotransplantation (discussed also elsewhere herein), and tumor development. Examples of methods and approaches include those described Lee K, et al., Proc Natl Acad Sci USA. 2014 May 20; 111(20):7260-5; and Schomberg et al. FASEB Journal, April 2016; 30(1): Suppl 571.1. 
     SNPs in the animals may be modified. Examples of methods and approaches include those described Tan W. et al., Proc Natl Acad Sci USA. 2013 Oct. 8; 110(41):16526-31; Mali P, et al., Science. 2013 Feb. 15; 339(6121):823-6. 
     Stem cells (e.g., induced pluripotent stem cells) may be modified and differentiated into desired progeny cells, e.g., as described in Heo Y T et al., Stem Cells Dev. 2015 Feb. 1; 24(3):393-402. 
     Profile analysis (such as Igenity) may be performed on animals to screen and identify genetic variations related to economic traits. The genetic variations may be modified to introduce or improve the traits, such as carcass composition, carcass quality, maternal and reproductive traits and average daily gain. 
     Further embodiments are illustrated in the following Examples which are given for illustrative purposes only and are not intended to limit the scope of the invention. 
     EXAMPLES 
     Now having described the embodiments of the present disclosure, in general, the following Examples describe some additional embodiments of the present disclosure. While embodiments of the present disclosure are described in connection with the following examples and the corresponding text and figures, there is no intent to limit embodiments of the present disclosure to this description. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of embodiments of the present disclosure. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ° C., and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20° C. and 1 atmosphere. 
     Example 1 
     Exemplary and informative polynucleotide sequences are included herein and can be found in SEQ ID NOS: 57-108 present in the sequence listing and as set forth in Appendices A-K of U.S. Provisional Patent Application No. 62/850,494, filed on May 20, 2019 entitled “Non-Class I Multi-Component Nucleic Acid Targeting Systems,” which are incorporated by reference as if expressed in their entireties herein. The tables below (Tables 14-23) set forth various features associated with one or more of SEQ ID NOS: 57-108. Sequence analysis and annotation was completed using Geneious Software. 
     
       
         
           
               
             
               
                 TABLE 14 
               
             
            
               
                   
               
               
                 Features relevant to one or more of SEQ ID NOs: 57-61 (Locus 
               
               
                 25_14_17 Orgainized) See also Appendix A of U.S. Provisional 
               
               
                 Patent Application No. 62/850,494. 
               
            
           
           
               
               
               
            
               
                 Feature Key 
                   
                 Sequence(s) 
               
               
                 ID/ 
                   
                 of reference 
               
               
                 Annotation 
                 Feature Location/Qualifiers 
                 or note 
               
               
                   
               
               
                 Misc_feature 
                 /Original Bases=“SEQ ID NO: 57” 
                 SEQ ID NO: 57 
               
               
                   
                 /label=“SEQ ID NO: 57” 
                   
               
               
                 CDS 
                 complement(&lt;1..7) 
                   
               
               
                   
                 /product=“CHAT domain-containing 
                   
               
               
                   
                 protein &amp; pfam 12770” 
                   
               
               
                   
                 /label=“CHAT domain-containing 
                   
               
               
                   
                 protein &amp; pfam 12770 CDS” 
                   
               
               
                   
               
               
                 CDS 
                 complement(7.. 192) 
                   
               
               
                   
                 /product=“hypothetical protein &amp; Hypo-rule applied” 
                   
               
               
                   
                 /label=“hypothetical protein &amp; Hypo-rule applied CDS” 
                   
               
               
                   
               
               
                 DR_5′ 
                 complement(94..104) 
                 SEQ ID NO: 58 
               
               
                   
                 /Mismatches=2 
                   
               
               
                   
                 /%_Identity=“81.81818181818181” 
                   
               
               
                   
                 /Motif=“GTTGCAGTGAG” (SEQ ID NO. 58) 
                   
               
               
                   
                 /annotation group=“DR: 21,808 &lt;- 21.818” 
                   
               
               
                   
                 label=“DR” 
                   
               
               
                   
               
               
                 mismatch 
                 102.. 103 
                   
               
               
                   
                 /Motif=“GTTGCAGTGAG” 
                   
               
               
                   
                 /annotation_group=“DR: 21,808 &lt;- 21.818” 
                   
               
               
                   
                 /label=“TT 
                   
               
               
                   
               
               
                 CDS 
                 589.903 
                   
               
               
                   
                 /product=“hypothetical protein &amp; Hypo-rule applied” 
                   
               
               
                   
                 /label=“hypothetical protein &amp; Hypo-rule applied CDS” 
                   
               
               
                   
               
               
                 DR_5′ 
                 complement(959..969) 
                 SEQ ID NO: 58 
               
               
                   
                 /Mismatches=2 
                   
               
               
                   
                 /%_Identity=“81.81818181818181” 
                   
               
               
                   
                 /Motif=“GTTGCAGTGAG” SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=“DR: 22,673 &lt;- 22,683” 
                   
               
               
                   
                 /label=“DR” 
                   
               
               
                   
               
               
                 mismatch 
                 964 
                   
               
               
                   
                 /Motif=“GTTGCAGTGAG” SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=“DR: 22,673 &lt;- 22.683” 
                   
               
               
                   
                 /label=“A” 
                   
               
               
                   
               
               
                 P2 
                 complement(966..1475) 
                   
               
               
                   
                 /product=“Helix-turn-helix domain- 
                   
               
               
                   
                 containing protein/Helix-turn-helix &amp; 
                   
               
               
                   
                 pfam13560,pfam01381” 
                   
               
               
                   
                 /label=“Helix-tum-helix domain-containing 
                   
               
               
                   
                 protein/Helix-turn-helix &amp; 
                   
               
               
                   
                 pfam13560,pfam01381 CDS” 
                   
               
               
                   
               
               
                 mismatch 
                 967 
                   
               
               
                   
                 /Motif=“GTTGCAGTGAG” SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=“DR: 22,673 &lt;- 22,683” 
                   
               
               
                   
                 /label=“T” 
                   
               
               
                   
               
               
                 Edit 
                 1476..&gt;6780 
                   
               
               
                   
                 /created_by=“altacth” 
                   
               
               
                   
                 /label=“From_14_organized” 
                   
               
               
                   
               
               
                 −35_Box 
                 1498..1503 
                   
               
               
                   
                 /created_by=“al taeth” 
                   
               
               
                   
               
               
                 −10_Box 
                 1519..1527 
                   
               
               
                   
                 /created_by=“altaeth” 
                   
               
               
                   
                 /label=“-10_Box” 
                   
               
               
                   
               
               
                 P1 
                 1755..3230 
                   
               
               
                   
                 /product=“-&gt; cas9(229,269)[32.8]: 
                   
               
               
                   
                 5-methylcytosine-specific restriction 
                   
               
               
                   
                 endonuclease McrA &amp; COG1403” 
                   
               
               
                   
                 /modified_by=“altaeth” 
                   
               
               
                   
                 /label=“IscB (Inactive RuvC)” 
                   
               
               
                   
               
               
                 −10_Box 
                 3282..3290 
                   
               
               
                   
                 /created_by=“altaeth” 
                   
               
               
                   
               
               
                 POI 
                 3286..6780 
                   
               
               
                   
                 /product=“-&gt; KOON_Cas14u(970.1075) 
                   
               
               
                   
                 [26.0]: hypothetical 
                   
               
               
                   
                 protein &amp; Hypo-rule applied” 
                   
               
               
                   
                 /modified by=“altaeth” 
                   
               
               
                   
                 /label=“VII cC” 
                   
               
               
                   
               
               
                 DR_5′ 
                 complement(3480..3490) 
                 SEQ ID NO: 58 
               
               
                   
                 /Mismatehes=1 
                   
               
               
                   
                 /%_Identity=“90.9090909090909” 
                   
               
               
                   
                 /Motif=“GTTGCAGTGAG” SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=“DR: 25,194 &lt;- 25,204” 
                   
               
               
                   
                 /label=“DR” 
                   
               
               
                   
               
               
                 mismatch 
                 480 
                 SEQ ID NO: 58 
               
               
                   
                 /Motif=“GTTGCAGTGAG” SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=“DR: 25,194 &lt;- 25,204” 
                   
               
               
                   
                 /label=“G” 
                   
               
               
                 Edit 
                 complement(6781..&gt;8311) 
                   
               
               
                   
                 /created_by=“altaeth” 
                   
               
               
                   
                 /label=“From_17_organized” 
                   
               
               
                 DR 
                 complement(7462..7498) 
                   
               
               
                   
                 /product=1 
                   
               
               
                   
                 /label=“1 DR” 
                   
               
               
                 DR_5′ 
                 7463..7473 
                 SEQ ID NO: 58 
               
               
                   
                 /Mismatches=2 
                   
               
               
                   
                 /%_Identity=“81.81818181818181” 
                   
               
               
                   
                 /Motif=“GTTGCAGTGAG” SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=“DR: 28,214 -&gt; 28,224” 
                   
               
               
                   
                 /label=“DR” 
                   
               
               
                   
               
               
                 mismatch 
                 7472..7473 
                 SEQ ID NO: 58 
               
               
                   
                 /Motif=“GTTGCAGTGAG” SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=“DR: 28,214 -&gt; 28,224” 
                   
               
               
                   
                 /label=“AG” 
                   
               
               
                 DR 
                 complement(7535..7571) 
                   
               
               
                   
                 /product=1 
                   
               
               
                   
                 /label=“1 DR” 
                   
               
               
                   
               
               
                 DR_5′ 
                 7536..7546 
                 SEQ ID NO: 58 
               
               
                   
                 /Mismatches=2 
                   
               
               
                   
                 /%_Identity=“81.81818181818181” 
                   
               
               
                   
                 /Motif=“GTTGCAGTGAG” SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=“DR: 28.287 -&gt; 28.297” 
                   
               
               
                   
                 /label=“DR” 
                   
               
               
                   
               
               
                 mismatch 
                 7545..7546 
                 SEQ ID NO: 58 
               
               
                   
                 /Motif=“GTTGCAGTGAG” SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=“DR: 28,287 -&gt; 28,297” 
                   
               
               
                   
                 /label=“AG” 
                   
               
               
                   
               
               
                 Misc_feeature 
                 7606{circumflex over ( )}7607 
                 SEQ ID NO: 59 
               
               
                   
                 /Original_Bases=“SEQ ID NO: 59” 
                   
               
               
                   
                 /label=“SEQ ID NO: 59” 
                   
               
               
                 DR 
                 complement(7756..7774) 
                   
               
               
                   
                 /product=0 
                   
               
               
                   
               
               
                 DR_5′ 
                 7756..7766 
                 SEQ ID NO: 58 
               
               
                   
                 /Mismatches=2 
                   
               
               
                   
                 /%_Identity=“81.81818181818181” 
                   
               
               
                   
                 /Motif=“GTTGCAGTGAG” SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=“DR: 28,730 -&gt; 28,740” 
                   
               
               
                   
                 /label=“DR” 
                   
               
               
                   
               
               
                 mismatch 
                 7765..7766 
                 SEQ ID NO: 58 
               
               
                   
                 /Motif=“GTTGCAGTGAG” SEQ ID NO: 58 
                   
               
               
                   
                 /annotation group=“DR: 28,730 -&gt; 28.740” 
                   
               
               
                   
                 /label=“AG” 
                   
               
               
                   
               
               
                 Misc_feature 
                 /Original_Bases=“SEQ ID NO: 60” 
                 SEQ ID NO: 60 
               
               
                   
                 /label=“SEQ ID NO: 60” 
                   
               
               
                   
               
               
                 ORIGIN 
                   
                 SEQ ID NO: 61 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 15 
               
             
            
               
                   
               
               
                 Features relevant to one or more of SEQ ID NOs: 62-63 (Locus 12_Orgainized) 
               
               
                 See also Appendix A of U.S. Provisional Patent Application No. 62/850,494. 
               
            
           
           
               
               
               
            
               
                 Feature Key 
                   
                 Sequcnce(s) of 
               
               
                 ID/Annotation 
                 Feature Location/Qualifiers 
                 reference or note 
               
               
                   
               
               
                 Misc_feature 
                   
                   
               
               
                 P2 
                 complement(3..371) 
                   
               
               
                   
                 /product=“Transcriptional regulator, 
                   
               
               
                   
                 contains XRE-family 
                   
               
               
                   
                 HTH domain &amp; COG1396” 
                   
               
               
                   
               
               
                 P1 
                 630.. 2120 
                   
               
               
                   
                 /product=“-&gt; cas9(236,274)[29.9]: 
                   
               
               
                   
                 5-methylcytosine-spccific restriction 
                   
               
               
                   
                 endonuclease McrA &amp; COG1403” 
                   
               
               
                   
                 /modified_by=“altaeth” 
                   
               
               
                   
                 /label=“IscB (Inactive RuvC)” 
                   
               
               
                   
               
               
                 POI 
                 2133.4628 
                   
               
               
                   
                 /product=“-&gt; KOON_Cas14u(646.752)[32.2]: hypothetical 
                   
               
               
                   
                 /protein &amp; Hypo-rule applied”/modified by=“altaeth” 
                   
               
               
                   
                 /label=“VII cC” 
                   
               
               
                   
               
               
                 −35_Box 
                 2191..2196 
                   
               
               
                   
                 /created_by=“altaeth” 
                   
               
               
                   
               
               
                 −10_Box 
                 2213..2221 
                   
               
               
                   
                 /created_by=“altaeth” 
                   
               
               
                   
               
               
                 DR 
                 5031..5067 
                   
               
               
                   
                 /product=0 
                   
               
               
                   
               
               
                 DR_5′ 
                 5031..5041 
                 SEQ ID NO: 58 
               
               
                   
                 /Mismatches=2 
                   
               
               
                   
                 /%_Identity=“81.81818181818181” 
                   
               
               
                   
                 /Motif=“GTTGCAGTGAG” SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=“DR: 27,898 -&gt; 27,908” 
                   
               
               
                   
                 /label=“DR” 
                   
               
               
                   
               
               
                 mismatch 
                 5040..5041 
                 SEQ ID NO: 58 
               
               
                   
                 /Motif=“GTTGCAGTGAG” SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=“DR: 27,898 -&gt; 27.908” 
                   
               
               
                   
                 /label=“AG” 
                   
               
               
                   
               
               
                 DR 
                 5105..5141 
                   
               
               
                   
                 /product=0 
                   
               
               
                   
               
               
                 DR_5′ 
                 5105..5115 
                 SEQ ID NO: 58 
               
               
                   
                 /Mismatches=2 
                   
               
               
                   
                 /%_Identity=“81.81818181818181” 
                   
               
               
                   
                 /Motif=“GTTGCAGTGAG” SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=“DR: 27,972 -&gt; 27,982” 
                   
               
               
                   
                 /label=“DR” 
                   
               
               
                   
               
               
                 Mismatch 
                 5114.5115 
                 SEQ ID NO: 58 
               
               
                   
                 /Motif=“GTTGCAGTGAG” SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=“DR: 27,972 -&gt; 27,982” 
                   
               
               
                   
                 /label=“AG” 
                   
               
               
                   
               
               
                 Misc_feature 
                 5177A5178 
                 SEQ ID NO:62 
               
               
                   
                 /Original_Bases=“SEQ ID NO: 62” 
                   
               
               
                   
                 /label=“SEQ ID NO: 62” 
                   
               
               
                   
                 /note=“Geneious_type: Editing History Deletion” 
                   
               
               
                   
               
               
                 DR 
                 5178..5214 
                   
               
               
                   
                 /product=0 
                   
               
               
                   
               
               
                 DR_5′ 
                 5178..5188 
                 SEQ ID NO: 58 
               
               
                   
                 /Mismatches=2 
                   
               
               
                   
                 /%_Identity =“81.81818181818181” 
                   
               
               
                   
                 /Motif=“GTTGCAGTGAG” SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=“DR: 29,142 -&gt; 29,152” 
                   
               
               
                   
                 /label=“DR” 
                   
               
               
                 mismatch 
                 5187..5188 
                 SEQ ID NO: 58 
               
               
                   
                 /Motif=“GTTGCAGTGAG” SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=“DR: 29,142 -&gt; 29,152” 
                   
               
               
                   
                 /label=“AG” 
                   
               
               
                   
               
               
                 DR 
                 5252..5288 
                   
               
               
                   
                 /product=3 
                   
               
               
                   
               
               
                 DR_5′ 
                 5252..5262 
                   
               
               
                   
                 /Mismatches=2 
                   
               
               
                   
                 /%_Identity=“81.81818181818181” 
                   
               
               
                   
                 /Motif=“GTTGCAGTGAG” 
                   
               
               
                   
                 /annotation_group=“DR: 29,216 -&gt; 29.226” 
                   
               
               
                   
                 /label=“DR” 
                   
               
               
                   
               
               
                 mismatch 
                 5261..5262 
                   
               
               
                   
                 /Motif=“GTTGCAGTGAG” 
                   
               
               
                   
                 /annotation_group=“DR: 29,216 -&gt; 29.226” 
                   
               
               
                   
                 /label=“AG” 
                   
               
               
                   
               
               
                 ORIGIN 
                 SEQ ID NO: 63 
                 SEQ ID NO: 63 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 16 
               
             
            
               
                   
               
               
                 Features relevant to one or more of SEQ ID NOs: 64-66 (Locus 
               
               
                 12_20inf Orgainized) See also Appendix A of U.S. Provisional Patent Application No. 
               
               
                 62/850,494. 
               
            
           
           
               
               
               
            
               
                 Feature Key 
                   
                 Sequence(s) of 
               
               
                 ID/Annotation 
                 Feature Location/Qualifiers 
                 reference or note 
               
               
                   
               
               
                 Edit 
                 1..729 
                   
               
               
                   
                 /created_by=″altaeth″ 
                   
               
               
                   
                 /modified_by=″altaeth″ 
                   
               
               
                   
                 /label=″Inferred From 20_organized with 88% Homology″ 
                   
               
               
                   
               
               
                 Misc_feature 
                   
                   
               
               
                 P2 
                 complement(630..1100) 
                   
               
               
                   
                 /product=″Transcriptional regulator, contains XRE-family 
                   
               
               
                   
                 HTH domain &amp; COG1396″ 
                   
               
               
                   
                 /label=″Transcriptional regulator, contains XRE-family HTH 
                   
               
               
                   
                 domain &amp; C0G1396 CDS″ 
                   
               
               
                   
               
               
                 P1 
                 1359..2849 
                   
               
               
                   
                 /product=″-&gt;cas9(236,274) 
                   
               
               
                   
                 5-methylcytosine-specific restriction endonuclease McrA &amp; 
                   
               
               
                   
                 COG1403″ 
                   
               
               
                   
                 /modified_by=″altaeth″ 
                   
               
               
                   
                 /label=″IscB (Inactive RuvC)″ 
                   
               
               
                   
               
               
                 POI 
                 2862..5357 
                   
               
               
                   
                 /product=″-&gt;KOON_Cas14u(646,752)[32.2]: hypothetical 
                   
               
               
                   
                 protein &amp; Hypo-rule applied″ 
                   
               
               
                   
                 /modified_by=″altaeth″ 
                   
               
               
                   
                 /label=″VII cC″ 
                   
               
               
                   
               
               
                 -35_Box 
                 2920..2925 
                   
               
               
                   
                 /created_by=″altaeth″ 
                   
               
               
                   
                 /label=″-35 Box″ 
                   
               
               
                   
               
               
                 -10_Box 
                 2942..2950 
                   
               
               
                   
                 /created_by=″altaeth″ 
                   
               
               
                   
                 /label=″-10 Box″ 
                   
               
               
                   
               
               
                 DR 
                 5760..5796 
                   
               
               
                   
                 /product=0 
                   
               
               
                   
                 /label=″0 DR″ 
                   
               
               
                   
               
               
                 DR_5′ 
                 5760..5770 
                 SEQ ID O: 58 
               
               
                   
                 /Mismatches=2 
                   
               
               
                   
                 /%_Identity=″81.81818181818181″ 
                   
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID O: 58 
                   
               
               
                   
                 /annotation_group=″DR: 27,898-&gt;27,908″ 
                   
               
               
                   
                 /label=″DR″ 
                   
               
               
                   
               
               
                 mismatch 
                 5769..5770 
                   
               
               
                   
                 /Motif=″GTTGCAGTGAG″ 
                   
               
               
                   
                 /annotation_group=″DR: 27,898-&gt;27,908″ 
                   
               
               
                   
                 /label=″AG″ 
                   
               
               
                   
               
               
                 DR 
                 5834..5870 
                   
               
               
                   
                 /product=0 
                   
               
               
                   
                 /label=″0 DR″ 
                   
               
               
                   
               
               
                 DR_5′ 
                 5834..5844 
                   
               
               
                   
                 /Mismatches=2 
                   
               
               
                   
                 /%_Identity=″81.81818181818181″ 
                   
               
               
                   
                 /Motif=″GTTGCAGTGAG″ 
                   
               
               
                   
                 /annotation_group=″DR: 27,972-&gt;27,982″ 
                   
               
               
                   
                 /label=″DR″ 
                   
               
               
                   
               
               
                 mismatch 
                 5843..5844 
                   
               
               
                   
                 /Motif=″GTTGCAGTGAG″ 
                   
               
               
                   
                 /annotation_group=″DR: 27,972-&gt;27,982″ 
                   
               
               
                   
                 /label=″AG″ 
                   
               
               
                   
               
               
                 Misc_feature 
                 5906{circumflex over ( )}5907 
                   
               
               
                   
                 /Original_Bases=″SEQ ID NO: 64″ 
                   
               
               
                   
                 /label=″SEQ ID NO: 64″ 
                   
               
               
                   
                 /note=″Geneious type: Editing History Deletion″ 
                   
               
               
                   
               
               
                 DR 
                 5907..5943 
                   
               
               
                   
                 /product=0 
                   
               
               
                   
                 /label=″0 DR″ 
                   
               
               
                   
               
               
                 dR_5′ 
                 5907..5917 
                 SEQ ID NO: 58 
               
               
                   
                 /Mismatches=2 
                   
               
               
                   
                 /%_Identity=″81.81818181818181″ 
                   
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=″DR: 29,142-&gt;29,152″ 
                   
               
               
                   
                 /label=″DR″ 
                   
               
               
                   
               
               
                 mismatch 
                 5916..5917 
                 SEQ ID NO: 58 
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=″DR: 29,142-&gt;29,152″ 
                   
               
               
                   
                 /label=″AG″ 
                   
               
               
                   
               
               
                 DR 
                 5981..6017 
                   
               
               
                   
                 /product=3 
                   
               
               
                   
                 /label=″3 DR″ 
                   
               
               
                   
               
               
                 DR_5′ 
                 5981..5991 
                   
               
               
                   
                 /Mismatches=2 
                   
               
               
                   
                 /%_Identity=″81.81818181818181″ 
                   
               
               
                   
                 /Motif=″GTTGCAGTGAG″ 
                   
               
               
                   
                 /annotation_group=″DR: 29,216-&gt;29,226″ 
                   
               
               
                   
                 /label=″DR″ 
                   
               
               
                   
               
               
                 mismatch 
                 5990..5991 
                 SEQ ID NO: 58 
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=″DR: 29,216-&gt;29,226″ 
                   
               
               
                   
                 /label=″AG″ 
                   
               
               
                   
               
               
                 CDS 
                 complement(6107..+226109) 
                   
               
               
                   
                 /created_by=″altaeth″ 
                   
               
               
                   
                 /label=″Protein of unknown function (DUF2493) &amp; pfam10686 
                   
               
               
                   
                 CDS″ 
                   
               
               
                   
               
               
                 Misc_feature 
                 /Original_Bases=″SEQ ID NO: 65″ 
                 SEQ ID NO: 65 
               
               
                   
                 /label=″SEQ ID NO: 65″ 
                   
               
               
                   
                 /note=″Geneious type: Editing History Deletion″ 
                   
               
               
                   
               
               
                 ORIGIN 
                 SEQ ID NO: 66 
                 SEQ ID NO: 66 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 17 
               
             
            
               
                   
               
               
                 Features relevant to one or more of SEQ ID NOs: 67-68 (Locus 15_Orgainized) 
               
               
                 See also Appendix A of U.S. Provisional Patent Application No. 62/850,494. 
               
            
           
           
               
               
               
            
               
                 Feature Key 
                   
                 Sequence(s) of 
               
               
                 ID/Annotation 
                 Feature Location/Qualifiers 
                 reference or note 
               
               
                   
               
               
                 Misc_feature 
                   
                   
               
               
                 CDS 
                 2..439 
                   
               
               
                   
                 /product=″hypothetical protein &amp; Hypo-rule applied″ 
                   
               
               
                   
               
               
                 CDS 
                 457..636 
                   
               
               
                   
                 /product=″hypothetical protein &amp; Hypo-rule applied″ 
                   
               
               
                   
               
               
                 CDS 
                 777..1058 
                   
               
               
                   
                 /product=″hypothetical protein &amp; Hypo-rule applied″ 
                   
               
               
                   
               
               
                 DR_5′ 
                 1061..1071 
                 SEQ ID NO: 58 
               
               
                   
                 /Mismatches=2 
                   
               
               
                   
                 /%_Identity=″81.81818181818181″ 
                   
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=″DR: 22,182-&gt;22,192″ 
                   
               
               
                   
                 /label=″DR″ 
                   
               
               
                   
               
               
                 mismatch 
                 1068 
                 SEQ ID NO: 58 
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=″DR: 22,182-&gt;22,192″ 
                   
               
               
                   
                 /label=″T″ 
                   
               
               
                   
               
               
                 mismatch 
                 1070 
                 SEQ ID NO: 58 
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=″DR: 22,182-&gt;22,192″ 
                   
               
               
                   
                 /label=″A″ 
                   
               
               
                   
               
               
                 CDS 
                 complement(1216..1542) 
                   
               
               
                   
                 /product=″hypothetical protein &amp; Hypo-rule applied″ 
                   
               
               
                   
               
               
                 P2 
                 complement(1616..2074) 
                   
               
               
                   
                 /product=″Transcriptional regulator, contains XRE-family 
                   
               
               
                   
                 HTH domain/Transcriptional regulator, contains XRE-family 
                   
               
               
                   
                 HTH domain &amp; COG1396, COG1396″ 
                   
               
               
                   
               
               
                 CDS 
                 2140..2283 
                   
               
               
                   
                 /product=″hypothetical protein &amp; Hypo-rule applied″ 
                   
               
               
                   
               
               
                 P1 
                 2344..3861 
                   
               
               
                   
                 /product=″-&gt;cas9(240,276)[32.3]: ORF @ 2343-3861″ 
                   
               
               
                   
                 /modified_by=″altaeth″ 
                   
               
               
                   
                 /label=″IscB (Inactive RuvC)″ 
                   
               
               
                   
               
               
                 POI 
                 3879..6368 
                   
               
               
                   
                 /product=″ORF @ 3878-6368″ 
                   
               
               
                   
                 /modified_by=″altaeth″ 
                   
               
               
                   
                 /label=″VII cC″ 
                   
               
               
                   
               
               
                 DR_5′ 
                 4614..4624 
                 SEQ ID NO: 58 
               
               
                   
                 /Mismatches=2 
                   
               
               
                   
                 /%_Identity=″81.81818181818181″ 
                   
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=″DR: 25,735-&gt;25,745″ 
                   
               
               
                   
                 /label=″DR″ 
                   
               
               
                   
               
               
                 mismatch 
                 4615 
                   
               
               
                   
                 /Motif=″GTTGCAGTGAG″ 
                   
               
               
                   
                 /annotation_group=″DR: 25,735-&gt;25,745″ 
                   
               
               
                   
                 /label=″T″ 
                   
               
               
                   
               
               
                 mismatch 
                 4623 
                   
               
               
                   
                 /Motif=″GTTGCAGTGAG″ 
                   
               
               
                   
                 /annotation_group=″DR: 25,735-&gt;25,745″ 
                   
               
               
                   
                 /label=″A″ 
                   
               
               
                   
               
               
                 DR_5′ 
                 complement(5147..5157) 
                 SEQ ID NO: 58 
               
               
                   
                 /Mismatches=2 
                   
               
               
                   
                 /%_Identity=″81.81818181818181″ 
                   
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=″DR: 26,268&lt;-26,278″ 
                   
               
               
                   
                 /label=″DR″ 
                   
               
               
                   
               
               
                 mismatch 
                 5151 
                 SEQ ID NO: 58 
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=″DR: 26,268&lt;-26,278″ 
                   
               
               
                   
                 /label=″G″ 
                   
               
               
                   
               
               
                 mismatch 
                 5155 
                 SEQ ID NO: 58 
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=″DR: 26,268&lt;-26,278″ 
                   
               
               
                   
                 /label=″T″ 
                   
               
               
                   
               
               
                 DR_5′ 
                 6364..6374 
                 SEQ ID NO: 58 
               
               
                   
                 /Mismatches=2 
                   
               
               
                   
                 /%_Identity=″81.81818181818181″ 
                   
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=″DR: 27,485-&gt;27,495″ 
                   
               
               
                   
                 /label=″DR″ 
                   
               
               
                   
               
               
                 mismatch 
                 6368..6369 
                 SEQ ID NO: 58 
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=″DR: 27,485-&gt;27,495″ 
                   
               
               
                   
                 /label=″CA″ 
                   
               
               
                   
               
               
                 DR 
                 6820..6856 
                   
               
               
                   
                 product=0 
                   
               
               
                   
               
               
                 DR_5′ 
                 6820..6830 
                 SEQ ID NO: 58 
               
               
                   
                 /Mismatches=2 
                   
               
               
                   
                 /%_Identity=″81.81818181818181″ 
                   
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=″DR: 27,941-&gt;27,951″ 
                   
               
               
                   
                 /label=″DR″ 
                   
               
               
                   
               
               
                 mismatch 
                 6829..6830 
                 SEQ ID NO: 58 
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=″DR: 27,941-&gt;27,951″ 
                   
               
               
                   
                 /label=″AG″ 
                   
               
               
                   
               
               
                 DR 
                 6893..6929 
                   
               
               
                   
                 /product=0 
                   
               
               
                   
               
               
                 DR_5′ 
                 6893..6903 
                 SEQ ID NO: 58 
               
               
                   
                 /Mismatches=2 
                   
               
               
                   
                 /%_Identity=″81.81818181818181″ 
                   
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=″DR: 28,014-&gt;28,024″ 
                   
               
               
                   
                 /label=″DR″ 
                   
               
               
                   
               
               
                 mismatch 
                 6902..6903 
                   
               
               
                   
                 /Motif=″GTTGCAGTGAG″ 
                   
               
               
                   
                 /annotation_group=″DR: 28,014-&gt;28,024″ 
                   
               
               
                   
                 /label=″AG″ 
                   
               
               
                   
               
               
                 Misc_feature 
                 6964{circumflex over ( )}6965 
                 SEQ ID NO: 67 
               
               
                   
                 /Original_Bases=″SEQ ID NO: 67″ 
                   
               
               
                   
                 /label=″SEQ ID NO: 67″ 
                   
               
               
                   
                 /note=″Geneious type: Editing History Deletion″ 
                   
               
               
                   
               
               
                 DR 
                 6965..7001 
                   
               
               
                   
                 /product=0 
                   
               
               
                   
               
               
                 DR_5′ 
                 6965..6975 
                 SEQ ID NO: 58 
               
               
                   
                 /Mismatches=2 
                   
               
               
                   
                 /%_Identity=″81.81818181818181″ 
                   
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=″DR: 28,304-&gt;28,314″ 
                   
               
               
                   
                 /label=″DR″ 
                   
               
               
                   
               
               
                 mismatch 
                 6974..6975 
                 SEQ ID NO: 58 
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=″DR: 28,304-&gt;28,314″ 
                   
               
               
                   
                 /label=″AG″ 
                   
               
               
                   
               
               
                 DR 
                 7039..7075 
                   
               
               
                   
                 /product=0 
                   
               
               
                   
               
               
                 DR_5′ 
                 7039..7049 
                 SEQ ID NO: 58 
               
               
                   
                 /Mismatches=2 
                   
               
               
                   
                 /%_Identity=″81.81818181818181″ 
                   
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=″DR: 28,378-&gt;28,388″ 
                   
               
               
                   
                 /label=″DR″ 
                   
               
               
                   
               
               
                 mismatch 
                 7048..7049 
                 SEQ ID NO: 58 
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=″DR: 28,378-&gt;28,388″ 
                   
               
               
                   
                 /label=″AG″ 
                   
               
               
                   
               
               
                 CDS 
                 7579..7737 
                   
               
               
                   
                 /product=″hypothetical protein &amp; Hypo-rule applied″ 
                   
               
               
                   
               
               
                 ORIGIN 
                 SEQ ID NO: 68 
                 SEQ ID NO: 68 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 18 
               
             
            
               
                   
               
               
                 Features relevant to one or more of SEQ ID NOs: 69-72 (Locus 20_Orgainized) 
               
               
                 See also Appendix A of U.S. Provisional Patent Application No. 62/850,494. 
               
            
           
           
               
               
               
            
               
                 Feature Key 
                   
                 Sequence(s) of 
               
               
                 ID/Annotation 
                 Feature Location/Qualifiers 
                 reference or note 
               
               
                   
               
               
                 CDS 
                 complement(&lt;1..5) 
                   
               
               
                   
                 /product=″Superfamily II DNA and RNA helicase/Superfamily 
                   
               
               
                   
                 II DNA or RNA helicase &amp; COG0513, COG1061″ 
                   
               
               
                   
                 /label=″Superfamily II DNA and RNA helicase/Superfamily II 
                   
               
               
                   
                 DNA or RNA helicase &amp; COG0513, COG1061  CDS″ 
                   
               
               
                   
               
               
                 Misc_feature 
                 /Original_Bases=″SEQ ID NO: 69″ 
                 SEQ ID NO: 69 
               
               
                   
                 /label=″SEQ ID NO: 69″ 
                   
               
               
                   
                 /note=″Geneious type: Editing History Deletion″ 
                   
               
               
                   
               
               
                 P2 
                 complement(635..1105) 
                   
               
               
                   
                 /product=″Transcriptional regulator, contains XRE-family 
                   
               
               
                   
                 HTH domain/Transcriptional regulator, contains XRE-family 
                   
               
               
                   
                 HTH domain &amp; COG1396, COG1396″ 
                   
               
               
                   
                 /label=″Transcriptional regulator, contains XRE-family HTH 
                   
               
               
                   
                 domain/Transcriptional regulator, contains XRE-family HTH 
                   
               
               
                   
                 domain &amp; COG1396, COG1396 CDS″ 
                   
               
               
                   
               
               
                 P1 
                 1366..2853 
                   
               
               
                   
                 /product=″-&gt;cas9(234,271)[29.0]: ORF @ 144025-145513″ 
                   
               
               
                   
                 /modified_by=″altaeth″ 
                   
               
               
                   
                 /label=″IscB (Inactive RuvC)″ 
                   
               
               
                   
               
               
                 POI 
                 2958..5336 
                   
               
               
                   
                 /product=″-&gt;KOON_Cas14u(632,713)[28.5]: hypothetical 
                   
               
               
                   
                 protein &amp; Hypo-rule applied″ 
                   
               
               
                   
                 /modified_by=″altaeth″ 
                   
               
               
                   
                 /label=″VII cC″ 
                   
               
               
                   
               
               
                 DR 
                 complement(5745..5781) 
                   
               
               
                   
                 /product=0 
                   
               
               
                   
                 /label=″0 DR 
                   
               
               
                   
               
               
                 DR_5′ 
                 5745..5755 
                 SEQ ID NO: 58 
               
               
                   
                 /Mismatches=2 
                   
               
               
                   
                 /%_Identity=″81.81818181818181″ 
                   
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=″DR: 27,787-&gt;27,797″ 
                   
               
               
                   
                 /label=″DR″ 
                   
               
               
                   
               
               
                 Mismatch 
                 5754..5755 
                 SEQ ID NO: 58 
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=″DR: 27,787-&gt;27,797″ 
                   
               
               
                   
                 /label=″AG″ 
                   
               
               
                   
               
               
                 DR 
                 complement(5819..5855) 
                   
               
               
                   
                 /product=0 
                   
               
               
                   
                 /label=″0 DR″ 
                   
               
               
                   
               
               
                 DR_5′ 
                 5819..5829 
                 SEQ ID NO: 58 
               
               
                   
                 /Mismatches=2 
                   
               
               
                   
                 /%_Identity=″81.81818181818181″ 
                   
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=″DR: 27,861-&gt;27,871″ 
                   
               
               
                   
                 /label=″DR″ 
                   
               
               
                   
               
               
                 mismatch 
                 5828..5829 
                 SEQ ID NO: 58 
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=″DR: 27,861-&gt;27,871″ 
                   
               
               
                   
                 /label=″AG″ 
                   
               
               
                   
               
               
                 Misc_feature 
                 5892{circumflex over ( )}5893 
                   
               
               
                   
                 /Original_Bases=″SEQ ID NO: 70″ 
                   
               
               
                   
               
               
                 DR 
                 complement(5893..5929) 
                   
               
               
                   
                 /product=0 
                   
               
               
                   
                 /label=″0 DR″ 
                   
               
               
                   
               
               
                 DR_5′ 
                 5893..5903 
                 SEQ ID NO: 58 
               
               
                   
                 /Mismatches=2 
                   
               
               
                   
                 /%_Identity=″81.81818181818181″ 
                   
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=″DR: 28,156-&gt;28,166″ 
                   
               
               
                   
                 /label=″DR″ 
                   
               
               
                   
               
               
                 mismatch 
                 5902..5903 
                 SEQ ID NO: 58 
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=″DR: 28,156-&gt;28,166″ 
                   
               
               
                   
                 /label=″AG″ 
                   
               
               
                   
               
               
                 DR 
                 complement(5968..6004) 
                   
               
               
                   
                 /product=0 
                   
               
               
                   
                 /label=″0 DR″ 
                   
               
               
                   
               
               
                 DR_5′ 
                 5968..5978 
                 SEQ ID NO: 58 
               
               
                   
                 /Mismatches=2 
                   
               
               
                   
                 /%_Identity=″81.81818181818181″ 
                   
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=″DR: 28,231-&gt;28,241″ 
                   
               
               
                   
                 /label=″DR″ 
                   
               
               
                   
               
               
                 mismatch 
                 5977..5978 
                 SEQ ID NO: 58 
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=″DR: 28,231-&gt;28,241″ 
                   
               
               
                   
                 /label=″AG″ 
                   
               
               
                   
               
               
                 CDS 
                 complement(6550..&gt;6556) 
                   
               
               
                   
                 /product=″Integrase &amp; COG0582″ 
                   
               
               
                   
                 /codon_start=2 
                   
               
               
                   
                 /label=″Integrase &amp; COG0582 CDS″ 
                   
               
               
                   
               
               
                 Misc_feature 
                 /Original_Bases=″SEQ ID NO: 71″ 
                 SEQ ID NO: 71 
               
               
                   
                 /label=″ SEQ ID NO: 71″ 
                   
               
               
                   
                 /note=″Geneious type: Editing History Deletion″ 
                   
               
               
                   
               
               
                 ORIGIN 
                 SEQ ID NO: 72 
                 SEQ ID NO: 72 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 19 
               
             
            
               
                   
               
               
                 Features relevant to one or more of SEQ ID NOs: 73-74 (Locus 30_Orgainized) 
               
               
                 See also Appendix A of U.S. Provisional Patent Application No. 62/850,494. 
               
            
           
           
               
               
               
            
               
                 Feature Key 
                   
                 Sequence(s) of 
               
               
                 ID/Annotation 
                 Feature Location/Qualifiers 
                 reference or note 
               
               
                   
               
               
                 Padding 
                 /label 
                   
               
               
                   
               
               
                 Misc_feature 
                 /Original_Bases=″SEQ ID NO: 73″ 
                 SEQ ID NO: 73 
               
               
                   
                 /label=″SEQ ID NO: 73″ 
                   
               
               
                   
                 /note=″Geneious type: Editing History Deletion″ 
                   
               
               
                   
               
               
                 CDS 
                 &lt;1..21 
                   
               
               
                   
                 /product=″two-component system, NtrC family, response 
                   
               
               
                   
                 regulator PilR &amp; KO:K02667″ 
                   
               
               
                   
               
               
                 Questional_CDS 
                 187..849 
                   
               
               
                   
                 /product=″ORF @ 3976-4639″ 
                   
               
               
                   
               
               
                 CDS 
                 complement(905..1105) 
                   
               
               
                   
                 product=″hypothetical protein &amp; Hypo-rule applied″ 
                   
               
               
                   
               
               
                 P2 
                 complement(1479..1958) 
                   
               
               
                   
                 /product=″Transcriptional regulator, contains XRE-family 
                   
               
               
                   
                 HTH domain/Transcriptional regulator, contains XRE-family 
                   
               
               
                   
                 HTH domain &amp; COG1396, COG1396″ 
                   
               
               
                   
               
               
                 P2 
                 2130..2720 
                   
               
               
                   
                 /product=″hypothetical protein &amp; Hypo-rule applied″ 
                   
               
               
                   
                 /modified_by=″altaeth″ 
                   
               
               
                   
                 /label=″Helix Turn Helix DNA Binding Protein″ 
                   
               
               
                   
               
               
                 POI 
                 2778..5099 
                   
               
               
                   
                 /product=″hypothetical protein &amp; Hypo-rule applied″ 
                   
               
               
                   
                 /modified_by=″altaeth″ 
                   
               
               
                   
                 /label=″VII cC″ 
                   
               
               
                   
               
               
                 DR_5′ 
                 complement(2844..2854) 
                 SEQ ID NO: 58 
               
               
                   
                 /Mismatches=2 
                   
               
               
                   
                 /%_Identity=″81.81818181818181″ 
                   
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=″DR: 25,066&lt;-25,076″ 
                   
               
               
                   
                 /label=″DR″ 
                   
               
               
                   
               
               
                 mismatch 
                 2849 
                 SEQ ID NO: 58 
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=″DR: 25,066&lt;-25,076″ 
                   
               
               
                   
                 /label=″A″ 
                   
               
               
                   
               
               
                 mismatch 
                 2854 
                 SEQ ID NO: 58 
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=″DR: 25,066&lt;-25,076″ 
                   
               
               
                   
                 /label=″G″ 
                   
               
               
                   
               
               
                 DR 
                 5516..5554 
                   
               
               
                   
                 /product=6 
                   
               
               
                   
               
               
                 DR_5′ 
                 5516..5526 
                 SEQ ID NO: 58 
               
               
                   
                 /Mismatches=0 
                   
               
               
                   
                 /%_Identity=100 
                   
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=″DR: 27,738-&gt;27,748″ 
                   
               
               
                   
                 /label=″DR 
                   
               
               
                   
               
               
                 DR 
                 5589..5627 
                   
               
               
                   
                 /product=6 
                   
               
               
                   
               
               
                 CDS 
                 complement(6173..6421) 
                   
               
               
                   
                 /product=″hypothetical protein &amp; Hypo-rule applied″ 
                   
               
               
                   
               
               
                 ORIGIN 
                 SEQ ID NO: 74 
                 SEQ ID NO: 74 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 20 
               
             
            
               
                   
               
               
                 Features relevant to one or more of SEQ ID NOs: 75-77 (Locus 4_Orgainized) 
               
               
                 See also Appendix A of U.S. Provisional Patent Application No. 62/850,494. 
               
            
           
           
               
               
               
            
               
                 Feature Key 
                   
                 Sequence(s) of 
               
               
                 ID/Annotation 
                 Feature Location/Qualifiers 
                 reference or note 
               
               
                   
               
               
                 Padding 
                 /label 
                   
               
               
                   
               
               
                 Misc_feature 
                 /Original_Bases=″SEQ ID NO: 75″ 
                 SEQ ID NO: 75 
               
               
                   
                 /label=″SEQ ID NO: 75″ 
                   
               
               
                   
                 /note=″Geneious type: Editing History Deletion″ 
                   
               
               
                   
               
               
                 CDS 
                 complement(&lt;1..217) 
                   
               
               
                   
                 /product=″hypothetical protein″ 
                   
               
               
                   
                 /label=″hypothetical protein CDS″ 
                   
               
               
                   
               
               
                 CDS 
                 complement(&lt;1..2) 
                   
               
               
                   
                 /product=″SNF2 family DNA or RNA helicase &amp; COG0553″ 
                   
               
               
                   
                 /label=″SNF2 family DNA or RNA helicase &amp; COG0553 CDS″ 
                   
               
               
                   
               
               
                 Questional_CDS 
                 complement(806..1288) 
                   
               
               
                   
                 /product=″ORF @ 12757-13240″ 
                   
               
               
                   
                 /label=″ORF @ 12757-13240 CDS″ 
                   
               
               
                   
               
               
                 P2 
                 complement(1182..1646) 
                   
               
               
                   
                 /product= transcriptional regulator with XRE-family HTH 
                   
               
               
                   
                 domain/transcriptional regulator with XRE-family HTH 
                   
               
               
                   
                 domain &amp; COG1396, COG1396″ 
                   
               
               
                   
                 /label=″transcriptional regulator with XRE-family HTH 
                   
               
               
                   
                 domain/transcriptional regulator with XRE-family HTH 
                   
               
               
                   
                 domain &amp; COG1396, COG1396 CDS″ 
                   
               
               
                   
               
               
                 P1 
                 657..3417 
                   
               
               
                   
                 /product=″ORF @ 13608-15369″ 
                   
               
               
                   
                 /modified_by=″altaeth″ 
                   
               
               
                   
                 /label=″IscB (Inactive RuvC)″ 
                   
               
               
                   
               
               
                 -35_Box 
                 1669..1674 
                   
               
               
                   
                 /created_by=″altaeth″ 
                   
               
               
                   
                 /label=″-35 Box″ 
                   
               
               
                   
               
               
                 -10_Box 
                 1690..1698 
                   
               
               
                   
                 /created_by=″altaeth″ 
                   
               
               
                   
                 /label=″-10 Box″ 
                   
               
               
                   
               
               
                 unsure 
                 1903..1905 
                   
               
               
                   
                 /created_by=″altaeth″ 
                   
               
               
                   
                 /label=″Possible Start″ 
                   
               
               
                   
               
               
                 DR_5′ 
                 3304..3314 
                 SEQ ID NO: 58 
               
               
                   
                 /Mismatches=2 
                   
               
               
                   
                 /%_Identity=″81.81818181818181″ 
                   
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=″DR: 24,894-&gt;24,904″ 
                   
               
               
                   
                 /label=″DR″ 
                   
               
               
                   
               
               
                 POI 
                 3410..5878 
                   
               
               
                   
                 /product=″-&gt;KOON_Cas14u(646,752)[30.1]: hypothetical 
                   
               
               
                   
                 protein″ 
                   
               
               
                   
                 /modified_by=″altaeth″ 
                   
               
               
                   
                 /label=″VII cC″ 
                   
               
               
                   
               
               
                 DR 
                 6306..6327 
                   
               
               
                   
                 /product=0 
                   
               
               
                   
                 /label=″0 DR″ 
                   
               
               
                   
               
               
                 DR_5′ 
                 6306..6316 
                 SEQ ID NO: 58 
               
               
                   
                 /Mismatches=2 
                   
               
               
                   
                 /%_Identity=″81.81818181818181″ 
                   
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=″DR: 27,896-&gt;27,906″ 
                   
               
               
                   
                 /label=″DR″ 
                   
               
               
                   
               
               
                 DR 
                 6380..6401 
                   
               
               
                   
                 /product=0 
                   
               
               
                   
                 /label=″0 DR″ 
                   
               
               
                   
               
               
                 DR_5′ 
                 6380..6390 
                 SEQ ID NO: 58 
               
               
                   
                 /Mismatches=2 
                   
               
               
                   
                 /%Identity=″81.81818181818181″ 
                   
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=″DR: 27,970-&gt;27,980″ 
                   
               
               
                   
                 /label=″DR″ 
                   
               
               
                   
               
               
                 DR 
                 6452..6473 
                   
               
               
                   
                 /product=0 
                   
               
               
                   
                 /label=″0 DR″ 
                   
               
               
                   
               
               
                 DR_5′ 
                 6452..6462 
                 SEQ ID NO: 58 
               
               
                   
                 /Mismatches=2 
                   
               
               
                   
                 /%_Identity=″81.81818181818181″ 
                   
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=″DR: 28,042-&gt;28,052″ 
                   
               
               
                   
                 /label=″DR″ 
                   
               
               
                   
               
               
                 CDS 
                 complement(6510..&gt;6521) 
                   
               
               
                   
                 /product=″predicted KAP-like P-loop ATPase/predicted 
                   
               
               
                   
                 KAP-like P-loop ATPase &amp; COG4928, COG4928″ 
                   
               
               
                   
                 /label=″predicted KAP-like P-loop ATPase/predicted 
                   
               
               
                   
                 KAP-like P-loop ATPase &amp; COG4928, COG4928 CDS″ 
                   
               
               
                   
               
               
                 Misc_feature 
                 /Original_Bases=″SEQ ID NO: 76″ 
                 SEQ ID NO: 76 
               
               
                   
                 /label=″SEQ ID NO: 76″ 
                   
               
               
                   
                 /note=″Geneious type: Editing History Deletion″ 
                   
               
               
                   
               
               
                 ORIGIN 
                 SEQ ID NO: 77 
                 SEQ ID NO” 77 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 21 
               
             
            
               
                   
               
               
                 Features relevant to one or more of SEQ ID NOs: 78-83 (Locus 
               
               
                 Chloroflexales_bacterium_ZM16-3_NODE_109_(reversed) See also Appendix A of U.S. 
               
               
                 Provisional Patent Application No. 62/850,494. 
               
            
           
           
               
               
               
            
               
                 Feature Key 
                   
                 Sequence(s) of 
               
               
                 ID/Annotation 
                 Feature Location/Qualifiers 
                 reference or note 
               
               
                   
               
               
                 Misc_feature 
                 /Original Bases=″SEQ ID NO: 78″ 
                 SEQ ID NO: 78 
               
               
                   
                 /label=″SEQ ID NO: 78″ 
                   
               
               
                   
                 /note=″Geneious type: Editing History Deletion″ 
                   
               
               
                   
               
               
                 P2 
                 complement(1280..1789) 
                   
               
               
                   
                 /created_by=″altaeth″ 
                   
               
               
                   
                 /modified_by=″altaeth″ 
                   
               
               
                   
                 /label=″XRE″ 
                   
               
               
                   
               
               
                 -35 Box 
                 1812..1817 
                   
               
               
                   
                 /created_by=″altaeth″ 
                   
               
               
                   
                 /modified_by=″altaeth″ 
                   
               
               
                   
                 /label=″-35 Box″ 
                   
               
               
                   
               
               
                 -10_Box 
                 1833..1841 
                   
               
               
                   
                 /created_by=″altaeth″ 
                   
               
               
                   
                 /modified_by=″altaeth″ 
                   
               
               
                   
                 /label=″-10 Box″ 
                   
               
               
                   
               
               
                 P1 
                 2048..3544 
                   
               
               
                   
                 /created_by=″altaeth″ 
                   
               
               
                   
                 /modified_by=″altaeth″ 
                   
               
               
                   
                 /label=″IscB (Inactive RuvC)″ 
                   
               
               
                   
               
               
                 POI 
                 3541..7101 
                   
               
               
                   
                 /created_by=″altaeth″ 
                   
               
               
                   
                 /modified_by=″altaeth″ 
                   
               
               
                   
                 /label=″VII cC″ 
                   
               
               
                   
               
               
                 DR_5′ 
                 complement(3795..3805) 
                 SEQ ID NO: 58 
               
               
                   
                 /Mismatches=1 
                   
               
               
                   
                 /%_Identity=″90.9090909090909″ 
                   
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=″DR: 25,254&lt;-25,264″ 
                   
               
               
                   
                 /label=″DR″ 
                   
               
               
                   
               
               
                 DR 
                 7791..7827 
                 SEQ ID NO: 80 
               
               
                   
                 /Mismatches=14 
                   
               
               
                   
                 /%_Identity=″62.16216216216216″ 
                   
               
               
                   
                 /Motif=″GTTGCAGTGGTATCAGCGCGCCAGAGGGCAGTGGAAG″ 
                   
               
               
                   
                 SEQ ID NO: 80 
                   
               
               
                   
                 /annotation_group=″DR: 30,743-&gt;30,779″ 
                   
               
               
                   
                 /label=″DR″ 
                   
               
               
                   
               
               
                 DR_Sample 
                 7791..7827 
                 SEQ ID NO: 81 
               
               
                   
                 /Mismatches=0 
                   
               
               
                   
                 /%_Identity=100 
                   
               
               
                   
                 /Motif=″GTTGCAGTGGTCGAAATCGTGTAGCGGCCAATGGAGG″ 
                   
               
               
                   
                 SEQ ID NO: 81 
                   
               
               
                   
                 /annotation_group=″DR: 7,791-&gt;7,827″ 
                   
               
               
                   
                 /label=″DR″ 
                   
               
               
                   
               
               
                 DR_5′ 
                 7791..7801 
                 SEQ ID NO: 58 
               
               
                   
                 /Mismatches=2 
                   
               
               
                   
                 /%_Identity=″81.81818181818181″ 
                   
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=″DR: 29,250-&gt;29,260″ 
                   
               
               
                   
                 /label=″DR″ 
                   
               
               
                   
               
               
                 DR 
                 7865..7901 
                 SEQ ID NO: 80 
               
               
                   
                 /Mismatches=14 
                   
               
               
                   
                 /%_Identity=″62.16216216216216″ 
                   
               
               
                   
                 /Motif=″GTTGCAGTGGTATCAGCGCGCCAGAGGGCAGTGGAAG″ 
                   
               
               
                   
                 SEQ ID NO: 80 
                   
               
               
                   
                 /annotation_group=″DR: 30,817-&gt;30,853″ 
                   
               
               
                   
                 /label=″DR″ 
                   
               
               
                   
               
               
                 DR_Sample 
                 7865..7901 
                 SEQ ID NO: 81 
               
               
                   
                 /Mismatches=0 
                   
               
               
                   
                 /%_Identity=100 
                   
               
               
                   
                 /Motif=″GTTGCAGTGGTCGAAATCGTGTAGCGGCCAATGGAGG″ 
                   
               
               
                   
                 SEQ ID NO: 81 
                   
               
               
                   
                 /annotation_group=″DR: 7,865-&gt;7,901″ 
                   
               
               
                   
                 /label=″DR″ 
                   
               
               
                   
               
               
                 DR_5′ 
                 7865..7875 
                 SEQ ID NO: 58 
               
               
                   
                 /Mismatches=2 
                   
               
               
                   
                 /%_Identity=″81.81818181818181″ 
                   
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=″DR: 29,324-&gt;29,334″ 
                   
               
               
                   
                 /label=″DR″ 
                   
               
               
                   
               
               
                 Misc_feature 
                 7939{circumflex over ( )}7940 
                 SEQ ID NO: 79 
               
               
                   
                 /Original_Bases=″SEQ ID NO: 79″ 
                   
               
               
                   
                 /label=″SEQ ID NO: 79″ 
                   
               
               
                   
                 /note=″Geneious type: Editing History Deletion″ 
                   
               
               
                   
               
               
                 DR 
                 7940..7976 
                 SEQ ID NO: 80 
               
               
                   
                 /Mismatches=14 
                   
               
               
                   
                 /%_Identity=″62.16216216216216″ 
                   
               
               
                   
                 /Motif=″GTTGCAGTGGTATCAGCGCGCCAGAGGGCAGTGGAAG″ 
                   
               
               
                   
                 SEQ ID NO: 80 
                   
               
               
                   
                 /annotation_group=″DR: 31,405-&gt;31,441″ 
                   
               
               
                   
                 /label=″DR″ 
                   
               
               
                   
               
               
                 DR_sample 
                 7940..7976 
                 SEQ ID NO: 81 
               
               
                   
                 /Mismatches=0 
                   
               
               
                   
                 /%_Identity=100 
                   
               
               
                   
                 /Motif=″GTTGCAGTGGTCGAAATCGTGTAGCGGCCAATGGAGG″ 
                   
               
               
                   
                 SEQ ID NO: 81 
                   
               
               
                   
                 /annotation_group=″DR: 8,453-&gt;8,489″ 
                   
               
               
                   
                 /label=″DR″ 
                   
               
               
                   
               
               
                 DR_5′ 
                 7940..7950 
                 SEQ ID NO: 58 
               
               
                   
                 /Mismatches=2 
                   
               
               
                   
                 /%_Identity=″81.81818181818181″ 
                   
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=″DR: 29,912-&gt;29,922″ 
                   
               
               
                   
                 /label=″DR″ 
                   
               
               
                   
               
               
                 DR 
                 8014..8050 
                 SEQ ID NO: 80 
               
               
                   
                 /Mismatches=14 
                   
               
               
                   
                 /%_Identity=″62.16216216216216″ 
                   
               
               
                   
                 /Motif=″GTTGCAGTGGTATCAGCGCGCCAGAGGGCAGTGGAAG″ 
                   
               
               
                   
                 SEQ ID NO: 80 
                   
               
               
                   
                 /annotation_group=″DR: 31,479-&gt;31,515″ 
                   
               
               
                   
                 /label=″DR 
                   
               
               
                   
               
               
                 DR_Sample 
                 8014..8050 
                 SEQ ID NO: 81 
               
               
                   
                 /Mismatches=0 
                   
               
               
                   
                 /%_Identity=100 
                   
               
               
                   
                 /Motif=″GTTGCAGTGGTCGAAATCGTGTAGCGGCCAATGGAGG″ 
                   
               
               
                   
                 SEQ ID NO: 81 
                   
               
               
                   
                 /annotation_group=″DR: 8,527-&gt;8,563″ 
                   
               
               
                   
                 /label=″DR″ 
                   
               
               
                   
               
               
                 DR_5′ 
                 8014..8024 
                 SEQ ID NO: 58 
               
               
                   
                 /Mismatches=2 
                   
               
               
                   
                 /%_Identity=″81.81818181818181″ 
                   
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=″DR: 29,986-&gt;29,996″ 
                   
               
               
                   
                 /label=″DR″ 
                   
               
               
                   
               
               
                 DR_5′ 
                 8086..8096 
                 SEQ ID NO: 58 
               
               
                   
                 /Mismatches=2 
                   
               
               
                   
                 /%_Identity=″81.81818181818181″  
                   
               
               
                   
                 /Motif=″GTTGCAGTGAG″ SEQ ID NO: 58 
                   
               
               
                   
                 /annotation_group=″DR: 30,058-&gt;30,068″ 
                   
               
               
                   
                 /label=″DR″ 
                   
               
               
                   
               
               
                 Misc_feature 
                 /Original_Bases=″SEQ ID NO: 82″ 
                 SEQ ID NO: 82 
               
               
                   
                 /label=″SEQ ID NO: 82″ 
                   
               
               
                   
                 /note=″Geneious type: Editing History Deletion″ 
                   
               
               
                   
               
               
                 ORIGIN 
                 SEQ ID NO: 83 
                 SEQ ID NO: 83 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 22 
               
             
            
               
                   
               
               
                 Features relevant to one or more of SEQ ID NOs: 88-94 (Locus 
               
               
                 VII_cA1_contig_for_synthesis) See also Appendix C of U.S. Provisional Patent 
               
               
                 Application No. 62/850,494. 
               
            
           
           
               
               
               
            
               
                 Feature Key 
                   
                 Sequence(s) of 
               
               
                 ID/Annotation 
                 Feature Location/Qualifiers 
                 reference or note 
               
               
                   
               
               
                 Padding 
                 /label 
                   
               
               
                   
               
               
                 CDS 
                 &lt;1..37 
                   
               
               
                   
                 /product=″Ribonucleotide reductase, alpha subunit &amp; 
                   
               
               
                   
                 COG0209″ 
                   
               
               
                   
                 /codon_start=2 
                   
               
               
                   
                 /label=″Ribonucleotide reductase, alpha subunit &amp; COG0209 
                   
               
               
                   
                 CDS″ 
                   
               
               
                   
               
               
                 Misc_feature 
                 /Original_Bases=″SEQ ID NO: 88″ 
                 SEQ ID NO: 88 
               
               
                   
                 /label=″SEQ ID NO: 88″ 
                   
               
               
                   
                 /note=″Geneious type: Editing History Deletion″ 
                   
               
               
                   
               
               
                 Tracr_Put 
                 382..396 
                 SEQ ID NO: 89 
               
               
                   
                 /Mismatches=4 
                   
               
               
                   
                 /%_Identity=″73.33333333333333″ 
                   
               
               
                   
                 /Motif=″GTTTCATTCCCAGGG″ SEQ ID NO: 89 
                   
               
               
                   
                 /annotation_group=″DR: 24,153-&gt;24,167″ 
                   
               
               
                   
                 /label=″Half DR″ 
                   
               
               
                   
               
               
                 mismatch 
                 382 
                 SEQ ID NO: 89 
               
               
                   
                 /Motif=″GTTTCATTCCCAGGG″ SEQ ID NO: 89 
                   
               
               
                   
                 /annotation_group=″DR: 24,153-&gt;24,167″ 
                   
               
               
                   
                 /label=″G″ 
                   
               
               
                   
               
               
                 mismatch 
                 388 
                 SEQ ID NO: 89 
               
               
                   
                 /Motif=″GTTTCATTCCCAGGG″ SEQ ID NO: 89 
                   
               
               
                   
                 /annotation_group=″DR: 24,153-&gt;24,167″ 
                   
               
               
                   
                 /label=″T″ 
                   
               
               
                   
               
               
                 mismatch 
                 391..392 
                 SEQ ID NO: 89 
               
               
                   
                 /Motif=″GTTTCATTCCCAGGG″ SEQ ID NO: 89 
                   
               
               
                   
                 /annotation_group=″DR: 24,153-&gt;24,167″ 
                   
               
               
                   
                 /label=″CC″ 
                   
               
               
                   
               
               
                 Tracr_Put 
                 441..455 
                 SEQ ID NO: 89 
               
               
                   
                 /Mismatches=4 
                   
               
               
                   
                 /%_Identity=″73.33333333333333″ 
                   
               
               
                   
                 /Motif=″GTTTCATTCCCAGGG″ SEQ ID NO: 89 
                   
               
               
                   
                 /annotation_group=″DR: 24,212-&gt;24,226″ 
                   
               
               
                   
                 /label=″Half DR″ 
                   
               
               
                   
               
               
                 mismatch 
                 448..451 
                 SEQ ID NO: 89 
               
               
                   
                 /Motif=″GTTTCATTCCCAGGG″ 
                   
               
               
                   
                 /annotation_group=″DR: 24,212-&gt;24,226″ 
                   
               
               
                   
                 /label=″TCCC″ 
                   
               
               
                   
               
               
                 DR 
                 complement(515..551) 
                 SEQ ID NO: 90 
               
               
                   
                 /Mismatches=8 
                   
               
               
                   
                 /%_Identity=″78.37837837837837″ 
                   
               
               
                   
                 /Motif=″GTTGCAATGTATAGTGCAGCGACCCTGGGAATGAAAC″ 
                   
               
               
                   
                 SEQ ID NO: 90 
                   
               
               
                   
                 /annotation_group=″DR: 24,286&lt;-24,322″ 
                   
               
               
                   
                 /label=″DR″ 
                   
               
               
                   
               
               
                 Tracr_Put 
                 515..529 
                 SEQ ID NO: 89 
               
               
                   
                 /Mismatches=2 
                   
               
               
                   
                 /%_Identity=″86.66666666666667″ 
                   
               
               
                   
                 /Motif=″GTTTCATTCCCAGGG″ SEQ ID NO: 89 
                   
               
               
                   
                 /annotation_group=″DR: 24,286-&gt;24,300″ 
                   
               
               
                   
                 /label= ″Half DR 
                   
               
               
                   
               
               
                 mismatch 
                 521 
                 SEQ ID NO: 90 
               
               
                   
                 /Motif=″GTTGCAATGTATAGTGCAGCGACCCTGGGAATGAAAC″ 
                   
               
               
                   
                 SEQ ID NO: 90 
                   
               
               
                   
                 /annotation_group=″DR: 24,286&lt;-24,322″ 
                   
               
               
                   
                 /label=″A″ 
                   
               
               
                   
               
               
                 mismatch 
                 521 
                 SEQ ID NO: 89 
               
               
                   
                 /Motif=″GTTTCATTCCCAGGG″ SEQ ID NO: 89 
                   
               
               
                   
                 /annotation_group=″DR: 24,286-&gt;24,300″ 
                   
               
               
                   
                 /label=″T″ 
                   
               
               
                   
               
               
                 mismatch 
                 524 
                 SEQ ID NO: 90 
               
               
                   
                 /Motif=″GTTGCAATGTATAGTGCAGCGACCCTGGGAATGAAAC″ 
                   
               
               
                   
                 SEQ ID NO: 90 
                   
               
               
                   
                 /annotation_group=″DR: 24,286&lt;-24,322″ 
                   
               
               
                   
                 /label=″G″ 
                   
               
               
                   
               
               
                 mismatch 
                 524 
                 SEQ ID NO: 89 
               
               
                   
                 /Motif=″GTTTCATTCCCAGGG″ SEQ ID NO: 89 
                   
               
               
                   
                 /annotation_group=″DR: 24,286-&gt;24,300″ 
                   
               
               
                   
                 /label=″C″ 
                   
               
               
                   
               
               
                 mismatch 
                 Motif=″GTTGCAATGTATAGTGCAGCGACCCTGGGAATGAAAC″ 
                 SEQ ID NO: 90 
               
               
                   
                 SEQ ID NO: 90 
                   
               
               
                   
                 /annotation_group=″DR: 24,286&lt;-24,322″ 
                   
               
               
                   
                 /label=″G″ 
                   
               
               
                   
               
               
                 mismatch 
                 539..540 
                 SEQ ID NO: 90 
               
               
                   
                 /Motif=″GTTGCAATGTATAGTGCAGCGACCCTGGGAATGAAAC″ 
                   
               
               
                   
                 /annotation_group=″DR: 24,286&lt;-24,322″ 
                   
               
               
                   
                 /label=″TA″ 
                   
               
               
                   
               
               
                 mismatch 
                 542 
                 SEQ ID NO: 90 
               
               
                   
                 /Motif=″GTTGCAATGTATAGTGCAGCGACCCTGGGAATGAAAC″ 
                   
               
               
                   
                 SEQ ID NO: 90 
                   
               
               
                   
                 /annotation_group=″DR: 24,286&lt;-24,322″ 
                   
               
               
                   
                 /label=″T″ 
                   
               
               
                   
               
               
                 mismatch 
                 544 
                 SEQ ID NO: 90 
               
               
                   
                 /Motif=″GTTGCAATGTATAGTGCAGCGACCCTGGGAATGAAAC″ 
                   
               
               
                   
                 /annotation_group=″DR: 24,286&lt;-24,322″ 
                   
               
               
                   
                 /label=″T″ 
                   
               
               
                   
               
               
                 mismatch 
                 548 
                 SEQ ID NO: 90 
               
               
                   
                 /Motif=″GTTGCAATGTATAGTGCAGCGACCCTGGGAATGAAAC″ 
                   
               
               
                   
                 SEQ ID NO: 90 
                   
               
               
                   
                 /annotation_group=″DR: 24,286&lt;-24,322″ 
                   
               
               
                   
                 /label=″G″ 
                   
               
               
                   
               
               
                 DR 
                 complement(589..625) 
                 SEQ ID NO: 90 
               
               
                   
                 /Mismatches=8 
                   
               
               
                   
                 /%_Identity=″78.37837837837837″ 
                   
               
               
                   
                 /Motif=″GTTGCAATGTATAGTGCAGCGACCCTGGGAATGAAAC″ 
                   
               
               
                   
                 SEQ ID NO: 90 
                   
               
               
                   
                 /annotation_group=″DR: 24,433&lt;-24,447″ 
                   
               
               
                   
                 /label=″DR″ 
                   
               
               
                   
               
               
                 Tracr_Put 
                 589..603 
                 SEQ ID NO: 89 
               
               
                   
                 /Mismatches=2 
                   
               
               
                   
                 /%_Identity=″86.66666666666667″ 
                   
               
               
                   
                 /Motif=″GTTTCATTCCCAGGG″ SEQ ID NO: 89 
                   
               
               
                   
                 /annotation_group=″DR: 24,433-&gt;24,447″ 
                   
               
               
                   
                 /label=″Half DR″ 
                   
               
               
                   
               
               
                 Misc_feature 
                 88{circumflex over ( )}589 
                 SEQ ID NO: 91 
               
               
                   
                 /Original_Bases=″SEQ ID NO: 91″ 
                   
               
               
                   
                 /lable=″SEQ ID NO: 91″ 
                   
               
               
                   
                 /note=″Geneious type: Editing History Deletion″ 
                   
               
               
                   
               
               
                 mismatch 
                 595 
                 SEQ ID NO: 90 
               
               
                   
                 /Motif=″GTTGCAATGTATAGTGCAGCGACCCTGGGAATGAAAC″ 
                   
               
               
                   
                 SEQ ID NO: 90 
                   
               
               
                   
                 /annotation_group=″DR: 24,433&lt;-24,469″ 
                   
               
               
                   
                 /label=″A″ 
                   
               
               
                   
               
               
                 mismatch 
                 595 
                 SEQ ID NO: 89 
               
               
                   
                 /Motif=″GTTTCATTCCCAGGG″ SEQ ID NO: 89 
                   
               
               
                   
                 /annotation_group=″DR: 24,433-&gt;24,447″ 
                   
               
               
                   
                 /label=″T″ 
                   
               
               
                   
               
               
                 mismatch 
                 598 
                 SEQ ID NO: 90 
               
               
                   
                 /Motif=″GTTGCAATGTATAGTGCAGCGACCCTGGGAATGAAAC″ 
                   
               
               
                   
                 SEQ ID NO: 90 
                   
               
               
                   
                 /annotation_group=″DR: 24,433&lt;-24,469″ 
                   
               
               
                   
                 /label=″G″ 
                   
               
               
                   
               
               
                 mismatch 
                 598 
                 SEQ ID NO: 89 
               
               
                   
                 /Motif=″GTTTCATTCCCAGGG″ SEQ ID NO: 89 
                   
               
               
                   
                 /annotation_group=″DR: 24,433-&gt;24,447″ 
                   
               
               
                   
                 /label=″C″ 
                   
               
               
                   
               
               
                 mismatch 
                 605 
                 SEQ ID NO: 90 
               
               
                   
                 /Motif=″GTTGCAATGTATAGTGCAGCGACCCTGGGAATGAAAC″ 
                   
               
               
                   
                 SEQ ID NO: 90 
                   
               
               
                   
                 /annotation_group=″DR: 24,433&lt;-24,469″ 
                   
               
               
                   
                 /label=″G 
                   
               
               
                   
               
               
                 mismatch 
                 613..614 
                 SEQ ID NO: 90 
               
               
                   
                 /Motif=″GTTGCAATGTATAGTGCAGCGACCCTGGGAATGAAAC″ 
                   
               
               
                   
                 SEQ ID NO: 90 
                   
               
               
                   
                 /annotation_group=″DR: 24,433&lt;-24,469″ 
                   
               
               
                   
                 /label=″TA″ 
                   
               
               
                   
               
               
                 mismatch 
                 616 
                 SEQ ID NO: 90 
               
               
                   
                 /Motif=″GTTGCAATGTATAGTGCAGCGACCCTGGGAATGAAAC″ 
                   
               
               
                   
                 SEQ ID NO: 90 
                   
               
               
                   
                 /annotation_group=″DR: 24,433&lt;-24,469″ 
                   
               
               
                   
                 /label=″T″ 
                   
               
               
                   
               
               
                 mismatch 
                 618 
                 SEQ ID NO: 90 
               
               
                   
                 /Motif=″GTTGCAATGTATAGTGCAGCGACCCTGGGAATGAAAC″ 
                   
               
               
                   
                 SEQ ID NO: 90 
                   
               
               
                   
                 /annotation_group=″DR: 24,433&lt;-24,469″ 
                   
               
               
                   
                 /label=″T″ 
                   
               
               
                   
               
               
                 mismatch 
                 622 
                 SEQ ID NO: 90 
               
               
                   
                 /Motif=″GTTGCAATGTATAGTGCAGCGACCCTGGGAATGAAAC″ 
                   
               
               
                   
                 SEQ ID NO: 90 
                   
               
               
                   
                 /annotation_group=″DR: 24,433&lt;-24,469″ 
                   
               
               
                   
                 /label=″G″ 
                   
               
               
                   
               
               
                 DR 
                 complement(663..699) 
                 SEQ ID NO: 90 
               
               
                   
                 /Mismatches=8 
                   
               
               
                   
                 /%_Identity=″78.37837837837837″ 
                   
               
               
                   
                 /Motif=″GTTGCAATGTATAGTGCAGCGACCCTGGGAATGAAAC″ 
                   
               
               
                   
                 /annotation_group=″DR: 24,507&lt;-24,543″ 
                   
               
               
                   
                 /label=″DR″ 
                   
               
               
                   
               
               
                 Tracr_Put 
                 663..677 
                 SEQ ID NO: 89 
               
               
                   
                 /Mismatches=2 
                   
               
               
                   
                 /%_Identity=″86.66666666666667″ 
                   
               
               
                   
                 /Motif=″GTTTCATTCCCAGGG″ SEQ ID NO: 89 
                   
               
               
                   
                 /annotation_group=″DR: 24,507-&gt;24,521″ 
                   
               
               
                   
                 /label=″Half DR″ 
                   
               
               
                   
               
               
                 mismatch 
                 669 
                 SEQ ID NO: 90 
               
               
                   
                 /Motif=″GTTGCAATGTATAGTGCAGCGACCCTGGGAATGAAAC″ 
                   
               
               
                   
                 SEQ ID NO: 90 
                   
               
               
                   
                 /annotation_group=″DR: 24,507&lt;-24,543″ 
                   
               
               
                   
                 /label=″A 
                   
               
               
                   
               
               
                 mismatch 
                 669 
                 SEQ ID NO: 89 
               
               
                   
                 /Motif=″GTTTCATTCCCAGGG″ SEQ ID NO: 89 
                   
               
               
                   
                 /annotation_group=″DR: 24,507-&gt;24,521″ 
                   
               
               
                   
                 /label=″T″ 
                   
               
               
                   
               
               
                 mismatch 
                 672 
                 SEQ ID NO: 90 
               
               
                   
                 /Motif=″GTTGCAATGTATAGTGCAGCGACCCTGGGAATGAAAC″ 
                   
               
               
                   
                 /annotation_group=″DR: 24,507&lt;-24,543″ 
                   
               
               
                   
                 /label=″G″ 
                   
               
               
                   
               
               
                 mismatch 
                 672 
                 SEQ ID NO: 89 
               
               
                   
                 /Motif=″GTTTCATTCCCAGGG″ SEQ ID NO: 89 
                   
               
               
                   
                 /annotation_group=″DR: 24,507-&gt;24,521″ 
                   
               
               
                   
                 /label=″C″ 
                   
               
               
                   
               
               
                 mismatch 
                 679 
                 SEQ ID NO: 90 
               
               
                   
                 /Motif=″GTTGCAATGTATAGTGCAGCGACCCTGGGAATGAAAC″ 
                   
               
               
                   
                 /annotation_group=″DR: 24,507&lt;-24,543″ 
                   
               
               
                   
                 /label=″G″ 
                   
               
               
                   
               
               
                 mismatch 
                 687..688 
                 SEQ ID NO: 90 
               
               
                   
                 /Motif=″GTTGCAATGTATAGTGCAGCGACCCTGGGAATGAAAC″ 
                   
               
               
                   
                 SEQ ID NO: 90 
                   
               
               
                   
                 /annotation_group=″DR: 24,507&lt;-24,543″ 
                   
               
               
                   
                 /label=″TA″ 
                   
               
               
                   
               
               
                 mismatch 
                 690 
                 SEQ ID NO: 90 
               
               
                   
                 /Motif=″GTTGCAATGTATAGTGCAGCGACCCTGGGAATGAAAC″ 
                   
               
               
                   
                 SEQ ID NO: 90 
                   
               
               
                   
                 /annotation_group=″DR: 24,507&lt;-24,543″ 
                   
               
               
                   
                 /label=″T″ 
                   
               
               
                   
               
               
                 mismatch 
                 692 
                 SEQ ID NO: 90 
               
               
                   
                 /Motif=″GTTGCAATGTATAGTGCAGCGACCCTGGGAATGAAAC″ 
                   
               
               
                   
                 SEQ ID NO: 90 
                   
               
               
                   
                 /annotation_group=″DR: 24,507&lt;-24,543″ 
                   
               
               
                   
                 /label=″T″ 
                   
               
               
                   
               
               
                 mismatch 
                 696 
                 SEQ ID NO: 90 
               
               
                   
                 /Motif=″GTTGCAATGTATAGTGCAGCGACCCTGGGAATGAAAC″ 
                   
               
               
                   
                 SEQ ID NO: 90 
                   
               
               
                   
                 /annotation_group=″DR: 24,507&lt;-24,543″ 
                   
               
               
                   
                 /label=″G″ 
                   
               
               
                   
               
               
                 CDS 
                 complement(821..1045) 
                   
               
               
                   
                 /product=″hypothetical protein″ 
                   
               
               
                   
                 /label=″hypothetical protein CDS″ 
                   
               
               
                   
               
               
                 -35_Box 
                 1120..1125 
                   
               
               
                   
                 /created_by=″altaeth″ 
                   
               
               
                   
                 /label=″-35 Box 
                   
               
               
                   
               
               
                 Misc_feature 
                 1138..1146 
                   
               
               
                   
                 /created_by=″altaeth″ 
                   
               
               
                   
                 /modified_by=″altaeth″ 
                   
               
               
                   
                 /label=″-10 Box″ 
                   
               
               
                   
                 /note=″Geneious type: RBS Binding Site 
                   
               
               
                   
               
               
                 POI 
                 1156..&gt;1185 
                   
               
               
                   
                 /product=″-&gt;cas9(319,439)[25.5]: Restriction 
                   
               
               
                   
                 endonuclease &amp; COG1403″ 
                   
               
               
                   
                 /modified_by=″altaeth″ 
                   
               
               
                   
                 /label=″VII_cA1″ 
                   
               
               
                   
               
               
                 ORF 
                 1156..2988 
                   
               
               
                   
                 /created_by=″skannan″ 
                   
               
               
                   
                 /label=″VII_cA1 human codon optimized″ 
                   
               
               
                   
               
               
                 Misc_feature 
                 1188..2988 
                 SEQ ID NO: 92 
               
               
                   
                 /Original_Bases=″SEQ ID NO: 92″ 
                   
               
               
                   
                 /label=″SEQ ID NO: 92″ 
                   
               
               
                   
                 /note=″Geneious type: Editing History Replacement″ 
                   
               
               
                   
               
               
                 CDS 
                 3089..3652 
                   
               
               
                   
                 /product=″hypothetical protein″ 
                   
               
               
                   
                 /label=″hypothetical protein CDS″ 
                   
               
               
                   
               
               
                 CDS 
                 3744..&gt;3752 
                   
               
               
                   
                 /product=″hypothetical protein″ 
                   
               
               
                   
                 /modified_by=″altaeth″ 
                   
               
               
                   
                 /label=″RNA Binding Domain″ 
                   
               
               
                   
               
               
                 Misc_feature 
                 /Original_Bases=″SEQ ID NO: 93″ 
                 SEQ ID NO: 93 
               
               
                   
                 /label=″SEQ ID NO: 93″ 
                   
               
               
                   
                 /note=″Geneious type: Editing History Deletion″ 
                   
               
               
                   
               
               
                 ORIGIN 
                 SEQ ID NO: 94 
                 SEQ ID NO: 94 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 23 
               
             
            
               
                   
               
               
                 Features relevant to one or more of SEQ ID NOs: 95-100 (Locus 
               
               
                 VII_cA2_contig_for_synthesis) See also Appendix C of U.S. Provisional Patent 
               
               
                 Application No. 62/850,494. 
               
            
           
           
               
               
               
            
               
                 Feature Key 
                   
                 Sequence(s) of 
               
               
                 ID/Annotation 
                 Feature Location/Qualifiers 
                 reference or note 
               
               
                   
               
               
                 padding 
                 /label 
                   
               
               
                   
               
               
                 Misc_feature 
                 /Original_Bases=″SEQ ID NO: 95″ 
                 SEQ ID NO: 95 
               
               
                   
                 /label=″SEQ ID NO: 95″ 
                   
               
               
                   
                 note=″Geneious type: Editing History Deletion″ 
                   
               
               
                   
               
               
                 CDS 
                 20..470 
                   
               
               
                   
                 /product=″hypothetical protein &amp; Hypo-rule applied″ 
                   
               
               
                   
                 /label=″hypothetical protein &amp; Hypo-rule applied CDS″ 
                   
               
               
                   
               
               
                 DR 
                 complement(783..819) 
                 SEQ ID NO: 90 
               
               
                   
                 /Mismatches=0 
                   
               
               
                   
                 /%_Identity=100 
                   
               
               
                   
                 /Motif=″GTTGCAATGTATAGTGCAGCGACCCTGGGAATGAAAC″ 
                   
               
               
                   
                 SEQ ID NO: 90 
                   
               
               
                   
                 /annotation_group=″DR: 24,011&lt;-24,047″ 
                   
               
               
                   
                 /label=″DR″ 
                   
               
               
                   
               
               
                 Tracr_Put 
                 783..797 
                 SEQ ID NO: 89 
               
               
                   
                 /Mismatches=0 
                   
               
               
                   
                 /%_Identity=100 
                   
               
               
                   
                 /Motif=″GTTTCATTCCCAGGG″ SEQ ID NO: 89 
                   
               
               
                   
                 /annotation_group=″DR: 24,011-&gt;24,025″ 
                   
               
               
                   
                 /label=″Half DR″ 
                   
               
               
                   
               
               
                 Misc_feature 
                 782{circumflex over ( )}783 
                 SEQ ID NO: 96 
               
               
                   
                 /Original_Bases=″SEQ ID NO: 96″ 
                   
               
               
                   
                 /label=″ SEQ ID NO: 96″ 
                   
               
               
                   
                 /note=″Geneious type: Editing History Deletion″ 
                   
               
               
                   
               
               
                 DR 
                 complement(858..894) 
                 SEQ ID NO: 90 
               
               
                   
                 /Mismatches=0 
                   
               
               
                   
                 /%_Identity=100 
                   
               
               
                   
                 /Motif=″GTTGCAATGTATAGTGCAGCGACCCTGGGAATGAAAC″ 
                   
               
               
                   
                 SEQ ID NO: 90 
                   
               
               
                   
                 /annotation_group=″DR: 24,456&lt;-24,492″ 
                   
               
               
                   
                 /label=″DR″ 
                   
               
               
                   
               
               
                 Tracr_Put 
                 858..872 
                   
               
               
                   
                 /Mismatches=0 
                   
               
               
                   
                 /%_Identity=100 
                   
               
               
                   
                 /Motif=″GTTTCATTCCCAGGG″ 
                   
               
               
                   
                 /annotation_group=″DR: 24,456-&gt;24,470″ 
                   
               
               
                   
                 /label=″Half DR″ 
                   
               
               
                   
               
               
                 misc_feature 
                 57858 
                 SEQ ID NO: 97 
               
               
                   
                 /Original_Bases=″SEQ D NO: 97″ 
                   
               
               
                   
                 /label=″SEQ D NO: 97″ 
                   
               
               
                   
                 /note=″Geneious type: Editing History Deletion″ 
                   
               
               
                   
               
               
                 DR 
                 complement(932..968) 
                 SEQ ID NO: 90 
               
               
                   
                 /Mismatches=0 
                   
               
               
                   
                 /%_Identity=100 
                   
               
               
                   
                 /Motif=″GTTGCAATGTATAGTGCAGCGACCCTGGGAATGAAAC″ 
                   
               
               
                   
                 SEQ ID NO: 90 
                   
               
               
                   
                 /annotation_group=″DR: 24,530&lt;-24,544″ 
                   
               
               
                   
                 /label=″DR″ 
                   
               
               
                   
               
               
                 Tracr_Put 
                 932..946 
                 SEQ ID NO: 89 
               
               
                   
                 /Mismatches=0 
                   
               
               
                   
                 /%_Identity=100 
                   
               
               
                   
                 /Motif=″GTTTCATTCCCAGGG″ SEQ ID NO: 89 
                   
               
               
                   
                 /annotation_group=″DR: 24,530-&gt;24,544″ 
                   
               
               
                   
                 /label=″Half DR″ 
                   
               
               
                   
               
               
                 Tracr_Put 
                 complement(968..982) 
                 SEQ ID NO: 89 
               
               
                   
                 /Mismatches=4 
                   
               
               
                   
                 /%_Identity=″73.33333333333333″ 
                   
               
               
                   
                 /Motif=″GTTTCATTCCCAGGG″ SEQ ID NO: 89 
                   
               
               
                   
                 /annotation_group=″DR: 24,566&lt;-24,580″ 
                   
               
               
                   
                 /label=″Half DR″ 
                   
               
               
                   
               
               
                 mismatch 
                 971..973 
                 SEQ ID NO: 89 
               
               
                   
                 /Motif=″GTTTCATTCCCAGGG″ SEQ ID NO: 89 
                   
               
               
                   
                 /annotation_group=″DR: 24,566&lt;-24,580″ 
                   
               
               
                   
                 /label=″CCA 
                   
               
               
                   
               
               
                 mismatch 
                 977 
                 SEQ ID NO: 89 
               
               
                   
                 /Motif=″GTTTCATTCCCAGGG″ SEQ ID NO: 89 
                   
               
               
                   
                 /annotation_group=″DR: 24,566&lt;-24,580″ 
                   
               
               
                   
                 /label=″A″ 
                   
               
               
                   
               
               
                 Tracr_Put 
                 complement(1076..1090) 
                 SEQ ID NO: 89 
               
               
                   
                 /Mismatches=4 
                   
               
               
                   
                 /%_Identity=″73.33333333333333″ 
                   
               
               
                   
                 /Motif=″GTTTCATTCCCAGGG″ SEQ ID NO: 89 
                   
               
               
                   
                 /annotation_group=″DR: 24,674&lt;-24,688″ 
                   
               
               
                   
                 /label=″Half DR″ 
                   
               
               
                   
               
               
                 mismatch 
                 1085..1087 
                 SEQ ID NO: 89 
               
               
                   
                 /Motif=″GTTTCATTCCCAGGG″ SEQ ID NO: 89 
                   
               
               
                   
                 /annotation_group=″DR: 24,674&lt;-24,688″ 
                   
               
               
                   
                 /label=″TCA″ 
                   
               
               
                   
               
               
                 mismatch 
                 1090 
                 SEQ ID NO: 89 
               
               
                   
                 /Motif=″GTTTCATTCCCAGGG″ SEQ ID NO: 89 
                   
               
               
                   
                 /annotation_group=″DR: 24,674&lt;-24,688″ 
                   
               
               
                   
                 /label=″G″ 
                   
               
               
                   
               
               
                 CDS 
                 1132..1347 
                   
               
               
                   
                 /product=″hypothetical protein &amp; Hypo-rule applied″ 
                   
               
               
                   
                 /label=″hypothetical protein &amp; Hypo-rule applied CDS″ 
                   
               
               
                   
               
               
                 -35_Box 
                 1336..1341 
                   
               
               
                   
                 /created_by=″altaeth″ 
                   
               
               
                   
                 /label=″-35 Box″ 
                   
               
               
                   
               
               
                 Misc_feature 
                 1359..1367 
                   
               
               
                   
                 /created_by=″altaeth″ 
                   
               
               
                   
                 /modified_by=″altaeth″ 
                   
               
               
                   
                 /label=″-10 Box″ 
                   
               
               
                   
                 note=″Geneious type: RBS Binding Site 
                   
               
               
                   
               
               
                 mutation 
                 1378 
                   
               
               
                   
                 /created_by=″altaeth″ 
                   
               
               
                   
                 /label=″T-&gt;A for ATG 
                   
               
               
                   
               
               
                 POI 
                 1378..&gt;1407 
                   
               
               
                   
                 /product=″5-methylcytosine-specific restriction 
                   
               
               
                   
                 endonuclease McrA &amp; COG1403″ 
                   
               
               
                   
                 /modified_by=″altaeth″ 
                   
               
               
                   
                 /label=″VII_cA2″ 
                   
               
               
                   
               
               
                 Misc_feature 
                 1378 
                   
               
               
                   
                 /Original_Bases=″T″ 
                   
               
               
                   
                 /label=″T″ 
                   
               
               
                   
                 /note=″Geneious type: Editing History Replacement″ 
                   
               
               
                   
               
               
                 ORF 
                 1378..3255 
                   
               
               
                   
                 /created_by=″skannan″ 
                   
               
               
                   
                 /label=″VII_cA2_human_codonopt″ 
                   
               
               
                   
               
               
                 Misc_feature 
                 1410..3255 
                 SEQ ID NO: 98 
               
               
                   
                 /Original_Bases=″SEQ ID NO: 98″ 
                   
               
               
                   
                 /label=″SEQ ID NO: 98″ 
                   
               
               
                   
               
               
                 CDS 
                 complement(3665..3880) 
                   
               
               
                   
                 /product= Protein of unknown function (DUF3006) &amp; 
                   
               
               
                   
                 pfam11213″ 
                   
               
               
                   
                 /label=″Protein of unknown function (DUF3006) &amp; pfam11213 
                   
               
               
                   
                 CDS″ 
                   
               
               
                   
               
               
                 CDS 
                 complement(3901..&gt;3974) 
                   
               
               
                   
                 /product=″competence protein ComEC &amp; KO:K02238″ 
                   
               
               
                   
                 /codon_start=3 
                   
               
               
                   
                 /label=″competence protein ComEC &amp; KO:K02238 CDS″ 
                   
               
               
                   
               
               
                 Misc_feature 
                 /Original_Bases=″SEQ ID NO: 99″ 
                 SEQ ID NO: 99 
               
               
                   
                 /label=″SEQ ID NO: 99″ 
                   
               
               
                   
                 /note=″Geneious type: Editing History Deletion″ 
                   
               
               
                   
               
               
                 ORIGIN 
                 SEQ ID NO: 100 
                 SEQ ID NO: 100 
               
               
                   
               
            
           
         
       
     
     Various modifications and variations of the described methods, pharmaceutical compositions, and kits of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it will be understood that it is capable of further modifications and that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure come within known customary practice within the art to which the invention pertains and may be applied to the essential features herein before set forth.