Patent Publication Number: US-2022235340-A1

Title: Novel crispr-cas systems and uses thereof

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims the benefit of U.S. Provisional Application No. 62/850,516, filed May 20, 2019, and U.S. Provisional Application No. 63/000,293, filed Mar. 26, 2020. The entire contents of the above-identified applications are hereby fully incorporated herein by reference. 
    
    
     STATEMENT AS TO FEDERALLY SPONSORED RESEARCH 
     This invention was made with government support under Grant Nos. HL141201 and HG009761 awarded by The National Institutes of Health. The government has certain rights in the invention. 
    
    
     TECHNICAL FIELD 
     The invention provides for systems, methods, and compositions for targeting nucleic acids. In particular, the invention provides DNA or RNA-targeting systems comprising a novel DNA or RNA-targeting CRISPR effector protein and at least one targeting nucleic acid component like a guide RNA. 
     REFERENCE TO AN ELECTRONIC SEQUENCE LISTING 
     The contents of the electronic sequence listing (“BROD-4230WP_ST25.txt”; 115,445 bytes; created on May 20, 2020) is herein incorporated by reference in its entirety 
     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. The bacterial CRISPR-Cas system has provided an improvement over earlier generations of genome engineering designer enzymes as it does not require alteration of the protein sequence to encode specificity, but rather allows the use of a single RNA polynucleotide (e.g., guide RNA) to direct the Cas protein to a given genomic locus. There remains a need however for Cas systems which allow further optimization and expansion of applications involving Cas proteins. 
     Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention. 
     SUMMARY 
     In one aspect, the present disclosure provides an engineered Cas protein comprising a region providing access to a location of target polynucleotide binding. 
     In some embodiments, the engineered Cas protein comprises no more than 600, no more than 700, or no more than 800 amino acids. In some embodiments, the protein lacks or substantially lacks a Rec1 and/or Rec2 domain or the structural equivalent thereof. In some embodiments, the protein lacks or substantially lacks a Rec lobe or structural equivalent thereof. In some embodiments, the protein comprises at least one nuclease domain. In some embodiments, the Cas protein comprises an HNH and a RuvC nuclease domain. In some embodiments, the RuvC domain comprises RuvCI, RuvCII, and/or RuvCIII, preferably all. 
     In some embodiments, the Cas protein targets DNA. In some embodiments, the Cas protein targets dsDNA. In some embodiments, the Cas protein comprises a region that has a 10-%45% identity to IscB. In some embodiments, the Cas protein comprises a region that has 20-25% identity to IscB. 
     In some embodiments, the Cas protein has at least 10%, at least 20%, at least 30%, at least 40% or at least 45% identity to SpCas9 or is at least 10%, preferably at least 20%, shorter than SpCas9. In some embodiments, the Cas protein is a Class 2, Type II CRISPR-Cas protein. 
     In some embodiments, one or both nuclease domains are catalytically inactive or modified to be catalytically inactive, or the protein is a nickase. In some embodiments, both nuclease domains are catalytically inactive. 
     In some embodiments, the Cas protein comprise a region that has at least 80% identity to IscB. In some embodiments, the region is at N-terminus of the Cas protein. 
     In another aspect, the present disclosure provides an engineered CRISPR-Cas system comprising the Cas protein herein and a guide molecule capable of forming a complex with the Cas protein and directing site-specific binding of the complex to a target sequence of a target polypeptide. 
     In some embodiments, the Cas protein and/or the guide molecule further comprise a functional domain. In some embodiments, the functional domain comprises base editing activity, nucleotide deaminase activity, methylase activity, demethylase activity, translation activation activity, translation repression activity, transcription activation activity, transcription repression activity, transcription release factor activity, chromatin modifying or remodeling 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, nucleic acid binding activity, detectable activity, or any combination thereof. In some embodiments, the functional domain is a nucleotide deaminase linked or fused to the Cas protein. In some embodiments, said deaminase is an adenosine deaminase or a cytidine deaminase. In some embodiments, the system or Cas protein further comprises one or more nucleic acid modifying proteins or domains. In some embodiments, the one or more DNA modifying proteins comprises DNA polymerase, recombinase, ribonucleotide reductase, methyltransferase, diadenosine tetraphosphate hydrolase, DNA helicase, or RNA helicase. 
     In some embodiments, the target sequence comprises a PAM sequence. In some embodiments, the PAM sequence is NGG. 
     In another aspect, the present disclosure provides a vector comprising the polynucleotide herein. 
     In another aspect, the present disclosure provides a vector system comprising two or more vectors herein. 
     In another aspect, the present disclosure provides a cell comprising a polynucleotide, vector, or vector system herein. In some embodiments, the cell is a eukaryotic cell, a prokaryotic cell, or a plant cell. 
     In another aspect, the present disclosure provides a plant or non-human animal comprising one or more polynucleotides, vectors, vector systems, or cells herein. 
     In another aspect, the present disclosure provides a method of targeting a polynucleotide, comprising contacting a sample that comprises the polynucleotide with the system or Cas protein, the polynucleotide, the vector, or the vector system herein. In some embodiments, the method further comprises detecting binding of the complex to the polynucleotide. In some embodiments, contacting results in modification of a gene product or modification of the amount or expression of a gene product. In some embodiments, the target sequence of the polynucleotide is a disease-associated target sequence. 
     In another aspect, the present disclosure provides a method of modifying an adenine or cytidine in a target polynucleotide sequence, comprising contacting said target polynucleotide with the system or Cas protein herein. 
     In another aspect, the present disclosure provides an antiviral composition comprising the system or Cas protein herein. 
     In another aspect, the present disclosure provides a method for treating, preventing, suppressing and/or alleviating viral pathogenesis, infection, propagation, and/or replication in a subject in need thereof, comprising administering to a subject in need thereof the composition herein. 
     These and other aspects, objects, features, and advantages of the example embodiments will become apparent to those having ordinary skill in the art upon consideration of the following detailed description of illustrated example embodiments. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       An understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention may be utilized, and the accompanying drawings of which: 
         FIGS. 1-8  show annotations on the sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 14. 
         FIG. 9  shows an exemplary method for identifying and characterizing novel CRISPR-Cas systems and other RNA-guided nucleases. 
         FIG. 10  shows exemplary CRISPR-Cas systems and other RNA-guided nucleases. 
         FIG. 11  shows the locus of an exemplary ProCas9, which is associated with various enzymes (e.g., nucleic acid modifying enzyme). 
         FIG. 12  shows screening of PAM of an exemplary ProCas9. 
         FIG. 13  shows processed crRNA and tracrRNA revealed by dRNA-seq. 
         FIG. 14  shows purification of an exemplary ProCas9 protein. 
     
    
    
     The figures herein are for illustrative purposes only and are not necessarily drawn to scale. 
     DESCRIPTION OF EMBODIMENTS 
     General Definitions 
     Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Definitions of common terms and techniques in molecular biology may be found in Molecular Cloning: A Laboratory Manual, 2 nd  edition (1989) (Sambrook, Fritsch, and Maniatis); Molecular Cloning: A Laboratory Manual, 4 th  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, 2 nd  edition 2013 (E. A. Greenfield ed.); Animal Cell Culture (1987) (R. I. Freshney, ed.); Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008 (ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 9780471185710); Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley &amp; Sons (New York, N.Y. 1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley &amp; Sons (New York, N.Y. 1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2 nd  edition (2011). 
     As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise. 
     The term “optional” or “optionally” means that the subsequent described event, circumstance or substituent may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. 
     The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints. 
     The term “about” in relation to a reference numerical value and its grammatical equivalents as used herein can include the numerical value itself and a range of values plus or minus 10% from that numerical value. For example, the amount “about 10” includes 10 and any amounts from 9 to 11. For example, the term “about” in relation to a reference numerical value can also include a range of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value. 
     As used herein, a “biological sample” may contain whole cells and/or live cells and/or cell debris. The biological sample may contain (or be derived from) a “bodily fluid”. The present invention encompasses embodiments wherein the bodily fluid is selected from amniotic fluid, aqueous humour, vitreous humour, bile, blood serum, breast milk, cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudates, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretion, vomit and mixtures of one or more thereof. Biological samples include cell cultures, bodily fluids, cell cultures from bodily fluids. Bodily fluids may be obtained from a mammal organism, for example by puncture, or other collecting or sampling procedures. 
     The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed. 
     The term “exemplary” is used herein to mean serving as an example, instance, or illustration. Any aspect or design described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other aspects or designs. Rather, use of the word exemplary is intended to present concepts in a concrete fashion. 
     Various embodiments are described hereinafter. It should be noted that the specific embodiments are not intended as an exhaustive description or as a limitation to the broader aspects discussed herein. One aspect described in conjunction with a particular embodiment is not necessarily limited to that embodiment and can be practiced with any other embodiment(s). Reference throughout this specification to “one embodiment”, “an embodiment,” “an example embodiment,” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” or “an example embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention. For example, in the appended claims, any of the claimed embodiments can be used in any combination. 
     A protein or nucleic acid derived from a species means that the protein or nucleic acid has a sequence identical to an endogenous protein or nucleic acid or a portion thereof in the species. The protein or nucleic acid derived from the species may be directly obtained from an organism of the species (e.g., by isolation), or may be produced, e.g., by recombination production or chemical synthesis. 
     All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference to the same extent as though each individual publication, published patent document, or patent application was specifically and individually indicated as being incorporated by reference. 
     It will be appreciated that the terms Cas enzyme, CRISPR enzyme, CRISPR protein, Cas protein and CRISPR Cas are generally used interchangeably and at all points of reference herein refer by analogy to CRISPR effector proteins further described in this application, unless otherwise apparent. 
     Overview 
     There is an unmet need for improved Cas proteins that allow their further development as polynucleotide binding proteins for a wide variety of applications. This invention caters to this need by providing new Cas proteins and methods to use said proteins. Addition of these novel Cas proteins to the current collection of polynucleotide targeting strategies and CRISPR-Cas applications will impact the efficiency of both known strategies to manipulate polynucleotides and strategies that may be developed in the future. Furthermore, previous hypothesized manipulation strategies that failed to deliver results in practice due to low efficiencies may become feasible due to these new Cas proteins. The current invention thus opens exciting new avenues for the manipulation of polynucleotides. 
     In one aspect, the present disclosure provides Cas proteins that may mediate increased exposure of the DNA:RNA duplex due to the absence of one or more domains of the Cas protein which typically blocks access thereto. In particular embodiments, the Cas proteins are Type II-like Cas proteins that are characterized by the absence of all or part of a Rec lobe, as a result of the absence of all or part of the Rec1 and/or Rec2 domains or the structural equivalents thereof in the Cas protein. For example, a Type II-like Cas protein may comprise a HNH domain and a RuvC domain, but does not have a Rec1 or Rec2 domain or structural equivalents thereof. The increased exposure of the DNA:RNA duplex is beneficial for the access and/or efficiency of functional domains fused to the Cas protein or provided in trans to the complex. The Cas proteins as described herein may thus enable direct efficient targeting of the DNA:RNA duplex by functional domains such as but not limited to nucleotide deaminases and/or prime editors. Furthermore, teachings as described herein provide the basis for a minimal CRISPR-Cas system, such as minimal Type II systems comprising a reduced Cas9 protein. 
     Cas Proteins 
     The present application describes nucleic acid-targeting systems, e.g., systems comprising novel RNA-guided endonucleases which are distinct from the CRISPR-Cas systems described previously. Therefore, the terminology of elements associated with these novel endonucleases are modified accordingly herein. The term “nucleic acid-targeting system”, wherein nucleic acid is DNA or RNA, (and in some aspects may also refer to DNA-RNA hybrids or derivatives thereof), refers collectively to transcripts and other elements involved in the expression of or directing the activity of DNA or RNA-targeting CRISPR-associated (“Cas”) genes, which may include sequences encoding a DNA or RNA-targeting Cas protein and a DNA or RNA-targeting guide RNA comprising a CRISPR RNA (crRNA) sequence and (in CRISPR-Cas9 system but not all systems) a trans-activating CRISPR-Cas system RNA (tracrRNA) sequence, or other sequences and transcripts from a DNA or RNA-targeting CRISPR locus. In certain example embodiments of the Cas DNA targeting RNA-guided endonuclease systems described herein, a tracrRNA sequence is not required. 
     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 target, e.g., 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 and is comprised within a target locus of interest. In some embodiments, a target sequence is located in the nucleus or cytoplasm of a cell. The herein described invention encompasses novel effector proteins of Class 2 CRISPR-Cas systems, of which Cas9 is an exemplary effector protein and hence terms used in this application to describe novel effector proteins, may correlate to the terms used to describe the CRISPR-Cas9 system. 
     The action of a naturally occurring CRISPR-Cas system is usually divided into three stages: (1) adaptation or spacer integration, (2) processing of the primary transcript of the CRISPR locus (pre-crRNA) and maturation of the crRNA which includes the spacer and variable regions corresponding to 5′ and 3′ fragments of CRISPR repeats, and (3) DNA (or RNA) interference. Two proteins, Cas1 and Cas2, that are present in the great majority of the known CRISPR-Cas systems are sufficient for the insertion of spacers into the CRISPR cassettes. These two proteins form a complex that is required for this adaptation process; the endonuclease activity of Cas1 is required for spacer integration whereas Cas2 appears to perform a nonenzymatic function. The Cas1-Cas2 complex represents the highly conserved “information processing” module of CRISPR-Cas that appears to be quasi-autonomous from the rest of the system. (See Annotation and Classification of CRISPR-Cas Systems. Makarova K S, Koonin E V. Methods Mol Biol. 2015; 1311:47-75). 
     The previously described Class 2 systems, e.g., Type II and the Type V, consisted of only three or four genes in the cas operon, namely the cas1 and cas2 genes comprising the adaptation module (the cas1-cas2 pair of genes are not involved in interference), a single multidomain effector protein that is responsible for interference but also contributes to the pre-crRNA processing and adaptation, and often a fourth gene with uncharacterized functions that is dispensable in at least some Type II systems (and in some cases the fourth gene is cas4 (biochemical or in silico evidence shows that Cas4 is a PD-(DE)xK superfamily nuclease with three-cysteine C-terminal cluster; possesses 5′-ssDNA exonuclease activity) or csn2, which encodes an inactivated ATPase). In most cases, a CRISPR array and a gene for a distinct RNA species known as tracrRNA, a trans-encoded small CRISPR RNA, are adjacent to Class 2 cas operons. The tracrRNA is partially homologous to the repeats within the respective CRISPR array and is essential for the processing of pre-crRNA that is catalyzed by RNAse III, a ubiquitous bacterial enzyme that is not associated with the CRISPR-Cas loci. In some embodiments, the system does not involve a tracr sequence. 
     Aspects of the invention relate to the identification and engineering of novel effector proteins associated with CRISPR-Cas systems. In a preferred embodiment, the effector protein comprises a single-subunit effector module. In a further embodiment the effector protein is functional in prokaryotic or eukaryotic cells for in vitro, in vivo or ex vivo applications. 
     An aspect of the invention encompasses computational methods and algorithms to predict new CRISPR-Cas systems and identify the components therein. 
     In one embodiment, a computational method of identifying novel Class 2 CRISPR-Cas loci comprises the following steps: detecting all contigs encoding the Cas1 protein; identifying all predicted protein coding genes within 20 kB of the cas1 gene; comparing the identified genes with Cas protein-specific profiles and predicting CRISPR arrays; selecting unclassified candidate CRISPR-Cas loci containing proteins larger than 500 amino acids (&gt;500 aa); analyzing selected candidates using PSI-BLAST and HHPred, thereby isolating and identifying novel CRISPR-Cas loci. In addition to the above mentioned steps, additional analysis of the candidates may be conducted by searching metagenomics databases for additional homologs. 
     In one aspect the detecting all contigs encoding the Cas1 protein is performed by GenemarkS which a gene prediction program as further described in “GeneMarkS: a self-training method for prediction of gene starts in microbial genomes. Implications for finding sequence motifs in regulatory regions.” John Besemer, Alexandre Lomsadze and Mark Borodovsky, Nucleic Acids Research (2001) 29, pp 2607-2618, herein incorporated by reference. 
     In one aspect the identifying all predicted protein coding genes is carried out by comparing the identified genes with Cas protein-specific profiles and annotating them according to NCBI Conserved Domain Database (CDD) which is a protein annotation resource that consists of a collection of well-annotated multiple sequence alignment models for ancient domains and full-length proteins. These are available as position-specific score matrices (PSSMs) for fast identification of conserved domains in protein sequences via RPS-BLAST. CDD content includes NCBI-curated domains, which use 3D-structure information to explicitly define domain boundaries and provide insights into sequence/structure/function relationships, as well as domain models imported from a number of external source databases (Pfam, SMART, COG, PRK, TIGRFAM). In a further aspect, CRISPR arrays were predicted using a PILER-CR program which is a public domain software for finding CRISPR repeats as described in “PILER-CR: fast and accurate identification of CRISPR repeats”, Edgar, R. C., BMC Bioinformatics, January 20; 8:18(2007), herein incorporated by reference. 
     In a further aspect, the case by case analysis is performed using PSI-BLAST (Position-Specific Iterative Basic Local Alignment Search Tool). PSI-BLAST derives a position-specific scoring matrix (PSSM) or profile from the multiple sequence alignment of sequences detected above a given score threshold using protein—protein BLAST. This PSSM is used to further search the database for new matches, and is updated for subsequent iterations with these newly detected sequences. Thus, PSI-BLAST provides a means of detecting distant relationships between proteins. 
     In another aspect, the case by case analysis is performed using HHpred, a method for sequence database searching and structure prediction that is as easy to use as BLAST or PSI-BLAST and that is at the same time much more sensitive in finding remote homologs. In fact, HHpred&#39;s sensitivity is competitive with the most powerful servers for structure prediction currently available. HHpred is the first server that is based on the pairwise comparison of profile hidden Markov models (HMMs). Whereas most conventional sequence search methods search sequence databases such as UniProt or the NR, HHpred searches alignment databases, like Pfam or SMART. This greatly simplifies the list of hits to a number of sequence families instead of a clutter of single sequences. All major publicly available profile and alignment databases are available through HHpred. HHpred accepts a single query sequence or a multiple alignment as input. Within only a few minutes it returns the search results in an easy-to-read format similar to that of PSI-BLAST. Search options include local or global alignment and scoring secondary structure similarity. HHpred can produce pairwise query-template sequence alignments, merged query-template multiple alignments (e.g. for transitive searches), as well as 3D structural models calculated by the MODELLER software from HHpred alignments. 
     In some embodiments, the novel CRISPR-Cas proteins are smaller compared to previously identified CRISPR-Cas proteins. The CRISPR-Cas systems described herein may allow an increased access to the site of target polynucleotide binding, which has several advantages. More particularly, it can allow easier access to the target polynucleotide for functional domains fused to the Cas protein or provided in trans. In certain embodiments, the RNA:DNA duplex formed by the Cas proteins of the present invention complexed with a guide RNA is substantially more exposed to the environment and/or functional domains present in proximity of the DNA:RNA complex than the duplexes formed by Cas proteins known in the art. In certain embodiments, the Cas proteins of the present invention confer a different degree of stability of the RNA:DNA duplex. In certain embodiments, the Cas proteins of the present invention enable direct targeting of the DNA:RNA complex by one or more functional domains. 
     In certain embodiments, the CRISPR-Cas system has no or limited target specificity. For example, a target polynucleotide does not need to have a specific sequence to be targeted by the CRISPR-Cas system. In certain embodiments, the Cas proteins of the invention do not have a PAM requirement, in that there is no sequence requirement outside of the target sequence which defines target specificity. In some cases, the target specificity of the CRISPR-Cas system may be determined by the sequence of the guide molecule only, not any sequence within the target polynucleotide. In alternative embodiments, the minimal CRISPR-Cas system has a target specificity, more particularly the binding of the Cas-protein-guide RNA complex is PAM-dependent. The Cas proteins of the invention may be modified to include PAM specificity (as described in Kleinstiver et al. 2015; Hirano et al. Mol. Cell 2016). 
     In certain embodiments, the Cas proteins correspond to a naturally occurring protein, a modified naturally occurring protein, functional fragment or truncated version thereof, or a non-naturally occurring protein. In certain embodiments, the Cas protein comprises one or more domains originating from other Cas proteins, more particularly originating from different organisms. In certain embodiments, the Cas protein may be designed by in silico approaches. Examples of in silico protein design have been described in the art and are therefore known to a skilled person. 
     In certain embodiments, the size of the Cas proteins is significantly smaller than the Cas proteins described in the art, especially standard known Type II CRISPR-Cas systems In particular embodiments, the size of the Cas proteins is less than 1000 amino acids. In further embodiments, the size comprises between about 600 and about 800 amino acids. In particular embodiments, the Cas proteins comprise no more than 800 amino acids. In some embodiments, the Cas proteins of the present invention comprise between 400 and 800 amino acids. In some embodiments, the Cas proteins comprise no more than 790 amino acids, no more than 780 amino acids, no more than 770 amino acids, no more than 760 amino acids, no more than 750 amino acids, no more than 740 amino acids, no more than 730 amino acids, no more than 720 amino acids, no more than 710 amino acids, no more than 700 amino acids, no more than 690 amino acids, no more than 680 amino acids, no more than 670 amino acids, no more than 660 amino acids, no more than 650 amino acids, no more than 640 amino acids, no more than 630 amino acids, no more than 620 amino acids, no more than 610 amino acids, no more than 600 amino acids, no more than 590 amino acids, no more than 580 amino acids, no more than 570 amino acids, no more than 560 amino acids, no more than 550 amino acids, no more than 540 amino acids, no more than 530 amino acids, no more than 520 amino acids, no more than 510 amino acids, no more than 500 amino acids, no more than 490 amino acids, no more than 480 amino acids, no more than 470 amino acids, no more than 460 amino acids, no more than 450 amino acids, than 440 amino acids, no more than 430 amino acids, than 420 amino acids, no more than 410 amino acids, or no more than 400 amino acids. 
     The present disclosure provides both novel Cas proteins and reduced versions of existing Cas proteins. A number of Cas proteins have been identified as part of nucleic acid-targeting systems, which can be envisaged in the context of the present invention. 
     In embodiments, the protein such as Cas as referred to herein also encompasses a homologs or an orthologs of Cas proteins described herein. The terms “ortholog” and “homolog” are well known in the art. By means of further guidance, a “homolog” 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 “ortholog” 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 particular embodiments, the homologue or orthologue of a Cas protein such 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 a Cas protein. In further embodiments, the homologue or orthologue of a Cas protein such 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 Cas protein. 
     In certain embodiments, the Cas protein lacks or substantially lacks a Rec domain. The term “Rec domain” as referred to herein is indicative for a protein domain which may be part of a Rec lobe of a Cas protein, such as Cas9. The Rec domain (or REC lobe) interacts with the repeat:anti-repeat duplex and includes the Rec 1 and Rec2 (alternatively commonly annotated as REC1 and REC2) domains. In a standard Type II Cas protein, REC1 is characterized by an elongated, α-helical structure comprising 25α-helices (α2-α5 and α12-α32) and two β-sheets (β6 and β10, and β7-β9), whereas REC2 adopts a six-helix bundle structure (α6-α11). A Dali search (Holm and Rosenstrom, 2010) revealed that the REC lobe does not share substantial structural similarity with other known proteins, indicating that it is a Cas9-specific, or a Cas protein specific, functional domain. Studies concerning the Rec lobe of Cas9 showed that it is one of the least conserved regions across the three Cas9 families within the Type II CRISPR system (IIA, IIB and IIC). In the currently accepted structure of a standard known Type II Cas protein, the REC2 domain does not contact the bound guide:target heteroduplex, and the deletion of either the repeat-interacting region (Δ97-150) or the anti-repeat-interacting region (Δ312-409) of the REC1 Cas9 domain abolished the DNA cleavage activity indicating that the recognition of the repeat:anti-repeat duplex by the REC1 domain is critical for the Cas9 function. 
     In certain embodiments, the Cas protein lacks or substantially lacks a Rec1 domain. In certain embodiments, the Cas protein lacks or substantially lacks a Rec 2 domain. In certain embodiment, the Cas protein lacks or substantially lacks both a Rec 1 domain and Rec 2 domain. In certain embodiments where the Cas protein is derived from a naturally occurring Cas protein, the Cas protein lacks a portion of the naturally occurring sequence of the protein corresponding to a Rec domain or a substantial portion of a Rec domain. In some embodiments, the Cas protein lacks a structural domain showing substantial similarity to a Rec domain. In some embodiments, the Cas protein amino acid sequence comprises mutations that induce at least partial unfolding of one or more Rec domains. In certain embodiments, at least one Rec domain may be swapped for a protein domain which is not part of a Cas protein known in the art. In further embodiments, the protein domain replacing one or more Rec domains may be substantially smaller than a naturally occurring Rec domain. In certain embodiments, the protein domain is about 10% smaller than the replaced Rec domain, or the sum of the replaced Rec domains, preferably about 15% smaller, about 20% smaller, about 25% smaller, about 30% smaller, about 35% smaller, about 40% smaller, about 45% smaller, about 50% smaller, about 55% smaller, about 60% smaller, about 65% smaller, about 70% smaller. In some embodiments, the Rec domain may be replaced with a structural module that is not a protein. It is hypothesized that the lack of a Rec domain may be beneficial for certain applications where a higher degree of tolerance is desired for mismatches between the guide molecule and the target polynucleotide. 
     Also intended are embodiments wherein a Cas protein has no Rec domain, the Cas protein may lack a domain showing structure similarity or sequence similarity to at least one Rec domain of the naturally occurring corresponding Cas protein. 
     In some embodiments, the Cas protein lacks or substantially lacks a Rec lobe compared to the corresponding natural Cas protein. In certain embodiments, where the Cas protein is a non-naturally occurring Cas protein, the protein lacks or substantially lacks a sequence similarity or structural similarity to a known Rec lobe in the art. 
     In some embodiments, the Rec lobe may be replaced with a structural module that is not a protein. For example, the structural module may be one or more amino acids that does not form a Rec lobe known in the art. 
     In certain embodiments, the Rec1 protein and/or Rec2 protein or a substantial portion hereof may be replaced with a suitable linker sequence. Linker sequences are well known in the art and are described elsewhere herein. In certain embodiments, the Rec lobe or a substantial portion of the Rec lobe is replaced with a protein linker sequence. In certain embodiments, the protein linker may be considered intrinsically disordered and therefor may not adopt a defined structure. In alternative embodiments, the introduced protein linker sequence may form a defined protein domain and/or protein structure. 
     In some embodiments, the Cas protein comprises at least one nuclease domain. In certain embodiments, the Cas protein comprises at least two nuclease domains. In certain embodiments, the nuclease domain is a HNH-like or RuvC-like domain. In certain embodiments, the one or more nuclease domains are only active upon presence of a cofactor. In certain embodiments, the cofactor is Magnesium (Mg). In embodiments where more than one nuclease domain is present and the substrate is a double stranded polynucleotide, the nuclease domains each cleave a different strand of the double stranded polynucleotide. 
     In certain embodiments, a least one nuclease domain shares a substantial structural similarity or sequence similarity to a RuvC domain described in the art. 
     For example, and as described in the art (e.g. Crystal structure of Cas9 in complex with guide RNA and target DNA, Nishimasu et al. Cell, 2014) the RuvC domain of Cas9 consists of a six-stranded mixed β-sheet (β1, β2, β5, β11, β14 and β17) flanked by α-helices (α33, α34 and α39-α45) and two additional two-stranded antiparallel β-sheets (β3/β4 and β15/β16). It has been described that the RuvC domain of Cas9 shares structural similarity with the retroviral integrase superfamily members characterized by an RNase H fold, such as  Escherichia coli  RuvC (PDB code 1HJR, 14% identity, root-mean-square deviation (rmsd) of 3.6 Å for 126 equivalent Cα atoms) and  Thermus thermophilus  RuvC (PDB code 4LD0, 12% identity, rmsd of 3.4 Å for 131 equivalent Ca atoms). RuvC nucleases have four catalytic residues (e.g., Asp7, Glu70, His143 and Asp146 in  T. thermophilus  RuvC), and cleave Holliday junctions through a two-metal mechanism. Asp10 (Ala), Glu762, His983 and Asp986 of the Cas9 RuvC domain are located at positions similar to those of the catalytic residues of  T. thermophilus  RuvC. There are key structural discrepancies between the Cas9 RuvC domain and the RuvC nucleases, which explain their functional differences. Unlike the Cas9 RuvC domain, the RuvC nucleases form dimers and recognize Holliday junctions. In addition to the conserved RNase H fold, the Cas9 RuvC domain has other structural elements involved in interactions with the guide:target heteroduplex (an end-capping loop between α42 and α43) and the PI domain/stem loop 3 (β-hairpin formed by β3 and β4). 
     In certain embodiments, at least one nuclease domain shares a substantial structural similarity or sequence similarity to a HNH domain described in the art. 
     For example, the HNH domain of Cas9 as described in the art (e.g. Crystal structure of Cas9 in complex with guide RNA and target DNA, Nishimasu et al. Cell, 2014) comprises a two-stranded antiparallel β-sheet (β12 and β13) flanked by four α-helices (α35-α38). It shares structural similarity with the HNH endonucleases characterized by a (3(3α-metal fold, such as phage T4 endonuclease VII (Endo VII) (PDB code 2QNC, 20% identity, rmsd of 2.7 Å for 61 equivalent Cα atoms) and  Vibrio vulnificus  nuclease (PDB code 1OUP, 8% identity, rmsd of 2.7 Å for 77 equivalent Ca atoms). HNH nucleases have three catalytic residues (e.g., Asp40, His41, and Asn62 in Endo VII), and cleave nucleic acid substrates through a single-metal mechanism. In the structure of the Endo VII N62D mutant in complex with a Holliday junction, a Mg2+ ion is coordinated by Asp40, Asp62, and the oxygen atoms of the scissile phosphate group of the substrate, while His41 acts as a general base to activate a water molecule for catalysis. Asp839, His840, and Asn863 of the Cas9 HNH domain correspond to Asp40, His41, and Asn62 of Endo VII, respectively, consistent with the observation that His840 is critical for the cleavage of the complementary DNA strand. The N863A mutant functions as a nickase, indicating that Asn863 participates in catalysis. The Cas9 HNH domain may cleave the complementary strand of the target DNA through a single-metal mechanism, as observed for other HNH superfamily nucleases. Although the Cas9 HNH domain shares a (3(3α-metal fold with other HNN endonucleases, their overall structures are distinct, consistent with the differences in their substrate specificities. 
     In certain embodiments, the Cas protein comprises at least a HNH or RuvC nuclease domain. In certain embodiments, the Cas protein comprises at least one reduced or minimal HNH or RuvC nuclease domain. In some embodiments, the Cas protein comprises two nuclease domains. In certain embodiments, the two nuclease domains are a HNH and a RuvC domain. In certain embodiments, the Cas protein comprises at least one nuclease domain substantially similar to a HNH or RuvC domain by sequence similarity. In certain embodiments, the Cas protein comprises at least one nuclease domain substantially similar to a HNH or RuvC domain by structural similarity. 
     In certain embodiments, the nuclease domain of the Cas protein has substantial structural and/or sequence similarity to a FokI domain. In further embodiments, the Cas proteins comprises multiple FokI or FokI-like domains. 
     In an embodiment, the Cas protein may be an ortholog of an organism of a genus which includes but is not limited to  Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma  and  Campylobacter . Species of organism of such a genus can be as otherwise herein discussed. 
     Further orthologs of known Cas proteins may be identified. Some methods of identifying orthologs of CRISPR-Cas system enzymes may involve identifying tracr sequences in genomes of interest. Identification of tracr sequences may relate to the following steps: Search for the direct repeats or tracr mate sequences in a database to identify a CRISPR region comprising a CRISPR enzyme. Search for homologous sequences in the CRISPR region flanking the CRISPR enzyme in both the sense and antisense directions. Look for transcriptional terminators and secondary structures. Identify any sequence that is not a direct repeat or a tracr mate sequence but has more than 50% identity to the direct repeat or tracr mate sequence as a potential tracr sequence. Take the potential tracr sequence and analyze for transcriptional terminator sequences associated therewith. 
     It will be appreciated that any of the functionalities described herein may be engineered into CRISPR enzymes from other orthologs, including chimeric enzymes comprising fragments from multiple orthologs. Examples of such orthologs are described elsewhere herein. Thus, chimeric enzymes may comprise fragments of CRISPR enzyme derived from, but not limited to,  Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma  and  Campylobacter . A chimeric enzyme can comprise a first fragment and a second fragment, and the fragments can be of CRISPR enzyme orthologs of organisms of genuses herein mentioned or of species herein mentioned; advantageously the fragments are from CRISPR enzyme orthologs of different species. 
     In certain embodiments, the Cas protein comprises a RuvC domain comprising RuvCI, RuvCII, or RuvCIII. In certain embodiments, the Cas protein comprises a RuvC domain comprising any selection of RuvCI, RuvCII, or RuvCIII. In certain embodiments, the Cas protein comprises RuvCI, RuvCII, and RuvCIII. In certain embodiments, either one of RuvCI, RuvCII, and/or RuvCIII may comprise at least one mutation to the naturally occurring sequence. 
     In certain embodiments, the Cas protein forms a CRISPR-Cas complex with a guide molecule and may target DNA or RNA. In certain embodiments, the CRISPR-Cas complex may target DNA. Intended herein by “targeting” is binding of the CRISPR-Cas complex to a polynucleotide sequence and optionally cleaving said polynucleotide sequence, wherein the polynucleotide sequence. A target sequence of the CRISPR-Cas system may be a portion of, equal to, or overspanning a polynucleotide sequence of interest. 
     In certain embodiments, the CRISPR-Cas complex may specifically target double stranded DNA. In certain embodiments, the Cas protein may both bind and cleave double stranded DNA. In certain embodiments, the Cas protein may bind to double stranded DNA without introducing a break to either or the strands. In certain embodiments, the Cas protein may open, disrupting the continuity of one of the two DNA strands, hereby introducing a nick of the double stranded DNA. 
     In an aspect of the invention, the Cas protein has, or comprises a region that has at least 10% sequence identity to IscB. In certain embodiments, the Cas protein, or comprises a region that has at least 12% sequence identity to IscB, at least 14% sequence identity to IscB, at least 16% sequence identity to IscB, at least 18% sequence identity to IscB, at least 20% sequence identity to IscB, at least 22% sequence identity to IscB, at least 24% sequence identity to IscB, at least 26% sequence identity to IscB, at least 28% sequence identity to IscB, at least 30% sequence identity to IscB, at least 32% sequence identity to IscB, at least 34% sequence identity to IscB, at least 36% sequence identity to IscB, at least 38% sequence identity to IscB, at least 40% sequence identity to IscB, at least 42% sequence identity to IscB, at least 44% sequence identity to IscB, at least 46% sequence identity to IscB, at least 48% sequence identity to IscB, at least 50% sequence identity to IscB, at least 52% sequence identity to IscB, at least 54% sequence identity to IscB, at least 56% sequence identity to IscB, at least 58% sequence identity to IscB, at least 60% sequence identity to IscB, at least 62% sequence identity to IscB, at least 64% sequence identity to IscB, at least 66% sequence identity to IscB, at least 68% sequence identity to IscB, at least 70% sequence identity to IscB, at least 72% sequence identity to IscB, at least 74% sequence identity to IscB, at least 76% sequence identity to IscB, at least 78% sequence identity to IscB, at least 80% sequence identity to IscB, at least 82% sequence identity to IscB, at least 84% sequence identity to IscB, at least 86% sequence identity to IscB, at least 88% sequence identity to IscB, at least 90% sequence identity to IscB, at least 92% sequence identity to IscB, or at least 95% sequence identity to IscB. In certain embodiments, the Cas protein, or comprises a region that has from 10% to 50%, from 10% to 45%, from 10% to 15%, from 15% to 20%, from 20% to 25%, from 25% to 30%, from 30% to 35%, from 35% to 40%, or from 40% to 45% sequence identity to IscB. 
     Examples of IsB include GeneBank accession Nos. EFH81639 and those described in FIGS. 1 and 3 of Kapitonov V V et al., ISC, a Novel Group of Bacterial and Archaeal DNA Transposons That Encode Cas9 Homologs, J Bacteriol. Dec. 28, 2015; 198(5):797-807. doi: 10.1128/JB.00783-15, which is incorporated by reference herein in its entirety. 
     In certain embodiments, the Cas protein may have a sequence identity to  Streptococcus pyogenes  Cas9. In certain embodiments, the Cas protein may have a sequence identity of 30% to the Cas9 sequence, preferably a sequence identity of 32%, preferably a sequence identity of 34%, preferably a sequence identity of 36%, preferably a sequence identity of 38%, preferably a sequence identity of 40%, preferably a sequence identity of 42%, preferably a sequence identity of 44%, preferably a sequence identity of 46%, preferably a sequence identity of 48%, preferably a sequence identity of 50%, preferably a sequence identity of 52%, preferably a sequence identity of 54%, preferably a sequence identity of 56%, preferably a sequence identity of 58%, preferably a sequence identity of 60%, preferably a sequence identity of 62%, preferably a sequence identity of 64%, preferably a sequence identity of 66%, preferably a sequence identity of 68%, preferably a sequence identity of 70%, preferably a sequence identity of 72%, preferably a sequence identity of 74%, preferably a sequence identity of 76%, preferably a sequence identity of 78%, preferably a sequence identity of 80%. In certain embodiments, the Cas protein may be at least 10% shorter than the Cas9 protein, preferably at least 12% shorter, at least 14% shorter, at least 16% shorter, at least 18% shorter, at least 20% shorter, at least 22% shorter, at least 24% shorter, at least 26% shorter, at least 28% shorter, at least 30% shorter, at least 32% shorter, preferably at least 34% shorter, preferably at least 36% shorter, preferably at least 38% shorter, preferably at least 40% shorter, preferably at least 42% shorter, preferably at least 50% shorter, at least 52% shorter, at least 54% shorter, at least 56% shorter, at least 58% shorter, at least 60% shorter, at least 62% shorter, at least 64% shorter, at least 66% shorter, at least 68% shorter, or at least 70% shorter. 
       Streptococcus pyogenes  Cas9 amino acid sequence: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 1) 
               
               
                 MDKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFGSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLADSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKERGHFLIEGDLNPDNSDVDKLFIQLVQIYNQLFEENP 
               
               
                   
               
               
                 INASRVDAKAILSARLSKSRRLENLIAQLPGEKRNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNSEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGAYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDRGMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGHSLHEQIANLAGSPAIKKGILQTVKIVDELVKV 
               
               
                   
               
               
                 IVIGHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEH 
               
               
                   
               
               
                 PVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKD 
               
               
                   
               
               
                 DSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDN 
               
               
                   
               
               
                 LTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKL 
               
               
                   
               
               
                 IREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIK 
               
               
                   
               
               
                 KYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE 
               
               
                   
               
               
                 ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTE 
               
               
                   
               
               
                 VQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKV 
               
               
                   
               
               
                 EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLP 
               
               
                   
               
               
                 KYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSP 
               
               
                   
               
               
                 EDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRD 
               
               
                   
               
               
                 KPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIH 
               
               
                   
               
               
                 QSITGLYETRIDLSQLGGD. 
               
            
           
         
       
     
     In certain embodiments, the Cas protein described herein may be an artificial Cas protein comprising domains of different Class 2 Cas proteins. In certain embodiments, the Cas protein comprises substantial structure similarity to a Class 2 CRISPR-Cas protein. Also intended are truncated versions of known Class 2, Type II proteins. 
     In certain embodiments, the Cas protein described herein may be a Cas9 protein, a portion thereof, or homolog thereof. In In certain embodiments, the Cas protein is a Cas protein occurring in or derived from  Corynebacter diphtheria, Eubacterium ventriosum, Streptococcus pasteurianus, Lactobacillus farciminis, Sphaerochaeta globus, Azospirillum  B510,  Gluconacetobacter diazotrophicus, Neisseria cinerea, Roseburia intestinalis, Parvibaculum lavamentivorans, Staphylococcus aureus, Nitratifractor salsuginis  DSM 16511 , Campylobacter lari  CF89-12,  Streptococcus thermophiles  LMD-9, and may include mutated Cas9 derived from these organisms. It is evident that these species are preferred and non-limiting species and that the Cas may be selected from any other Cas9 containing species. The Cas may be a Cas9 homolog or ortholog. The Cas protein may comprise a sequence that is homologous to a portion Cas9. 
     In certain example embodiments, the Cas protein is encoded by the following polynucleotide sequence or a portion thereof: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 2) 
               
               
                 acttcgccctcacccgcccggcctagctgatcaaaacgcctccgcccagc 
               
               
                   
               
               
                 cacgggagtgcagaaggcagctactccgctgccgcaacaaaggcgcgaag 
               
               
                   
               
               
                 gcttcggcctctcggaaggaattgaccatttcctgatgaatatggtatta 
               
               
                   
               
               
                 taagaagccatagtaatcccaaagggaaggaggcaacgcatgaggagctt 
               
               
                   
               
               
                 atgtcacggtccgcccgtcgcgccctgccgactctgaggtcagcaacatg 
               
               
                   
               
               
                 accgactgcaacgcttgcttgccgctgcacaattggtggatacagttgag 
               
               
                   
               
               
                 tttcactctgaggtcagcaacatgaccgactgcaacggtcgagcgcaacg 
               
               
                   
               
               
                 cccgcagtcaggccaccaacgggtttcactctgaggtcagcaacatgacc 
               
               
                   
               
               
                 gactgcaacccgggtagcagcatgataccgctaccggcgcatgacggttt 
               
               
                   
               
               
                 cactctgaggtcagcaacatgaccgactgcaaccgaccattctacggggt 
               
               
                   
               
               
                 tctgatgccccccaggggcgttggcgcagcagcgcccctcgcaaattgtg 
               
               
                   
               
               
                 actctgcgccaaactgccgcccatttgcgaaacagcgctttgctgtacct 
               
               
                   
               
               
                 cagagtggaatcagccccagcgagcgactcaatcgagtccacggagtatc 
               
               
                   
               
               
                 gaacccaataggcatagactccgtcaccagctacgtttgccggaatgctc 
               
               
                   
               
               
                 aagtagcacctgtggatgttgctccagtccgcagccctgcaagtctctgt 
               
               
                   
               
               
                 ggtaggacaactcgcccggtcgctgctctgtgcggcggcagggaaggtaa 
               
               
                   
               
               
                 taccccaccgcgaggtactgaagtgggtaacgtcccagagacatgtgcta 
               
               
                   
               
               
                 ccggcaaacatgggcgaggagcaaactcacccgtcttcggacggagtagg 
               
               
                   
               
               
                 cggtcgccttcgggcagactgccgaacccccgtaagggggaggacatata 
               
               
                   
               
               
                 atgcgcgtcttcgtgcaaaacgccgatgcgactccgctgatgccctgcca 
               
               
                   
               
               
                 tccggccagggcgcgcaagctcttgcgcaaggaccgcgcgcaggtcgtca 
               
               
                   
               
               
                 atatgcacccatttgtcattcgcctgactgagcaaatccaggacccaggg 
               
               
                   
               
               
                 atgcagaccgtggagctcggagtggatgacggagctaagaatgtgggcct 
               
               
                   
               
               
                 cgctgttgttcagcggcgtagcaagcgccccgatgtcgtgatctttgagg 
               
               
                   
               
               
                 gcgtaatcgaactccgcacagacatgaagaaaggcttggacgagagacgt 
               
               
                   
               
               
                 gccatgcgccgaggacgccgcagccgtatccgccaccgccaaccgcgctt 
               
               
                   
               
               
                 tgacaaccgaccgcgtgccaaatgcaaggtctgcggccgcaacacccctg 
               
               
                   
               
               
                 agggtcaggccctgtgccgcccccacgccgctgagggccatcacaagtac 
               
               
                   
               
               
                 gcacacctcgagaagaagccgggatggatacctcccagcatcaaggcccg 
               
               
                   
               
               
                 gaaggaccagaccctccgcaccgttcgccaactgctacgctggttgccca 
               
               
                   
               
               
                 tttccacagcccatctggaggttgccttctttgacacccaggccttgagt 
               
               
                   
               
               
                 gagcccaccctcactggcgagcagtatcaatatggcccgaacttcggaca 
               
               
                   
               
               
                 tcgcaatcgcaaggctgcagtcctcttcctctacaagcacacctgtcagt 
               
               
                   
               
               
                 attgtggcgcaaccgagggccgcatggagatagaccacatagttccacgc 
               
               
                   
               
               
                 ggcgccggcggcaccgacaccatcaccaacttgacctgtgcttgtgtcga 
               
               
                   
               
               
                 gtgcaatcgcaagaaggggaaccgcactcccgaagcagcggggatgaagc 
               
               
                   
               
               
                 tgcgcagatcgccaagagctatcgctctgcgcttgcgcgatgcggccgtt 
               
               
                   
               
               
                 gttcaggcggggaaaagctatctggagtatcacctccgcgacatgattcc 
               
               
                   
               
               
                 ggaggtgcggctggtgctgggctggatgacgaactggtggatgaagaaga 
               
               
                   
               
               
                 tgaatctgcccaagcacgagagcgatgggaagacgaagctgcactacacg 
               
               
                   
               
               
                 gatgccgtggctatggtcttacgccaacgccgagctacaacagccagaat 
               
               
                   
               
               
                 gtccgcagtggtgtatcgcatcgaggcccgccgccgccagacccgacaga 
               
               
                   
               
               
                 tgtttaagacggaaccgtattcgttcaagcgtaagcccccaatggccgac 
               
               
                   
               
               
                 tgcgtactgccggctcgcaagggaggtaagcgcaggctgctcaagaccac 
               
               
                   
               
               
                 cccgaatgaccaccttctcgcctgggtggacgatgcgggcaaacgcaaca 
               
               
                   
               
               
                 agcaggtagtaccgaacaggcgatatcccgacgccgacatgcctgtgctg 
               
               
                   
               
               
                 ccggcaatggcgcaagcagtgctacggttcgacagaaacgatattgtccg 
               
               
                   
               
               
                 cgtcaaagggcgtctcgctcgtgtatccgcggttttcaccaacggctctc 
               
               
                   
               
               
                 tgaaggttcaacccaccgacgcgaagcagcctctgtcggtgtccccctgg 
               
               
                   
               
               
                 acggcaagacttctcgccaaggcgcggcccgtcacgttcctgccctgtcc 
               
               
                   
               
               
                 agttccccccgtatccagcgcctggtagccagcgaggaggccggacacaa 
               
               
                   
               
               
                 tgaaatgcagcattgacggctgccccggcgagtacgaggaacgcaagatc 
               
               
                   
               
               
                 gtccacaccgtccgacatcacgggcaggtagtggtcattgatggcgtgcc 
               
               
                   
               
               
                 ggcagaggtctgttcggtgtgcagtgatgtcctactgaggccggagacgg 
               
               
                   
               
               
                 tccggcgcattgaagagttgctgcagagcaagacggccccgacgagcact 
               
               
                   
               
               
                 gccccgctctaccagtacgtatgaccacggcttgcatttgccgcaccccg 
               
               
                   
               
               
                 acgccccggctgctgccgcggcatattccatctcccgtgatcggacaggc 
               
               
                   
               
               
                 gagacgcctatcctaccttcttacgtcttctcactatcctgctctatcgt 
               
               
                   
               
               
                 cgcggcgaggcgggggatctcggggttggtgctgacgcggcgcgt 
               
            
           
         
       
     
     In some examples, the Cas protein has the following sequence: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 3) 
               
               
                 MRVFVQNADATPLMPCHPARARKLLRKDRAQVVNMHPFVIRLTEQIQDPG 
               
               
                   
               
               
                 MQTVELGVDDGAKNVGLAVVQRRSKRPDVVIFEGVIELRTDMKKGLDERR 
               
               
                   
               
               
                 AMRRGRRSRIRHRQPRFDNRPRAKCKVCGRNTPEGQALCRPHAAEGHHKY 
               
               
                   
               
               
                 AHLEKKPGWIPPSIKARKDQTLRTVRQLLRWLPISTAHLEVAFFDTQALS 
               
               
                   
               
               
                 EPTLTGEQYQYGPNFGHRNRKAAVLFLYKHTCQYCGATEGRMEIDHIVPR 
               
               
                   
               
               
                 GAGGTDTITNLTCACVECNRKKGNRTPEAAGMKLRRSPRAIALRLRDAAV 
               
               
                   
               
               
                 VQAGKSYLEYHLRDMIPEVRLVLGWMTNWWMKKMNLPKHESDGKTKLHYT 
               
               
                   
               
               
                 DAVAMVLRQRRATTARMSAVVYRIEARRRQTRQMFKTEPYSFKRKPPMAD 
               
               
                   
               
               
                 CVLPARKGGKRRLLKTTPNDHLLAWVDDAGKRNKQVVPNRRYPDADMPVL 
               
               
                   
               
               
                 PAMAQAVLRFDRNDIVRVKGRLARVSAVFTNGSLKVQPTDAKQPLSVSPW 
               
               
                   
               
               
                 TARLLAKARPVTFLPCPVPPVSSAW 
               
            
           
         
       
     
     In certain example embodiments, the Cas protein is encoded by the following polynucleotide sequence or a portion thereof: 
     
       
         
           
               
               
            
               
                 (SEQ ID NO: 4) 
                   
               
               
                 gatctacgggattacaagcctcctgtatgggaatgatagcagcgggcagatggcactgccgggtaacctggcggacctcc 
                   
               
               
                   
               
               
                 atgtgccgcagatcgcaaaaatcatttgccggtgcattttttatagagcaccggtcccggattacctggtatcatgggct 
               
               
                   
               
               
                 atagccaatgcaagcaagccgcacatttataaaaaacgataccactggaggaacgtgctttcccttgcatgtgctttcct 
               
               
                   
               
               
                 aaaatacaacctggcactgcaaaagccaggggaatcggaggttttgccattggacatcatgaagggctgcaaagacaggg 
               
               
                   
               
               
                 attacttatacggcaggttgcttgccgtggctgaccggatcgaataccggacttttaataacgagcatgacagcaggaga 
               
               
                   
               
               
                 ctgacgaacgcaaaacgctatatgcagagattttcagtccagccatgtcaaacctggcagctgatcgaacagaggatcca 
               
               
                   
               
               
                 gccatacctgcagaaactgaaaatgcctgaacgtctccattacctgaaactcatagacagcatcatgggtgaaatggatc 
               
               
                   
               
               
                 ccgctgatttcagtgataaccgcccattgaaagggctttacctggtcggattccaccaccagtcttacagtttttatgat 
               
               
                   
               
               
                 aatgcagggacgaatgtgacagacaaggaaacaggggaggattaattcatgtctaagttaaaagggaaaattgatttcac 
               
               
                   
               
               
                 cttatttgtaaccgtaaactatgcaaacccgaatggggatccgctgaatgggaaccagccaaggactaacctgaaaggct 
               
               
                   
               
               
                 acggcgagatctctgacgtatgcatcaaaaggaaaatacggaaccgtatgcaggatttagggtacaaggtctttgtccag 
               
               
                   
               
               
                 tcggacgacagggcggacgacgggcataccagcttaaaggaaagggctgtcagctgccaggaactcaaaaaagagctcgg 
               
               
                   
               
               
                 caaaaaaagaggggctgaccccaaaatttgtgcggaaatcgcatgcagggagtggctggatgtacgttccttcgggcagg 
               
               
                   
               
               
                 tgttcgcatttaaaggcagcaacgcatccctggggatccgtggacctgtgacaatccagacagccattagcctttcccct 
               
               
                   
               
               
                 gtggaaattgaaagcatgcagatcacgaagagtgcgaataatgaaccggtggacgggcggtcatccgatactatggggat 
               
               
                   
               
               
                 gaaacatttcgtgacattcggcgtatacaaaattgtaggcagcgtgactacccagcttgcagaaaagactgaattttcac 
               
               
                   
               
               
                 aggaagacgccgcaatcctgaaagagtgtttacggacgctttttgtaaatgatgcatcctcagcacgccctgatgggagc 
               
               
                   
               
               
                 atggatgttgccaggatgtactggtggcagcatacggaaaataccccagttgtaaccagccataggatccaggatgcctt 
               
               
                   
               
               
                 ccattatgaaaaaccggcagaccctgaaagatttgatgattaccttgtgtattgggaaccgattgacggatgtattactc 
               
               
                   
               
               
                 cagagatctttgagtcaataactcctgactaagtcaggagcttgcttcggtgagttcctgtcccgccaagcgggttattg 
               
               
                   
               
               
                 agcagaaccaagacctgccgttcactccggggtaacgccaagccccggacactggcacaggcaggccaaggttatggcaa 
               
               
                   
               
               
                 cacaacaggggtatacccctgacttacagtatgaaaggaatgttttatggtttatgtattggacatggaaggtaagccat 
               
               
                   
               
               
                 tgatgcccactacccggcacggatgggtccgcagggccttaaagtccgggagggcgaaagcagtacagaccttaccgttt 
               
               
                   
               
               
                 acgatccggctgcagtatagcctggatgattctgcattacaggatattaccctggggatcgaccccgggcgcaccaatat 
               
               
                   
               
               
                 cggggttgccgcagtccgggaagacggtacctgcctgtatgccgcccactgtgaaacccgcaacaggcaggtccggaaac 
               
               
                   
               
               
                 agatggatgaccgccgtatgcaccgccaggcttcgcgccggggcgaacggctccgcaggaagcgccgggcaaagcggaac 
               
               
                   
               
               
                 ggtacattaaagcaggtgacattttccatgacagcaatgggtccccgccccaacatcaataacaggggcgaatttttcag 
               
               
                   
               
               
                 gatgctgccgggatgcaaggaagtttctgtttacaaggatatccggaatacagaagcccggttccagaaccgggcacggc 
               
               
                   
               
               
                 cggaaggctggcttagcccgactgcggggcacctgctccgcacgcatctgaacctggtacgcaagatacagcggatcctg 
               
               
                   
               
               
                 cctgtttccagggtttcgttggaactgaaccggttttcctttatggagatggaagccggtgggaagctcccgcattgggc 
               
               
                   
               
               
                 atatcagtgcggcccgctgtatggaaaggggggcgtacaggacgccatttctgagatgcaggacgggaaatgcctgttgt 
               
               
                   
               
               
                 gtgggaaatcccctatcgatcactgccaccaccttacccagcgcgcatggggcggaacggaccggctggcgaacctggtt 
               
               
                   
               
               
                 gggctttgcagcggatgccataaaaagatccatacggatatggccgccagcaggaaactggaggcaaaggccggcaggcg 
               
               
                   
               
               
                 gaataaaggcttccgggcgctgtccaccctgaaccagatcatcccatcccttgcagacagcctggagggaatgtttggaa 
               
               
                   
               
               
                 accggttttatattgtcaatggatgggacacaaaacagttccgtgaggatcatgggatagaaaagacccatgaacaggat 
               
               
                   
               
               
                 gcatactgcatagcgtgttctactatgcctggggtgcggaatgtttccccggtagtggcaacattccaggtccggcagta 
               
               
                   
               
               
                 ccgccgccatgaccgtgcccttgtcaaaagacagacagaacggtgctattacctggggaagacgaaggttgctgtcaacc 
               
               
                   
               
               
                 gcaggaagcggatggaccagaaaacagattccctggaagactggtaccaggacatgcggacggaatatggggataagacc 
               
               
                   
               
               
                 gctgacgggatgcgttcccggctgaaagtaaagaaaagccagaggtcctacaataacccagggaggttgctccctggcgc 
               
               
                   
               
               
                 aaaattccattatggtgggaaaacctatgtcatggaatcgcagatgacaaacggacaatactaccgtgctgtaggacagg 
               
               
                   
               
               
                 gcaagaaaaatttccctgcagcaaaatccaggatcctatgcaggaaccaggggcttgtgactgttggggtcagctattga 
               
               
                   
               
               
                 cccgcattcatctcccggttctaccgggagttttctgcttaagtgtttaaataaattgaccgtgtcaatatgacaccctc 
               
               
                   
               
               
                 tataaatcgcatcttatatattaggtgtgcgagttgaaacgtgacatattaatagaagataaaaaatatcgtatcttaat 
               
               
                   
               
               
                 ataggtgcgcgagttaaaatgcaaaaaacgcaaaattatcaattggtttttatcgatcgcatcttaatataggtgcgcga 
               
               
                   
               
               
                 gttgaagcgtggaagccatgtgttaaatgggaagaagcatctgtcagggaaacctgatgggtgcttttttctgcaaaaat 
               
               
                   
               
               
                 tgctatggaaaaagaaatatgtgaaaaaaatctgtg 
               
            
           
         
       
     
     In some examples, the Cas protein has the following sequence: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 5) 
               
               
                 MVYVLDMEGKPLMPTTRHGWVRRALKSGRAKAVQTLPFTIRLQYSLDDSA 
               
               
                   
               
               
                 LQDITLGIDPGRTNIGVAAVREDGTCLYAAHCETRNRQVRKQMDDRRMHR 
               
               
                   
               
               
                 QASRRGERLRRKRRAKRNGTLKQVTFSMTAMGPRPNINNRGEFFRMLPGC 
               
               
                   
               
               
                 KEVSVYKDIRNTEARFQNRARPEGWLSPTAGHLLRTHLNLVRKIQRILPV 
               
               
                   
               
               
                 SRVSLELNRFSFMEMEAGGKLPHWAYQCGPLYGKGGVQDAISEMQDGKCL 
               
               
                   
               
               
                 LCGKSPIDHCHHLTQRAWGGTDRLANLVGLCSGCHKKIHTDMAASRKLEA 
               
               
                   
               
               
                 KAGRRNKGFRALSTLNQIIPSLADSLEGMFGNRFYIVNGWDTKQFREDHG 
               
               
                   
               
               
                 IEKTHEQDAYCIACSTMPGVRNVSPVVATFQVRQYRRHDRALVKRQTERC 
               
               
                   
               
               
                 YYLGKTKVAVNRRKRMDQKTDSLEDWYQDMRTEYGDKTADGMRSRLKVKK 
               
               
                   
               
               
                 SQRSYNNPGRLLPGAKFHYGGKTYVMESQMTNGQYYRAVGQGKKNFPAAK 
               
               
                   
               
               
                 SRILCRNQGLVTVGVSY 
               
            
           
         
       
     
     In certain example embodiments, the Cas protein is encoded by the following polynucleotide sequence or a portion thereof: 
     
       
         
           
               
               
            
               
                 (SEQ ID NO: 6) 
                   
               
               
                 aaacgatacataaccaagtgttgcaccccgcacggggtgcattagattgaaatcatcgggtcacagataacccggacgtg 
                   
               
               
                   
               
               
                 cgctgtgctgtcgcaccccgcacggggtgcgttgattgaaatgattgaaatcgcaacgtcggaatcagcagggcacttgt 
               
               
                   
               
               
                 cttgcgttgcaccccgcatggggtgcaactggtgtaggtataatatcctagcacgaacgaaataaatcaatatgtgttaa 
               
               
                   
               
               
                 gatatcttgcatcaaaagcagtctttatgagaaatcgtaaagactgctttttgtttgtgtgatgcaaaccgttacaaaag 
               
               
                   
               
               
                 acggaaacttatagcacagcccacaagtgggcagaaaggaaaaatatgctaacagaaaacatcaaacgaaaaattaagga 
               
               
                   
               
               
                 cgttgtaaaagaaatcaataattacaacgaaatcaaaatcatgccggacgacacaatcattgatatggaccagacagtac 
               
               
                   
               
               
                 gttggaaccgtgataccgttgctcgtctaaatgctaccgttggttcgcgtcaggcagaaatcggaacaaaagtggtagca 
               
               
                   
               
               
                 aagatagttgaccttattaacacgttccttttagaacaaaagtgcgaacatatcgagcaggcagcaaaaaacatcatctt 
               
               
                   
               
               
                 tgagtctatgcacgaagaaatcggtgatgtcttcctccacgaccacgacgacaactacgattacctgcgcagttgtgatg 
               
               
                   
               
               
                 actatagtttggaagggcaaatcaccaaaattgtcaacaaaaacgatgcttttgtggatatgctcaacagcatcgaaaga 
               
               
                   
               
               
                 gagcgcaagtctctaaatacgcaaaaagtaacgcaggatgcaccttggatgaatattcgcgctccgttcacctttacatt 
               
               
                   
               
               
                 tgctggaatcgaatattcaagcatttttgccgctattatctgctacaaatatggcgacaggttgaacaaagttggctgca 
               
               
                   
               
               
                 atacgtggaaagcatccaatattctctcttatatccggaaagaaaaaatcaaaacgagtcctgtttggaaaaacatcccg 
               
               
                   
               
               
                 gctcgccgcaaaaaaatcgaggatatcatcacggctgcagcttgtgctgggctgtacgagaaactctttgaaacaggaaa 
               
               
                   
               
               
                 caagaggctgattgcggatgacagtatcatcggagaatacggaagcatcctgacaaaggtacgagatgatgcatataagc 
               
               
                   
               
               
                 aaatgcgaatcgactttattgatgctaaagggtatttgcaaaaataggtggtttgaataaaactatctgtttttacaagt 
               
               
                   
               
               
                 atcacaggcatttggatgtttgttgcgcctattgattttcccagttgatagctccgcacgagtggcattgtctcgtacaa 
               
               
                   
               
               
                 taccgctcacagcaaacacccagccaagggaaacacaacctcctgtttcaacaggagagacttacagtaaaaggaggtga 
               
               
                   
               
               
                 cggatgtgtccactatttatgtactcaacaaagacggtaaacctttgatgcctacgactcgctgtggtcatgtacgtcgt 
               
               
                   
               
               
                 ctgcttaaagaacaaaaagcacgagccgtagcatcaaaaccgtttaccattcaactgttgtatgaaactgacaatatagt 
               
               
                   
               
               
                 acagccactttacttgggcatcgaccccggtagaaccaatatcggcgttgctgtagtcaaagcagacggcacggcagttt 
               
               
                   
               
               
                 ttaccgcgcatttggaaactcgtaacaaggaaattccgaaactaatgaaaaagcgtaaagattcccgccgcgcaagacgt 
               
               
                   
               
               
                 accaacggcagacgatgccgccgtcaacggagagccaaagcaaatggcaccatttctaaaaagtgcgtaaagcaagccac 
               
               
                   
               
               
                 tgctcaaaatggcagtgccagcaagcgtgcaaagaaaattggtgtcatcaagcgccatcttccggattgtgagaaagaag 
               
               
                   
               
               
                 tcctttgcatcggcatcaaaaataaagaagcaaagttcaacaatcgcgcaagaccggaaggctggctcacgcctaccgca 
               
               
                   
               
               
                 aatcagttgctacagacccacgtcaatttggtaaagaaaattcaaaagtttcttcccatcagtgatatcgtgcttgaagt 
               
               
                   
               
               
                 caacaaatttgcgttcatgcagttggataatcctaacattcagaaatggcagtatcagcaaggtccgctctatcaaaaag 
               
               
                   
               
               
                 caaaccttgaagaagccgtctctgaaatgcaggaacatcattgcttgttttgcaagaagccgattgagcattaccaccat 
               
               
                   
               
               
                 gtagtgcctcagcacaaaaacggcagcaatacaatcagcaacatcgttggcttatgcacaaagcatcacgaccttgtaca 
               
               
                   
               
               
                 taaggatactgaatggcatgaaaaacttactaaaaagaaaacaggtctcaacaaaaagtacggtgcgttaagtgtattga 
               
               
                   
               
               
                 atcaaatcataccggcactaacaaaagaattgagctctattttcccaaaacacttttttgtagcaacaggaaaaagcaca 
               
               
                   
               
               
                 tacgattatcgtgcagcgcacggcgtaagtaaagaccactggctcgatgcctattgcattgcttgctccgttttgcctaa 
               
               
                   
               
               
                 cgatgtttgcgacagcagcatcaataatcgcgtgccgtatgagcttaaacagtttcgtcgtcacgatagaagagcactgc 
               
               
                   
               
               
                 acaaagaaaatatgagccgtgtgtacacgctcaacggtaaaaaggtggcaacaaatcgccacaaagccattgagcagact 
               
               
                   
               
               
                 actgacagtttggaagagtttcgccaaaacaacctagataatgtatgtaaactcaaggtaaaagagcatcatccggaata 
               
               
                   
               
               
                 tcgaaatcctaaacgcaacttccccggctgtgtgtttcttgttggtaaacaaactcatgtaatgcaaggaaccagcggct 
               
               
                   
               
               
                 cacacaacggtaaagcagatggatattacgacacaaacggcaactcgtattcatctggtaaatgtaagtttgttgccaaa 
               
               
                   
               
               
                 aacgaaggaattatattcacataaattagtagaccacctattttcaaatagaaaatcacctaattttgcaaataccaatg 
               
               
                   
               
               
                 ctaaaaaaggaagcaacactctataacagatattaagcaaaacgccgtccacctcttggtgggcggtttttttattgcct 
               
               
                   
               
               
                 taaagcggatgcagagagacacaacatatgttataatagagttatattattgagagttcgagtctctctgggcatattta 
               
               
                   
               
               
                 gctatggagagaatgtcaaaaaggtacaggtccatattttgtcacaagtgcaataaggagtcacaaatgagtaagcacat 
               
               
                   
               
               
                 actaggctcggaccgcattctacacgaaggtgctggctaccgtaacaagtacacacttaaaaaccataagcctccggttg 
               
               
                   
               
               
                 gcagcagagaaaacccgtccaacccaaagcaagaggggacagacgcagtgtacatcccagataccgccaaatggtgtagc 
               
               
                   
               
               
                 aaaaagtaaaccacaagttgattgacccaccacgcagatgtggtataatataatcagaacgaaacgaaaggagacaaccg 
               
               
                   
               
               
                 aagatgctgtgcaagactgttaatgctatgtcgtttgctgagtatagttatgaatctgaattcgagtcctacgaatccag 
               
               
                   
               
               
                 ctttgtttcctataccaatcgacaagcaaaaaacagaccatgtacagatgcggtgcgtctctaaacgataactgcatttt 
               
               
                   
               
               
                 cacacgctgcttgtcgagtcatttcggcaggcagcgtttttttgttgcctgtagtacagaaaggcagcaagaaaatgaac 
               
               
                   
               
               
                 gttccaacaatcgatatccagcagacaggtgccaacatcaaggcactccgaaaagcggcaggcatcaaggtcaaggatgt 
               
               
                   
               
               
                 ggcagacacgctcggtgtatccacacaggcagttgccaaatggcaggcaggcactgcacttcctaccatcgacaaccttg 
               
               
                   
               
               
                 tgattctcgccgcgatgctcgatacgaaaatcgatgacatcctcgtcatagcataaaccctcgccgcaggattgcggcta 
               
               
                   
               
               
                 tatggccgaatagacgaattggttaagtcgcaagcccttcaagcttgagagtatgggttcaagccccatttcggtcacca 
               
               
                   
               
               
                 tctgcttctgtagctcagttggtagagcagtaggttgaagccctatgtgtcgctggttcgattccagccgggagcaccac 
               
               
                   
               
               
                 gaggcttaatgcctccttatatgtgccggtatgcaagtggttaaagtacgcggtctgtaaaaccgttccgttacggttcg 
               
               
                   
               
               
                 ctggttcgaatccagcccggcacaccataaggccccttcgacaagttggtctaagtcaccacactctcaatgtggagtca 
               
               
                   
               
               
                 gcagttcgagtctgctaggggtcaccaacgcaccctgcatagggtgtttacatgcagaggtcgcctaacggtatggcaac 
               
               
                   
               
               
                 ggaccgctaatccgtcgcgaggcaaaacggcactcactacgaagtgccaatcaatccctcgcctgcgagttcgaatctcg 
               
               
                   
               
               
                 ctctctgcgccatatgcatgtgtgtccgagtggctgatggaactggtccagaaaaccagcggtcagaaatggcccgtagg 
               
               
                   
               
               
                 ttcgaatcctaccacatgcgccaaagagcctatgcttggtgtttatcaagcataggctcttttgttttggactaaataaa 
               
               
                   
               
               
                 aaaatcgaagaagcagcatctatcatgcgtagtaaaaaaacggataatgctgtttgcttgatggatattcccgatgattt 
               
               
                   
               
               
                 tgggaaagatgacccggatttgtacgaaaaaacatatgacgcagcaatcgcttttgcaaaagaatttccagacacattat 
               
               
                   
               
               
                 ggtgctatggaaatcatgacttgagttatgtatgggggaagctagaaacaggatacaatccagaacttcgagatttggta 
               
               
                   
               
               
                 tgtcaaaagatagaggagctgaaagaggttcttccatcgccaactcagcttgcatatatccaccgaattgacaaaactct 
               
               
                   
               
               
                 gtttatgcatggcggtctctcaaacttctacgtacatcgttgggtaacgccaagcagtcaaaaagcgattggtcgtacca 
               
               
                   
               
               
                 tcaaagaaatcaataatatgtatgggttttgcacaagcaaacagtttacaggaggccatgacgattgcgctcgactatac 
               
               
                   
               
               
                 gcaatctcaaatgacagaaaaaaacgtcaagaaaactatcagagataatctgtattatatcccttcggttcgcacagggt 
               
               
                   
               
               
                 acacccactaaaaataataaatatccatccaactaaaggagctgcctaccgtttggtgggcggctctttttattgccgca 
               
               
                   
               
               
                 aaagcggatgcagagtaacacagtatatgctataataaagtcatcaaagtggtgcgaaccttaagctaacataaaattgc 
               
               
                   
               
               
                 tgggagcttcgcaccaaaaagtaccagatagagggtggttccaatgcagtaaataggcgagtgcttcagtgtgtgaagca 
               
               
                   
               
               
                 cataattagcaaaactgtacatcctacgtgctaccaagatacgcttgaattgtatagttttgctgtcgcaccccgcacgg 
               
               
                   
               
               
                 ggtgtattagattgaaatgattgaaataaacggcgtgtccttatcggtacacactgcgccgtaagcgatgggcgcaaaac 
               
               
                   
               
               
                 ggttgcctgcatattccacaacgccccagcccacgatcgcatagccggggtcgatgcccaaaacccgcatagtatcccct 
               
               
                   
               
               
                 ttccctgtgtttgaatctattcaggaaacgccgcaccggcaaaagggtgcactatccccatttaactgtatcagtatacc 
               
               
                   
               
               
                 acaaagccgtgtatgcggcaaggaaaagcccttccgcatttgcaaatttcttttgcggtggtgcgttctatttcatgtat 
               
               
                   
               
               
                 tttctgaattttttcatttttttcgaaaaaagggcttgctttttctgcttggatttggtatagtatacaagtcgcaagga 
               
               
                   
               
               
                 catgcgcggttagctcagctggtagagcatctgcttgacgtgcaggaggtcacaggttcgagtcctgtaccgcgcaccat 
               
               
                   
               
               
                 aaccggacaccatttttgatacaatacgtatcttgactggtgtccggttttttattaaggtttagattcttcagggcctt 
               
               
                   
               
               
                 taccctttattatgatttaacgctaactcttattgcaaaaatgaaatctatttttcacagagcgccttccggttacttta 
               
               
                   
               
               
                 cttttcggcaacagaggtttttatgcacccgcgggtgtttgtgatatttcggggtgagatatccggcccgttgtggggca 
               
               
                   
               
               
                 tcgcccctctactttgggcaaggcacatcgcccatttttattgttcgctccgggtgggtttcggcggggtcgggtgaccc 
               
               
                   
               
               
                 cggccctaccagccattttatggttttccgcccatcatcaaatgctctgtggggaacggtattgaccgttccgaaagttt 
               
               
                   
               
               
                 gcaaagatccaacatctcaaaatcaaatcacttcgtctattccctatcccaccagccaaagaattcccattcggggcact 
               
               
                   
               
               
                 accacaa 
               
            
           
         
       
     
     In some examples, the Cas protein has the following sequence: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 7) 
               
               
                 MPTTRCGHVRRLLKEQKARAVASKPFTIQLLYETDNIVQPLYLGIDPGRT 
               
               
                   
               
               
                 NIGVAVVKADGTAVFTAHLETRNKEIPKLMKKRKDSRRARRTNGRRCRRQ 
               
               
                   
               
               
                 RRAKANGTISKKCVKQATAQNGSASKRAKKIGVIKRHLPDCEKEVLCIGI 
               
               
                   
               
               
                 KNKEAKENNRARPEGWLTPTANQLLQTHVNLVKKIQKFLPISDIVLEVNK 
               
               
                   
               
               
                 FAFMQLDNPNIQKWQYQQGPLYQKANLEEAVSEMQEHHCLFCKKPIEHYH 
               
               
                   
               
               
                 HVVPQHKNGSNTISNIVGLCTKHHDLVHKDTEWHEKLTKKKTGLNKKYGA 
               
               
                   
               
               
                 LSVLNQIIPALTKELSSIFPKHFEVATGKSTYDYRAAHGVSKDHWLDAYC 
               
               
                   
               
               
                 IACSVLPNDVCDSSINNRVPYELKQFRRHDRRALHKENMSRVYTLNGKKV 
               
               
                   
               
               
                 ATNRHKAIEQTTDSLEEFRQNNLDNVCKLKVKEHHPEYRNPKRNFPGCVE 
               
               
                   
               
               
                 LVGKQTHVMQGTSGSHNGKADGYYDTNGNSYSSGKCKFVAKNEGIIFT 
               
            
           
         
       
     
     In certain example embodiments, the Cas protein is encoded by the following polynucleotide sequence or a portion thereof: 
     
       
         
           
               
               
            
               
                 (SEQ ID NO: 8) 
                   
               
               
                 tacgagatactgatcggtctcgtgggctcggagatgtgtataagagacagtgactcatttgttcaactatgacgtgagaa 
                   
               
               
                   
               
               
                 ataaaaaaagttgattattctgtcaaagatgaataaatatgtatggcataaataaaaaatctgtagtttccttattctgg 
               
               
                   
               
               
                 aggtgctatggatcatgcgaacaaaacaggaatttgaatttttcgggatccgattaaaacaactcagaaaaagtaaggga 
               
               
                   
               
               
                 atcacgcaagaagaattggcgtacagaatcaactgttctacaacatatatcagcaaactggaaaacgggaaatctatttg 
               
               
                   
               
               
                 cagtatggaacggttgtttgaaatctcggacgttttgcaatgcgatgtatcggaattattgttgggaacaaatcgcaaca 
               
               
                   
               
               
                 gccaaatctatttacatactgaattttctgaacagtttcagaaactgtcttcacatgacaaagaaattatttgtatgctc 
               
               
                   
               
               
                 atgaaatgtatgctggaaaagaccgaggagggtatccctgaatatgccgttccgtgatttctcatctgacgaaagggata 
               
               
                   
               
               
                 tgacaagatacttcgagtattcagctataccattatacaaatacgcattgaagctctcagacgatccacaaatagcggaa 
               
               
                   
               
               
                 gatatcgtccaaaatacattcatgaaactgatccggtactttgataatgttcgtggattttcaaatgagcggttcttcct 
               
               
                   
               
               
                 ttatgcaaaacgtgtactggtccatgaattcggcgccgaactgaaaataaaaagcacaatatctttgatcacggatatgg 
               
               
                   
               
               
                 atatgatccaagataaagaagaaaatatcgatttcatagatgcgatcttgacaagagacgaaataaaacgatgtataaat 
               
               
                   
               
               
                 caacttaatccaaaatacaaaatcatcgtatatctccgatactttgaaaataaaaccagtacccagatcggacagatatt 
               
               
                   
               
               
                 gaatatcccggataatcatgtcagaaccatacttagccgagccaggaatgaactgaaaagaaaatataaggaaataaagg 
               
               
                   
               
               
                 aaggaaataccgggcatgacaaataaagatctggatattttgaaaaataaaaaggcagaacaagaattctttgatatggc 
               
               
                   
               
               
                 cgcaagagaaattgataaggaattggaccaagtatttatagaagcccaaaacctgccggacccggacccggaaatcttac 
               
               
                   
               
               
                 aaaggatccaaaaaaacttaaacgcagaaatgtataaacagactgtggcttggtatatcaaaaaatgggcgagcggaatc 
               
               
                   
               
               
                 ggaaaagtcgcagcatgtatcgttttcgtgtcaggagtaatattcacaggcgcttatttacatgtcgaggcgaccaggaa 
               
               
                   
               
               
                 ccaagttaataatttctttctggaattgtttgatgaatacgctgtgatccatcacggcgaagatgatcgggaatctggtg 
               
               
                   
               
               
                 tcacgatgcctgcatcgtggtcgggtccgtttagtgttggctgggtaccacaacggtttacggatgtctttgcacaggat 
               
               
                   
               
               
                 atgacatatagctgggcgctatattacgatgatttgaatagcgataacgatctggcaatttatgtctgggatcctgatat 
               
               
                   
               
               
                 tgcaccaacgatcaatacagaaggggaaaataaaatcagtgaacaggaaaataaaatgacggaatactatcataatcctt 
               
               
                   
               
               
                 caagaaatacctattctgttgtatctgtacaaaacggatatatgatttatgtgaaaaatgccaatacccaagaagaagca 
               
               
                   
               
               
                 gaaaaaatattgaaaaatatttctttctgatgttgcgatttacgttgtttttcgttcttattatttggttcgatccaata 
               
               
                   
               
               
                 tttttgaaccagaaaggacgataacaatgaaaaaactgatttgtgtgtggctggctgcagttatgatgttctgcttctct 
               
               
                   
               
               
                 acctccgtatttgcagcagatacagcagagtcgatctcgaattcacacatcgaggcccgatacaatgctttcgacacggt 
               
               
                   
               
               
                 ttgggtatacttgacggaaacgcaaccggggatccttcatgtggaaggcggagccgggacatccagcagcagcaattacg 
               
               
                   
               
               
                 tcgagatccgtgttgtgatctgccagtaccagaactcgcaacaagggatggtcccggtagatggatttgaatggacatct 
               
               
                   
               
               
                 gcatctcagtttggaacatctctgcaggcgaatcgctcggtttcacgcggcagttaccaggcgatcatctatgcaaaatg 
               
               
                   
               
               
                 ctaccagaacggggtcctggtcgatgacgttgaggtagagagcagcatcgtaaacgttgtgagctgatacggttcctaaa 
               
               
                   
               
               
                 cgaccctgtatggaggaatcaatgaatagatggtcctgtacttaccagtaagacaaaatctatatcatgtggatttgggg 
               
               
                   
               
               
                 aattatgtctcttatggattgaaagcgctttctctgtcgattttttctatccgatccattaccttcgtgtcggatgtgac 
               
               
                   
               
               
                 acccgataggacccggatctttcatttggcatggctttgttccataggacaacttgacccaaatcaattgttggatgtga 
               
               
                   
               
               
                 tcgaagatttcatctgacaaatcttaattttcacttcactattaaaattggatcgaaccagcggtgtcagaagattttct 
               
               
                   
               
               
                 ggcgccgcttttttattgcactttgttgcaaagtatttatactggattcataagataaatcaaattgctacaggcacttt 
               
               
                   
               
               
                 agttttatcttcataatccgcaaaggtcatctcaaacagagatggccttttatcttttgcaagaaaggaaatttgtatga 
               
               
                   
               
               
                 acgtaaatcaatctctctatgagaaaatgaccgctgaacagaataagttccgggattggttgaaaggccaaaatccgcag 
               
               
                   
               
               
                 gagatcctggatcatgcctacgagtacacgatccgggaagacatcctgatggcgatggaggaacttgacctgcctgaaaa 
               
               
                   
               
               
                 ccaggccgctgcattgctggcgtcaccgtctccgttggccgatgtctacaaggagttttccaaccgggaaacgccttaca 
               
               
                   
               
               
                 tggatgtggtacgggacagtatcgaacagcgagccgaggctgcaatggacgctcaacgcaaattgccgatttaccagcac 
               
               
                   
               
               
                 aatgctgcttatgctcgtgaacagggtgaactggatttgtaccgggagtcccgccgcgtcaacatcgcctgtaagaaggc 
               
               
                   
               
               
                 tatcgaagcatccatctcgaaatatcaccatggcaaaggactgatcaaagaatatgtgcttgatgtgatcaaacagttcg 
               
               
                   
               
               
                 gctactcccgtaccctctatgtgctggcaaacaccgcacagcagaaagattgggatgggcgaatttgtaaggaaaataag 
               
               
                   
               
               
                 gaatgggctaagtccgtaaaaatcccggagaatccggattgctttggcagtgatcgaaaccgggattttgtgttagacag 
               
               
                   
               
               
                 tcatcccgctctggttgacctgtttttgacccaggccttggcgctgacgctccacgatgtctgcgacttttaataaggag 
               
               
                   
               
               
                 gaaaaataaaatgtcggctaaaaatcagtacacgaaaatgatttcccgtgatgggaaaattgtgggtgaaatcaaaaacc 
               
               
                   
               
               
                 tgcacagtcgcccttgccctatggctgggtgtaagggacatcgtatccatgtcctttggccggatgggaaaagcacctat 
               
               
                   
               
               
                 ccgtgctcaaacggatgcaaggaaattgatcccaataccttgcaaattttgtaaaaaagagagggtggctttattttggc 
               
               
                   
               
               
                 cgccaacatttttagaaaaacatagaaaggatgtgaacggcgcattcctcctatgacttcagtcttgggaggaatgccac 
               
               
                   
               
               
                 gaatttatgaaaaacataagtttaaccgagaaagaaaaaaaacgtctctcttatacaaaaccgtatggtctgtggaaagt 
               
               
                   
               
               
                 cactacagaaggagattgtgaaggtcgctcatcgagaaatctgggtatctttgaaggataccttgacgacatcgcatttt 
               
               
                   
               
               
                 atctggcagacaaagcgtattatactttggaattcgaaaaaatagatattctccgtatttcgcataaaaaagtgaatgcg 
               
               
                   
               
               
                 gaacgaagcgaagttaatgtttcgcttgacatatcttcaggaacatggggtatgtcgagtgaagagcgcgtcctggaatt 
               
               
                   
               
               
                 caaaaagctgctatccgggagacacgttcgcgtcacggaaggcgatacttatgcttccgtaaagttgtgcaaggacggaa 
               
               
                   
               
               
                 tttaacagaaatttgccgtggaaaaaagtacagaaaggatgtgagcggtgcagaatacccgtgacttcgtcacggagaaa 
               
               
                   
               
               
                 atgcaccaagtttatgaaacataacaaaattgtccgattgatttttgttgcagttctttgcagcatcgttatcggttctc 
               
               
                   
               
               
                 tatctggctgtgcgcagttggaaaacttactaaacactgccagggaaaaattggtcggttccgatttcacgatcacacag 
               
               
                   
               
               
                 tatgaccatatgggcaacccaaccatgaaaatccacggggacagtgtttccgttggcctgcttgaaaaccaatcgaacct 
               
               
                   
               
               
                 tgatattgagactacagggttcgaatcagaagttctcgaactgaccgttgacggaaatcaagttctaactgttggcgata 
               
               
                   
               
               
                 cctgtgttattgccgaagagggtctcgatatgatcacggatttctccgacatcaatacagacatcgatacggctgacggt 
               
               
                   
               
               
                 ctgcctgcttttattgctggtgatcggttggtaaatgattttcgaaactctatcggtaaaaatatggtcgtcgtaattaa 
               
               
                   
               
               
                 atcccagatgggtatgccaatcggaatttatcagggcgacgaggtctatgtgactgtgcctgatagtcttccaaaaacaa 
               
               
                   
               
               
                 cgcagctaactattgacggaaaacagctgtacatccatcgtgccaactatacgattatcgaaggggacatgttggataac 
               
               
                   
               
               
                 gctgcttgatttgcaaaaggaccctgcatatgagtcaggagcaatcctgacaagggtgtagggtacaggcatcatgggca 
               
               
                   
               
               
                 ttgctcatgaggaacgtacgtaaccttgcaggtcagctgcgaggggatgcactgccgggtttttccagctcggtagggtc 
               
               
                   
               
               
                 agggaatcatctgcatagcgtacgctcagccaggggaaacattaccttccgcaaggaagagtcttattgaagggagtagc 
               
               
                   
               
               
                 gtcatggctacagtttatgtattgtcaaagactggtaaacctttgatgcccacaactcgctgcgaccatgtgcgcatact 
               
               
                   
               
               
                 tcttaaacagaagaaagcacgggtcgtgaatctcaaaccgtttaccatccaactgttgtatgactgtaaggaaggtactc 
               
               
                   
               
               
                 agcccattgtgctgggtattgaccctggtcgtaccaacatcgggctttctgctgttcgaaaagatacaggtgaacctgta 
               
               
                   
               
               
                 ttcactgcgcagatggagacccgcaacaaagaaattcctaaactgatgagagaccgaaaggctttttgtcaaaaacatcg 
               
               
                   
               
               
                 gtgcttcggccgccgcaaagtacgtcaacgcagggcatctgcacataacaccaattcgtcaaagtgcgcaaaacaagagg 
               
               
                   
               
               
                 ttgcacaaaacggtggcgttagcaaacaggctcaaaaagttggcgtaatcaaacgacaacttccgggttgtgaaaaaccg 
               
               
                   
               
               
                 gttctttgtatcggaattaaaaacaaggaggctcgttttaataaccgcctacgtccgaaaggatggctaacgcctacagc 
               
               
                   
               
               
                 aaatcatttgttccagactcacataaacgttgttaataaggcaaagaaatttcttccaataacagatgtggttttggaag 
               
               
                   
               
               
                 tcaacaaatttgtattcatggcgttggacaatcctcacattcagaaatggatgtatcagcgtggaccgctcaaaggctac 
               
               
                   
               
               
                 ggcagcgttgaagaggcggtttctgtacagcaaggtggtcattgcatcttctgtaaaaaggagattacaaactaccacca 
               
               
                   
               
               
                 cattgtcccacaaggtaaacgcgggtctaatactattagcaacattattggcctttgctctatgcaccacgacttagttc 
               
               
                   
               
               
                 ataaggacagtacgtgggaacagaaactcaaaaccaaaaagcagggcatgaataaaaagtacggcgctttaagtgttttg 
               
               
                   
               
               
                 aatcaaattattcctaaactttgtgattctcttagtgccgagtttagtgagcatttctatgtgacggatggaagaagtac 
               
               
                   
               
               
                 caaggcttttcgtgatgcctacaacatcaagaaagaccattatcttgatgctttctgcattgcctgtagtattctttcgg 
               
               
                   
               
               
                 tagaagatgttaaggttccttgcgaaagcaatgtgttccttatccttcagttccgtcgtcatgacaggcgcgcttgtcac 
               
               
                   
               
               
                 caggagcgagttgaccggaagtattatcttgacggcaaacgcgttgccactaatcgtcacaaagctattgaacagatgaa 
               
               
                   
               
               
                 cgatagccttgaagaatatgtaaccaatggtggctgcgtcgataaactgactgcgccaaaacatccgccgttatacagac 
               
               
                   
               
               
                 ggaaaagccgcattatgccgggaacagtatttttggtcggcaacaaaactaaggtgatgattgcatcgcaaggcacgcat 
               
               
                   
               
               
                 aatggcgttcccaactactatcgtttcaccaatggtttaagagctacaccaaaaaactgtaaaccaatttatcaaaacac 
               
               
                   
               
               
                 cggcatcgtgtttgtttgattttgtatcaatcacggtgctgtaagggagaaaaatatgacgaacaagaacacatggacag 
               
               
                   
               
               
                 agacagactctgactgctgtcagtatgtccattattttgatgagatccttggaccgactgggactctttttgagttcgtt 
               
               
                   
               
               
                 caaatcacaggattgccgaatggtcagtacgggatctctcacgctgtcattgatatcgaatgctatgagcagaaagatat 
               
               
                   
               
               
                 ccttgatgcgcttaatctttacgggtataaatccatggacgatttcgtccaggaaatctctccgtacaagattgaaaaga 
               
               
                   
               
               
                 agaaagacggaacgcttgatccggaatccgaacactacattatcgacaacgagcaaatcgctgaaatgctcttcgagatc 
               
               
                   
               
               
                 ggtgctttcgactcccttctcgataacgtagtctttgacacgttcgaagatgctgaacagtatcttacgaaatttttcgt 
               
               
                   
               
               
                 ttgacgtcctcccacccctcacggagtgggattccccaaaccgaccaggagacagaggtcttccattaaacgagatcctg 
               
               
                   
               
               
                 ccgtgaagcaggtaagacagacaggcgcagccacaaaatggctgcgcctgttctatttagattcacgctccccatacggg 
               
               
                   
               
               
                 gagcgacggcatattgctcaaatgtatggctggctgcgagacatttggattcacgctccccatacggggagcgacattga 
               
               
                   
               
               
                 catcatgatgctttatggtgcacgctgaatttagattcacgctccccatacggggagcgacgttttgttttgcgatatca 
               
               
                   
               
               
                 ctactgccacgcttaatttagattcacgctccccatacggggagcgaccgtgtccttccgcacatacttgacaatggaca 
               
               
                   
               
               
                 gatttatattcacgctccccatacggggagcgacccgtgggttatcggtcaagatggtggcagccttatttagattcacg 
               
               
                   
               
               
                 ctccccatacggggagcgaacacctagcttcgggcggaaaggacacactatgaaatttagattcacgctccccatacggg 
               
               
                   
               
               
                 gagcgaccactcataattttgcggcactgcacgacatatttatttagattcacgctccccatacggggagcgacagcaat 
               
               
                   
               
               
                 cctgcacagtccattctctgctaagggaagcaatatttaacggaaatgcacaaaactgcattgatttcatcgctattata 
               
               
                   
               
               
                 ccacaaaaacaaaatacagtcaatcttaaagcggtgcgatcggacaatgaaaactgtgtttgcttgtggttcgcacaagg 
               
               
                   
               
               
                 aaaattttaatactttctttttgttgcaaagtaagtatattaaatc 
               
               
                   
               
               
                 aaaattttaatactttctttttgttgcaaagtaagtatattaaatc 
               
            
           
         
       
     
     In some examples, the Cas protein has the following sequence: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 9) 
               
               
                 MATVYVLSKTGKPLMPTTRCDHVRILLKQKKARVVNLKPFTIQLLYDCKE 
               
               
                   
               
               
                 GTQPIVLGIDPGRTNIGLSAVRKDTGEPVFTAQMETRNKEIPKLMRDRKA 
               
               
                   
               
               
                 FCQKHRCFGRRKVRQRRASAHNTNSSKCAKQEVAQNGGVSKQAQKVGVIK 
               
               
                   
               
               
                 RQLPGCEKPVLCIGIKNKEARFNNRLRPKGWLTPTANHLFQTHINVVNKA 
               
               
                   
               
               
                 KKFLPITDVVLEVNKFVFMALDNPHIQKWMYQRGPLKGYGSVEEAVSVQQ 
               
               
                   
               
               
                 GGHCIFCKKEITNYHHIVPQGKRGSNTISNITGLCSMHHDLVHKDSTWEQ 
               
               
                   
               
               
                 KLKTKKQGMNKKYGALSVLNQIIPKLCDSLSAEFSERFYVTDGRSTKAFR 
               
               
                   
               
               
                 DAYNIKKDHYLDAFCIACSILSVEDVKVPCESNVFLILQFRRHDRRACHQ 
               
               
                   
               
               
                 ERVDRKYYLDGKRVATNRHKAIEQMNDSLEEYVTNGGCVDKLTAPKHPPL 
               
               
                   
               
               
                 YRRKSRIMPGTVFLVGNKTKVMIASQGTHNGVPNYYRFTNGLRATPKNCK 
               
               
                   
               
               
                 PIYQNTGIVFV 
               
            
           
         
       
     
     In certain example embodiments, the Cas protein is encoded by the following polynucleotide sequence or a portion thereof: 
     
       
         
           
               
               
            
               
                 (SEQ ID NO: 10) 
                   
               
               
                 atacatcccggaaatccgggattgtgacgatttcgcgtttcccattatgtcctatattaaattccttaagatgccaggca 
                   
               
               
                   
               
               
                 tcgctcttggggtcataaaatacagatacaaacggctagacggctatgtatatcacatggcaaatattttcatggaccag 
               
               
                   
               
               
                 gatctgaacatttggattattgactggaaacactatccgggaatcttctggtcttataataaaaattggaaggtatatga 
               
               
                   
               
               
                 ggtgattctgtgaaagtcaacggcctaatacggtcaggttgaaactttactgcgtggtcctgccaggcgtaattatgata 
               
               
                   
               
               
                 ggtcaacggcctaatacggtcaggttgaaacgaattcgactatggcatccatgaaggcaacaaagtcgtcaacggcctaa 
               
               
                   
               
               
                 tacggtcaggttgaaacccatgattcagaaaaggaaaaactcaattaaattaacgcttttgttgtcggcttctcatttcg 
               
               
                   
               
               
                 attgtcgtcgatctccgatgatccaaaaatcccaggggatcgacgatcccgcgccgttgctgttttcattgacagatttc 
               
               
                   
               
               
                 aaccaatttacctcaaaggctctgctcatttttcggtacttttccccagatcgacgatgtttcaatctgaccgtgttgct 
               
               
                   
               
               
                 tgacctgcttgcggatcaggctacgttgacagctttagcagctttcacgctgctacgttatttcagttatcagacccggg 
               
               
                   
               
               
                 gatgcttctccagttcccggcactctggcggcgctgtaaaagtcctggaggcagggacggtcaaccgcaggacgaccgga 
               
               
                   
               
               
                 cgtttcaggcaagctggaataacattggcggggaggaattttaccatacgaaagtatgaggtagtttatgttagtgcatg 
               
               
                   
               
               
                 ttttaagtaaagaaggaaagcctttgatgccaacgcatccagcgaaagccaggaagttgctgaagctaggtaaggcaagg 
               
               
                   
               
               
                 cccgcgaaggcgaagacaggctatttcactgtacaattgacttacgacacagcaagttatatccagcctgtaacggttgg 
               
               
                   
               
               
                 cgtggacttaggaagcaacacagttcctatagctgccattgccaacggtaaggttgtctatgccaaagagaaactactga 
               
               
                   
               
               
                 ggcgtgatatatcagccaagctaaaagctaggggcgaatatagaagacagaggcgcggacgactgcgacacagaaagccg 
               
               
                   
               
               
                 cgttttgataacagagttaagaagaaatgtgcgcggtgcggtgtcaacaatgttccgcgtacctggaaaaagattaagcg 
               
               
                   
               
               
                 ccaaaacggcaagtcaaagaagagagtattagttggcagggctaatttatgtcgcaaatgtcaaggcgagaaaggcttgc 
               
               
                   
               
               
                 accggcagccgcatctactaccaccctcggtgaaggcgcgggcagatgccattttagcggatattgagaagttttgcaga 
               
               
                   
               
               
                 agtctaccggtcgctaagatagttgtagagatagcttatttcgatacgcagaaaatggcgaaccctgatattgaaggcgt 
               
               
                   
               
               
                 tgaataccaaaaaggcacacttgaaggcgaagagataaggtcgtatgtgttcaatgtattcaagcacaaatgcgcttatt 
               
               
                   
               
               
                 gcaaaggcgctagtggggataaaatacttgaaatagaccatgtgcgcccgaaaagcaaaaaaggcagcgataagttgagt 
               
               
                   
               
               
                 aacctggtcgctagttgcaggcaatgtaacatagcaaaaggcagcatgacgttagatcaatgggctaaaaggctacaagc 
               
               
                   
               
               
                 aagtccttgcgagcttgataaaaaacgtttgtcaagcctgaagcacatcaagaaacgcagtgatataaagaagggctttc 
               
               
                   
               
               
                 aatatagtgccttgactcagagttacaagagctatctacttcacgaattagctcagcgtcataaggataagcgtttctct 
               
               
                   
               
               
                 acaacatacggctatgccaccaagtttgccagaaaggcaatggggcttgagaagtcacagataaatgatgcgatggttat 
               
               
                   
               
               
                 agcatccgaaggcagaatgttcccgacacccaagtattacttattagagcgttgtctcaaaaaacgcagggctgctgagt 
               
               
                   
               
               
                 atataagcccgcataaagaaggcacgccggttgttaggaggccttggtctaatgcgaagtatgggtttaggctatgggat 
               
               
                   
               
               
                 aaggtggaagcagaagcgaagcagggctatgtagctgccttacgcgagagtggcagttttagggttcataccttgtatgg 
               
               
                   
               
               
                 ggataaaatatttggtggaaagtcttacaagaagcttaggttgataactgagtgcaattctaactatatgcgcgagtgga 
               
               
                   
               
               
                 agatggtaagccaagagcccatgcaaatggaactcacctttcccgcatgatcataacgggagttcagaatgcaatacgct 
               
               
                   
               
               
                 aatccaaagttaataggaagcaagataatagactgtgtgccgttgacgggtccggcgggcgaggtggtccaggggtacat 
               
               
                   
               
               
                 cacagcccggcggctaagcgggagcttccgggtgggagcacttgacggtcgggagattaccgggggaaagagctacaaga 
               
               
                   
               
               
                 aactaacgcttctaaagccgtgtcgcagcaactatcaatgtcaagtcaacagcctggcatgtttagattgaaacgggagg 
               
               
                   
               
               
                 ggaggtgaatcgtatgcacttaaaaaagactgagctgatattgagtttctgctttggattgtatatgactctcaggtgag 
               
               
                   
               
               
                 taaggatgataaggatgtcgtggagaattggctgtgggagaatccagattactgcgacctagttgaattgtgaaggaggt 
               
               
                   
               
               
                 gaatagaatgggtgatataatccgatacgagcatcatggtgcagaagtgtgcgttgatgaggacctgaagggcaaacaca 
               
               
                   
               
               
                 gaagtcactgcctttgcttcaggtgtgccaagttctgtcctgagaatagggaaaagagctgtccgagggcgaacttgctg 
               
               
                   
               
               
                 tatgcgtactgtgtggcatttgacatggtaacaccggtatacgaatgtcctgagtttgaggaggcagaataatgagaagc 
               
               
                   
               
               
                 ggtctccacagctttatcgtta 
               
            
           
         
       
     
     In some examples, the Cas protein has the following sequence: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 11) 
               
               
                 MLVHVLSKEGKPLMPTHPAKARKLLKLGKARPAKAKTGYFTVQLTYDTAS 
               
               
                   
               
               
                 YIQPVTVGVDLGSNTVPIAAIANGKVVYAKEKLLRRDISAKLKARGEYRR 
               
               
                   
               
               
                 QRRGRLRHRKPRFDNRVKKKCARCGVNNVPRTWKKIKRQNGKSKKRVLVG 
               
               
                   
               
               
                 RANLCRKCQGEKGLHRQPHLLPPSVKARADAILADIEKFCRSLPVAKIVV 
               
               
                   
               
               
                 EIAYFDTQKMANPDIEGVEYQKGTLEGEEIRSYVFNVFKHKCAYCKGASG 
               
               
                   
               
               
                 DKILEIDHVRPKSKKGSDKLSNLVASCRQCNIAKGSMTLDQWAKRLQASP 
               
               
                   
               
               
                 CELDKKRLSSLKHIKKRSDIKKGEQYSALTQSYKSYLLHELAQRHKDKRF 
               
               
                   
               
               
                 STTYGYATKFARKAMGLEKSQINDAMVIASEGRMFPTPKYYLLERCLKKR 
               
               
                   
               
               
                 RAAEYISPHKEGTPVVRRPWSNAKYGFRLWDKVEAEAKQGYVAALRESGS 
               
               
                   
               
               
                 FRVHTLYGDKIFGGKSYKKLRLITECNSNYMREWKMVSQEPMQMELTFPA 
               
            
           
         
       
     
     In certain example embodiments, the Cas protein is encoded by the following polynucleotide sequence or a portion thereof: 
     
       
         
           
               
               
            
               
                 (SEQ ID NO: 12) 
                   
               
               
                 ctaccccgcaaggaaggagccgccgaccgatgtcttgttaggaaaactaatccttcggaaattagtcgagaaggggctcg 
                   
               
               
                   
               
               
                 cccccccaaaagccaacctgtcgtggcaatgcatcgaaagcctgaccatcaccccgtggatatgggatcgcttcgccgag 
               
               
                   
               
               
                 cacgggatcgaagtcgcgctcggcgagatggtcaggcggcggggcgaagagaaatcaaaaaaaaatcgaacggggggttg 
               
               
                   
               
               
                 acttttggataatagccttcataatctcgcccatcgatagccgagcaacgaagttggatcaggacgtcattcttcgaatt 
               
               
                   
               
               
                 cgaaaacgcggggattgctcgaatgggtggatgatatgttggatcaggacgtcacttttcgaattcgaaaacgcaagtcg 
               
               
                   
               
               
                 aagttggcgacttgatcaaatagttggatcaggacgtcacttttcgaattcgaaaactaaagaccgctcggctgatactt 
               
               
                   
               
               
                 gacggaagttggatcaggacgtcacttttcgaattcgaaaaccgatctcgcctaagtgattgattcagcgaagaaattgt 
               
               
                   
               
               
                 agaatcgcagttgcaaaaaatgatgtcctgatccaataaggggcgcagcccccgcaccatttttccccgatctacggatc 
               
               
                   
               
               
                 ggatcggcgctgccttcgaaaagccgacatggaaaacccccagtttatcggggacgatgtaaacctgagtcgccacggcg 
               
               
                   
               
               
                 ctcatctcgcccgctctacggaccgggcttcgctggaaactgggagcggtaaccatgaaacaactcacactcgatgtcgg 
               
               
                   
               
               
                 catcgcttcgatcggctgggcgattgtcagtaaaaaagggaacgtcaaagccggatcacgcatctttcccgacgccaaat 
               
               
                   
               
               
                 ctgggcgggaaagcaaccaatcccgacgggcggctcggctgatgcgccgaggatatcggcgcaaagcaaagcgccgagcc 
               
               
                   
               
               
                 gacacgctcgccatcattcgctccatccaccccggcttcgaccccgaagggcaccctgacatcgagcgcgaagctctgat 
               
               
                   
               
               
                 caaagccatcattacccccggcgcccccaccccatcgctcgaccaactcgcttgcgcgttccagcgattcgccaagtctc 
               
               
                   
               
               
                 ggtggccgcaatattcgagaactctcccaaaacgaaccgagcgggaagaccagttcatccaagtttgggtgatcgccgag 
               
               
                   
               
               
                 cgaacctatcccgaccgctttacccccgacgtcgcaacccgcttgcttcaggcgatattctttcagcgccctatcaaaga 
               
               
                   
               
               
                 cggcgaccgcgcgaaatgtcaactattcaggcatcacggtgacaaagccccgctcgtcggctggacgcacgaacccgaac 
               
               
                   
               
               
                 tccaacgattcgccatcttgtccgatctgtccaatctcactatcgggatcggctcgactgataacctgctttgcgaatat 
               
               
                   
               
               
                 cccgacatcatcgaagacttggagacccgatgcttcgagaccggcatgagctggagagagatagccgaacacgtcaagga 
               
               
                   
               
               
                 agtcatcggcaaaggggtggtgtttcgaggcattgacgggcagaaaaaagtcgggcggaacgggatcgggcctgccaaac 
               
               
                   
               
               
                 tcgaaacaatcgacgaagaaggcaactctaccaagagcaccgcttcgatgtcggtcgaagcggcggtaatgatctatcac 
               
               
                   
               
               
                 caaatgaaagcggatcgatgccgagcggcaaccgctaaaaaaacgctgatcgacgcaggggctctttcagcccccctaac 
               
               
                   
               
               
                 cgccaaagatatcaaacgtggcgatcgcactctcaccatcaccgaactgatggatatggcgggcaggattaccgacccga 
               
               
                   
               
               
                 ctatcagggcgatctaccaccaagtcgagatgttggtcaacgagttgatcgcccgcttcggtaaacccgaacgcatcgtt 
               
               
                   
               
               
                 atcgaagcccaaaaggaaatcgggcgaagcatcgaagacatcgagaaagcgatggcaagagagcgagaaaagcatatcga 
               
               
                   
               
               
                 acggcaacgagagaatcgagcccgcaacgccgcaatgggtaccaaagcccgctttgcccgcctgtgcgccatcaggggtg 
               
               
                   
               
               
                 atcgatgctttatcagcgaccgacccgcagccgaagtcggccacctgatcgccgattcgatcgggggcacgcttgaaatg 
               
               
                   
               
               
                 gctaacctgatcccgatcgaccctgccatcaataaagagatgggcaaccgcactccctacgaagcctttcgaaagactga 
               
               
                   
               
               
                 gtattggtcgatcatccagcgcaaactgcaagcgcttgaagacgaagtgaaggctttgaaaccgccgaaagggacaaaag 
               
               
                   
               
               
                 gaacggcgtggacgatctatcaccgagccaagcatcagtttgattttttcgcttggcgatttcaatcgaatgcgagagaa 
               
               
                   
               
               
                 acccatcaaaggaactttcgccccggctcgcttgacgacctgcggtggatcgaaaacttgctctttctcggcgttgcccc 
               
               
                   
               
               
                 gatttgcgacaatatccgaatcgtcagtgggcgaacaaccgagcgcattcgccgagagatacttggaatggacaaagacc 
               
               
                   
               
               
                 gccgagaccatcgccaccatgcgctcgatgcgcttgctatcatgctcgccaatcctttgaagccgtgggatttgaaatcg 
               
               
                   
               
               
                 agcaattcgctcggtatcccgctcggccgaatcaaacaagccttcgccgacgctgtcgtctcgcaaaagcaagaccactc 
               
               
                   
               
               
                 gcttcgcactgcgttgcataaagagaacgcgatcccgaagaccaagcggggggctgcatatcgaaaaatcggaacggggg 
               
               
                   
               
               
                 cgagcgaacgcgtagtcgacacccaatcaaaggcctattgcgaagtttgggcgttgcctaacgggaagtgggaagcggtc 
               
               
                   
               
               
                 gtagtgtcgagtttcgacgctgcgcaaaagaactaccggcaagggattgaccatcgcccccatcccgccgctcgactggt 
               
               
                   
               
               
                 tatgcgcttgttcaaatccgatttgctcggtatcgggggcaagatataccgggttcaggaactactcggttctggctcga 
               
               
                   
               
               
                 tctacttggtcgatcatcgattcgctggcactattcgagacgcccgcgcagtttgtaagacgggcgtcaatgtcgatttc 
               
               
                   
               
               
                 tttagcaaagggggtgattcattgcgcaaggcgggggctcgtcttgtttcgattcgtaagagttgggtgggatcgtgagt 
               
               
                   
               
               
                 cacttttcgaattcggaaacttttccgaccgacaagtactcgatcatctagttggatcaggacgtcatgcttcgaattcg 
               
               
                   
               
               
                 aaaacgtcagtcgggtgacctacgagcgatagggggttggatcaggacgtcacttttcgaattcgaaaaccattcgatgc 
               
               
                   
               
               
                 cgatgcctacaccgcaaagagttggatcaggacgtcatgcttcgaattcgaaaaccttcgtttcaataacc 
               
            
           
         
       
     
     In some examples, the Cas protein has the following sequence: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 13) 
               
               
                 MKQLTLDVGIASIGWAIVSKKGNVKAGSRIFPDAKSGRESNQSRRAARLM 
               
               
                   
               
               
                 RRGYRRKAKRRADTLAIIRSIHPGFDPEGHPDIEREALIKAIITPGAPTP 
               
               
                   
               
               
                 SLDQLACAFQRFAKSRWPQYSRTLPKRTEREDQFIQVWVIAERTYPDRFT 
               
               
                   
               
               
                 PDVATRLLQAIFFQRPIKDGDRAKCQLFRHHGDKAPLVGWTHEPELQRFA 
               
               
                   
               
               
                 ILSDLSNLTIGIGSTDNLLCEYPDIIEDLETRCFETGMSWREIAEHVKEV 
               
               
                   
               
               
                 IGKGVVFRGIDGQKKVGRNGIGPAKLETIDEEGNSTKSTASMSVEAAVMI 
               
               
                   
               
               
                 YHQMKADRCRAATAKKTLIDAGALSAPLTAKDIKRGDRTLTITELMDMAG 
               
               
                   
               
               
                 RITDPTIRAIYHQVEMLVNELIARFGKPERIVIEAQKEIGRSIEDIEKAM 
               
               
                   
               
               
                 AREREKHIERQRENRARNAAMGTKARFARLCAIRGDRCFISDRPAAEVGH 
               
               
                   
               
               
                 LIADSIGGTLEMANLIPIDPAINKEMGNRTPYEAFRKTEYWSIIQRKLQA 
               
               
                   
               
               
                 LEDEVKALKPPKGTKGTAWTIYHRAKHQFDFFAWRFQSNARETHQRNFRP 
               
               
                   
               
               
                 GSLDDLRWIENLLFLGVAPICDNIRIVSGRTTERIRREILGMDKDRRDHR 
               
               
                   
               
               
                 HHALDALAIMLANPLKPWDLKSSNSLGIPLGRIKQAFADAVVSQKQDHSL 
               
               
                   
               
               
                 RTALHKENAIPKTKRGAAYRKIGTGASERVVDTQSKAYCEVWALPNGKWE 
               
               
                   
               
               
                 AVVVSSFDAAQKNYRQGIDHRPHPAARLVMRLFKSDLLGIGGKIYRVQEL 
               
               
                   
               
               
                 LGSGSIYLVDHRFAGTIRDARAVCKTGVNVDFFSKGGDSLRKAGARLVSI 
               
               
                   
               
               
                 RKSWVGS 
               
            
           
         
       
     
     In certain example embodiments, the Cas protein is encoded by the following polynucleotide sequence or a portion thereof: 
     
       
         
           
               
               
            
               
                 (SEQ ID NO: 14) 
                   
               
               
                 attgcaggatacgatactgttttttataactcgtcaaatctggataaactgattgagattgcttcaagagaaagtcgcac 
                   
               
               
                   
               
               
                 aattttaaccagaaatagcggtttcataaaaaaagagagaaaggttttagaggaaagaggtatcccacatcttttattgg 
               
               
                   
               
               
                 catctgacagtgttgaagaacagcttaaagagacagtagattattttaaacttgacctgaaggagagttcttttacactc 
               
               
                   
               
               
                 tgtgtggattgtaataagccgttttttaaaatatcaaaagaggatgctttgggtaaggttcctgagtttgtttatcaaaa 
               
               
                   
               
               
                 ttacaatgaatttgtccaatgcacaaactgtaaaaagatattctgggaagggacacatagggaaaggatggaggaaatgc 
               
               
                   
               
               
                 ttaaaagagtgtattgataaactaatgaaaggttatattgtttatataacctatctttccgttttgtccaagacggtaga 
               
               
                   
               
               
                 aatgccttatatcttttaataaagaggagatatcattcctttttaaataatttgtttctaaatagtgacagtagttcatt 
               
               
                   
               
               
                 gccattctgtttgcatccctgtatctttctttttcctctgataataaatccggatttagtatagaattttcaaacaactg 
               
               
                   
               
               
                 tctctttatactttttgcagggactctgccggaattaatactgctgctgtagaaaatggctgtgatatatttatcaacct 
               
               
                   
               
               
                 cagcctgaagctccatctcaaagatagttatctgtcttttatggatggcattccatactgcatatatgaaatggcttatc 
               
               
                   
               
               
                 tcctctgtggcaagaaaaaaatctgaaagatttattttgcagaaatctttttgcagatgaatattttttagattagatag 
               
               
                   
               
               
                 tgtctctctgccgagatatacagctattttgagttcatcgtcattctggcataaaaatagataaccttttgatgaggggc 
               
               
                   
               
               
                 ttttaatcctacaggatttttgattgtttgaattattagaaagcgggtcatttatgaggtaattttctatattaaggaat 
               
               
                   
               
               
                 gtctccactttatatattttttctattatcttctgtatccgggataacataaaatctattcgcccgctccctctttggga 
               
               
                   
               
               
                 gggagtgggttggggatgagagggaataaaatatgcctactgtatatacttgctgcctttggatattataggaataaccc 
               
               
                   
               
               
                 ctttttctttcaggagttgggcatccctatcactgtttgtcttgagccacctttcatatattctcaataaatcgctgttt 
               
               
                   
               
               
                 gtgttgatggatgccttatcgctgacctctgcaattacatctatgaattctttaaatctctctgatagctcactgaagag 
               
               
                   
               
               
                 ctcactgaaaagctccccatttccgtatctcttggtaatatatgccaggttgctgtaggctctccctccgataactatat 
               
               
                   
               
               
                 aataatcaatatcaataagttttttcttaaggctgtcggagaaaaagcccgatacaaacagtgaaaaatctccaacctgc 
               
               
                   
               
               
                 ttaaatttccttatcctctcattgtattctgaatttatggcactttctaaaagcattataagaggctcatcagatgcctc 
               
               
                   
               
               
                 ttcagggaatgatttatcaatttcaacatactcagagagaagattcactaaatagaatgccactgtctcatcagtgtcta 
               
               
                   
               
               
                 tatcctgatggtctatagccgtatccaccagttccttgaagaattcagcagtgtttttatgtgttgttattttggctgcc 
               
               
                   
               
               
                 atatattttctctaatttaaaattatgccatactttttaagccattcagtttcgtcaaatttatccgaaatatattcttc 
               
               
                   
               
               
                 aaaggcagcctcattttttgtaagtattctgccgtcaatgtaatctccttttggttttgattcatttggattatcttcta 
               
               
                   
               
               
                 atgatttctccgctgtatttatgaagtcgtttaccttgattatcaaggcaagcattgcagatggttctatcatattcctt 
               
               
                   
               
               
                 tttttaaggtcaaagagaatgcccagcacatctttagattttaggagcgcctttgtaattgcccttgagagcccatcaag 
               
               
                   
               
               
                 ctctttgttgtctttttcattatctctcattatatcctcctgattatttatcggtattatttttaataatataattaagc 
               
               
                   
               
               
                 aaaaaatataccatgattatcacttaattttaattcattgaaaaagaatgagatagctgatatattgaaataaataaggc 
               
               
                   
               
               
                 tggaaattatgtcctgattgtgtcaaatttatccagatttaagaaaaaatgttatgatattcgccaatgaaaaaatacgg 
               
               
                   
               
               
                 tgagaaagcaattgttagtgcaaaattagaagcgtgggataatccctatccggatagggattataagattgaaataagtt 
               
               
                   
               
               
                 tcccggagtttacatgcctctgccccagttcaggctatcctgattttgctacattcgccataaattacatccccgataag 
               
               
                   
               
               
                 tgtataatagaactcaaatcactgaaactatatctaaacagttttagaaatcagtatatctcacatgaggcggtaacaaa 
               
               
                   
               
               
                 taaaatctatgatgctttaaataagactttgaaaccgcgttttatagaggtagtgggggattttaatgaaagggggaatg 
               
               
                   
               
               
                 taaagacagttataagagttacatcaaaacagtagtctaatagtctaaaagcctaagagtctaagagttgtttcctttta 
               
               
                   
               
               
                 gactcttagactgttagactgatttatttggttgactcctcaaggtctaacatcattgtattgccgacatatttcttact 
               
               
                   
               
               
                 gattaccctttttatcctttgtagtttctttgtgacctcttttattctcattgcactctgaattgcagtattaagcatct 
               
               
                   
               
               
                 cctttatatcttcattatttgtattcatcagggctaattcaatactgctgacaatgcctgtcaggggattatttatctca 
               
               
                   
               
               
                 tggtttaaagttaccgctaactgagaaagtagagattctttatccttagaaagtatagtaagcgaattaatagaatttga 
               
               
                   
               
               
                 tgttaataaatttaattcacttgctaattgttttgacataatgttgaccaccctctaaaaaatttactaatttatgttgc 
               
               
                   
               
               
                 acttactatgccataatctattattatgctaacatattgatattataagtgttttgacttttgaatacccaaaatctcta 
               
               
                   
               
               
                 ttgtaaatatgtcaaagttatgacttaatgggaaaaatattccgattaaatctatcatgccttaatatctttatcggatt 
               
               
                   
               
               
                 tatctaagtttttataaagtctttttttaggcgggctattctatatagagattcctcaatcctctcttcttttatctcac 
               
               
                   
               
               
                 cactctctaatgtccttaatatagaattgaacatctctatctgtttattatggctatggcagattaagagtatatcaacc 
               
               
                   
               
               
                 cctgctttaacagactgaattgcagaaattttatcgctgtaattatttgagattgccttcatttccaaatcatctgtaat 
               
               
                   
               
               
                 tactacatcattaaacccaagtttttttcttaaaatctcatttattattttctctgacatagtagcaggataatcaggag 
               
               
                   
               
               
                 ctaaagcagggtaaagaacatgggctgtcataattgcccttaagcccattttaaatgtattaataaagggtttaagctct 
               
               
                   
               
               
                 atctcctctaatctttctattttgtgatttacgataggaagttcaaggtgggaatcagagaaagtatctccatgtccggg 
               
               
                   
               
               
                 gaaatgtttacctacaggaattatatttttttcactaaatgcctttatgacatgcccaccgagtctggatacaacatcag 
               
               
                   
               
               
                 ggtcggaactgaaagacctgtcgccaatgatagggtttttgggatttgtatttacatcaagtacaggtgacatattcata 
               
               
                   
               
               
                 tttacacctatttctataagctcatctgccattaaaagggcatttgaccttactgcatcttctgaacctgctcttcccaa 
               
               
                   
               
               
                 ctcgccataagaaggatactgtctgaaaggaggtttgagcctgaacactcttccaccttcctgatcaattgcgattaata 
               
               
                   
               
               
                 gagggattttatggcttttaattgataattcctgaagggaattgcataaatctttaacctgtgacggagactctatattt 
               
               
                   
               
               
                 cttgaaaataggattacccccccgaccccataatcagttattattccttttaactcatctgacatagtagtcccatgaaa 
               
               
                   
               
               
                 tcccaccataaacatctgcccgattttttcttttaaagatattattgacatggtatcaatctaaaataacaaggagtgat 
               
               
                   
               
               
                 tttgcaatcataatcttacaggatttggtattgacaagtaaatagatttacctataatgaagatgaaattattgaacctt 
               
               
                   
               
               
                 aataaaacctgacaatgcaaggacaaaaacttggtatagatttaggcggtaagcatgtcggtcttgctgttgtaagaaca 
               
               
                   
               
               
                 ccgataaacgaggtggcacattactgcactattgaactcagagaagacattaaggataagatggatgagaggaggtctct 
               
               
                   
               
               
                 tcggagggcgaggagaaacaggctctggcatagggaagcgaggtttgacaataggcaattaagggtgaaatgcaaatata 
               
               
                   
               
               
                 ttgataaagatacaggcgaaatctgcggagctaatactccaaagaaatccaatgtaaaacatcttctacttgagaatata 
               
               
                   
               
               
                 ctcgtcaatcttaaaatagctgatgaatctaaagaggaaatcagaagaagagggctggacagagacacaaacaaaagtga 
               
               
                   
               
               
                 attacagacaatccttgagaaattttcaataaataccttcctgaaaaaacagattaaagacatcattcttgaaaaggggg 
               
               
                   
               
               
                 aagggagggctgtcttttgcagagagcatatcccctttcattatgaacaggttgcaacagaggctgagagtttctggctg 
               
               
                   
               
               
                 tcaaattcaataagggctaaacaggaccagatactctcccgccttaaaagaatagcaaaggattttaagatagatgaggt 
               
               
                   
               
               
                 ggttattgaaagggcgaactttgatttgcaaaagctccagagacctgatgagatagaagcacctgaagattacatgaagg 
               
               
                   
               
               
                 gtcctaacttcgggcacagaaacaggtttgaggcattgaagcaggaatatggcaaccgatgctgtttctgcggaaagaag 
               
               
                   
               
               
                 ggtggagatgaagtaaagctgaagatagggcatctctatccgaaggctaaagatgagataaacaggtgggaaaaccttat 
               
               
                   
               
               
                 aactatatgtgaaaaatgtaatgcgaagcagggtaaaaggacaccagaggaggcagggatggaatttgtaattgtaaagg 
               
               
                   
               
               
                 agaaggtttttaatcctgcagcaggaagggtaatacccataaaaagagaactcaagccgaagcccataaatgaatcaaag 
               
               
                   
               
               
                 gttaataaatatatgacccatactgatattggcataaggaggctcaaaagagaaatccagaatatttttggaagcatacc 
               
               
                   
               
               
                 tataagagaaacatacggctatatcacatcgtattttagaaataaatgggagcttgaaaaagaacattataatgatgctg 
               
               
                   
               
               
                 tagtcatagcctctgacaaagaagatttgaatataaaacctgtatttaaagatgcagtccctcagacaattaaatcatct 
               
               
                   
               
               
                 atcaagggcgggaaactctttgatacaaatcccctccagtttagtgatggaaagttttaccagaacataacccttatagg 
               
               
                   
               
               
                 cagaaaggcagggatgcgttcatcaaaacataaaaggggtcagaggaatatcaggaactatggctcaatttatatggatg 
               
               
                   
               
               
                 agattgaacttataacctcagaatggaagaaaaaggttctctgcgaattaagagataaacttggttatgtaaaaggagat 
               
               
                   
               
               
                 aagaataagtcttttaagcctgaggaactgatgaatgcaaatctgcctttcaggactgtaactattgacaaaaggggtgt 
               
               
                   
               
               
                 aggagaatcttcaacccgcttaatcaataacaatgtattccgtgcctcagctgaagtaaatacgcatataatggtctatt 
               
               
                   
               
               
                 caaataatgacggtagaatgaaggcatttgcagtaaaaaatcctaagatatttaaagatgccggactccctcatgatttt 
               
               
                   
               
               
                 caaaaaaagatattcattgtaaaaaagggggatattgttacatggaaaaaaagtgaagatggaattgccgtaacaggcag 
               
               
                   
               
               
                 ggtgaccaaatgtttgacaaaaaatggggtaattgatataaaggacatgaataataaaatacactcagggaaaaaccctg 
               
               
                   
               
               
                 tgtatattgaaaagatagtatctcctgaaaggggtgctatttttgagagaaaatctctttctgctctttgaaaattagat 
               
               
                   
               
               
                 attaaagattgaaaacagcctgagtgttgaaacagacactaagttgttgggaacaggtaaagaactacggcggggtatct 
               
               
                   
               
               
                 tgaatggttaccagctccgccctcttgtagtttttgagtaggaagactcgcccctttggggaaggaaaatggtcggtaag 
               
               
                   
               
               
                 ccactgataaggccatctacaactcatagacatgccctgtccgacaaccttggcaagggaaaccattaaattatctaata 
               
               
                   
               
               
                 tcatattttaatctaaatacagtggcattaaaaccagaattagtaattgagaagatttcaatctaacacagtggcattaa 
               
               
                   
               
               
                 aactacgacctatcctaattattgatacagcgaaacaatatttcaatctaacacagtggcattaaaacccataaaaaggg 
               
               
                   
               
               
                 ctatcttttataatgggttatcatttgaagggggagttttaccctatcaacctataataatgacttcctcctatgcaatg 
               
               
                   
               
               
                 ggaatgttataattataaaaatatcaggagattaatatgacaaacacattggaaatagacagagatatatataatatact 
               
               
                   
               
               
                 tattaattcctttggtgaaaatacgctgagggaaaaaatagatgatattctcctgtccgcgatggatagcttgctggaaa 
               
               
                   
               
               
                 aatacactcgtaacatattggtatttgaagaaaagtatggggtctcttttaaagaatttgaaaaaatgtgggatgaaggg 
               
               
                   
               
               
                 aaaattgataataaacataaccacgaaatagaaggggattttattgattgggaaatgttagagatggaaaaaaaagagtt 
               
               
                   
               
               
                 gttatcagcactgtccagactcaaaggctttaaaaaatgaacaaccccaatgttgatgaattcctatcatcactaaagac 
               
               
                   
               
               
                 ctttctaaagaactattttacacaatataaaattgatttcttgataaaaacaccgaaatcccttaaagccaatattcatc 
               
               
                   
               
               
                 ttaatgagaaattttttattgcagttcgatataatgccagaaatgggagaatggactttgctttaatacaggataacaaa 
               
               
                   
               
               
                 agaatttttggatatgataatttaaaggaatggcactatcacccttataaaaacccatcagagcatatctcatgcgataa 
               
               
                   
               
               
                 accatctacagataaaatcttatatgaaattaaaaaagtttttgaagatgctaaatgaggcatgaaaaaacgaaggagaa 
               
               
                   
               
               
                 acctcatagaatgaaaaaaataataattgcaattacaggtgcgagtggtgctgtatatgcaaaatacctttttgatttcc 
               
               
                   
               
               
                 tttgcaaaaaaggtattgacctgcatattatcatctcagaaaatgcaaaaggaatattaaaggatgagacagggataggc 
               
               
                   
               
               
                 gagaattattttaaaaagaagaaagtatcaatatatgaaaattcaaatctaaatgtccggatagcaagcggctcttttaa 
               
               
                   
               
               
                 atttgatggtatggtcgtcatccctgcaagtatggggactcttgggagaattgcgaatggttattccaataatctaataa 
               
               
                   
               
               
                 gcagggttgctgatgttgcattaaaagagagaaggaagctgataatagtcccacgtgagacccccttaaatgatattcat 
               
               
                   
               
               
                 ataaaaaatatgctcaccttaagccgtgccggtgcagtaatactccctgcatcacctgccttctatcataagccgaaagg 
               
               
                   
               
               
                 tattgacgatatagcaaaatttatcacagcaagaattctcaatcagcttgatatagataatgacctcatccctccatatg 
               
               
                   
               
               
                 caagagagggttaagataatactattttacctttgcacccaccttgacatccccatcaaatccaataagaaccaacctcc 
               
               
                   
               
               
                 cgtctccacctgctgccaataccatcccctgcgattccgtgcccatcagttttgcagacttaaggtttgtcaccagaaca 
               
               
                   
               
               
                 attttttttccgattaattcttcaggtgtataactttcagcaatcccagcaacaacttgtctttcctctgtccctatatc 
               
               
                   
               
               
                 tactttgagttttaggagtttttttgatttctcaatcttctctgcctgttttatctcaccaactctcaaatcaacctttg 
               
               
                   
               
               
                 caaattcatcaatacttattaactgcctctcctcaactgcccctgccgattctgttactgttgctgttactttatcacca 
               
               
                   
               
               
                 atttccattttctcctccaccctcggaaaaagctgtctccctattttaatttcaatgcctgatttaattccaccccattt 
               
               
                   
               
               
                 tattgattctttaaaatcataattttctattctatccttaatccccaattgattccatatttcctgccctgtctctggca 
               
               
                   
               
               
                 taaatggatatatatagacagcaataatacgcagactttcagctaaagtatataaggtatttgatagtgtcccctcatcc 
               
               
                   
               
               
                 ttttcactccacggtgcagacttttgagcatattcattcatatctccgatgatcttccaaatgtaagaaagtgcatgagg 
               
               
                   
               
               
                 aaattcgagtttatacataaaactgtcaaaactgtttgcagcataactctcaaaaccttttagagcaggatcgtatttaa 
               
               
                   
               
               
                 aataaccctgaatattctgctccatacccctatttttagcatcaatttcaggtggaatcttaccctccctatattttttt 
               
               
                   
               
               
                 atcatattgagagtcctgcttaatagattgcccaagtcatttgcaaggtcactgtttatcctcccaatcaatgccctctg 
               
               
                   
               
               
                 ggaaaaatcaccgtcaagtccaaagggaacttctctcatcaaaaaatacctgaacgcatctaccccatatttatctatca 
               
               
                   
               
               
                 attcatgcggatttacaaaattccctctggactttgacatcttctcaccgttcacagtccaccagccgtgtgcaaatata 
               
               
                   
               
               
                 ttttccgggagtggcaagtccaatgccttgagcattgttgaccagtaaactgaatgggtggtgagtatatctttccccag 
               
               
                   
               
               
                 aaggtgttggtctgcgggccaccactctttgtcattgggggcaagatattttgtagcagaatagtaattaacgagtgcat 
               
               
                   
               
               
                 caaaccagacataggtaacatagttctcattaaagggcagtggaataccccatgaaagcctctgctttggtcttgagata 
               
               
                   
               
               
                 cagaggtctcccagtatgttattttttagaaaaccaagaacctcatttttacgggcttcaggaagtatataattagaatt 
               
               
                   
               
               
                 tttttcaatatattcttttaaatcttcttgatactttgacatcagaaaaaagtaattttcttcttctatgtgttccacag 
               
               
                   
               
               
                 gacggccgcagtcggggcagttgccaccttttatttccttctctgtccagaacctctcatcagggatacaataccatccc 
               
               
                   
               
               
                 aaataactccttttttctatcttcctttcatcaaataacttctgcaaaatatcctgcacaatcttaatatgcccttcatc 
               
               
                   
               
               
                 agttgtgcgtataaaggcattgtttgagatgttgagtcttttccagagatttttaaaattctctaccattaaatccgcat 
               
               
                   
               
               
                 gttcttttggtttaagacccctatctctggctgccttatctaccttctgtccatgctcatctgtccctgtcagaaaaaac 
               
               
                   
               
               
                 acctgatagtcgcagagccttttatatcgtgccataacatccgccgctacagtggtataggcatgccctatgtgtggaat 
               
               
                   
               
               
                 atcatttacatagtagataggtgtggttatatagaactttttcatcgtaaaattataaactccaaatcccaaattacaaa 
               
               
                   
               
               
                 taatatccaatatccaaaattctaaataatttggtcattgtgatttggttattatttgattattgatatttgggtattgg 
               
               
                   
               
               
                 aatctgttttatcccgtcaactgtaatctcttaattccctttgcttgtcctgaattttcatctatatcaattacaacagc 
               
               
                   
               
               
                 attaaattgaccttcaccgcccgccatctcaaacttctttggcatctttgtaagaaacttctctaatgcaagttcctttc 
               
               
                   
               
               
                 taattcctatgaccgagttggatggccctgtcattcccacatcggtaatataagcagtaccgttaggcaatatcttttca 
               
               
                   
               
               
                 tctgcagtctggacatgtgtatgtgtcccgattacagcacttaccctcccgtccagataccagcccattgcaattttttc 
               
               
                   
               
               
                 tgatgtcgcctcggcatgcatatcaactattgtaacctttatc 
               
            
           
         
       
     
     In some examples, the Cas protein has the following sequence: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 15) 
               
               
                 MQGQKLGIDLGGKHVGLAVVRTPINEVAHYCTIELREDIKDKMDERRSLR 
               
               
                   
               
               
                 RARRNRLWHREARFDNRQLRVKCKYIDKDTGEICGANTPKKSNVKHLLLE 
               
               
                   
               
               
                 NILVNLKIADESKEEIRRRGLDRDTNKSELQTILEKFSINTFLKKQIKDI 
               
               
                   
               
               
                 ILEKGEGRAVFCREHIPFHYEQVATEAESFWLSNSIRAKQDQILSRLKRI 
               
               
                   
               
               
                 AKDFKIDEVVIERANFDLQKLQRPDEIEAPEDYMKGPNFGHRNRFEALKQ 
               
               
                   
               
               
                 EYGNRCCFCGKKGGDEVKLKIGHLYPKAKDEINRWENLITICEKCNAKQG 
               
               
                   
               
               
                 KRTPEEAGMEFVIVKEKVFNPAAGRVIPIKRELKPKPINESKVNKYMTHT 
               
               
                   
               
               
                 DIGIRRLKREIQNIFGSIPIRETYGYITSYFRNKWELEKEHYNDAVVIAS 
               
               
                   
               
               
                 DKEDLNIKPVFKDAVPQTIKSSIKGGKLFDTNPLQFSDGKFYQNITLIGR 
               
               
                   
               
               
                 KAGMRSSKHKRGQRNIRNYGSIYMDEIELITSEWKKKVLCELRDKLGYVK 
               
               
                   
               
               
                 GDKNKSFKPEELMNANLPFRTVTIDKRGVGESSTRLINNNVFRASAEVNT 
               
               
                   
               
               
                 HIMVYSNNDGRMKAFAVKNPKIFKDAGLPHDFQKKIFIVKKGDIVTWKKS 
               
               
                   
               
               
                 EDGIAVTGRVTKCLTKNGVIDIKDMNNKIHSGKNPVYIEKIVSPERGAIF 
               
               
                   
               
               
                 ERKSLSAL 
               
            
           
         
       
     
     In certain example embodiments, the Cas protein is encoded by the following polynucleotide sequence or a portion thereof: 
     
       
         
           
               
               
            
               
                 (SEQ ID NO: 16) 
                   
               
               
                 cgggggcggtagtttgatcaaatcaaggaggaacgagacttccatccactgccggcgggcgatgggggttgcaacaagga 
                   
               
               
                   
               
               
                 aacggatatgaaagagcttagcgcgccactcttccatccactgccggcgggcgatgggggttgcaaccccatctatggaa 
               
               
                   
               
               
                 gtcgcctcagccaccgatccttggggatcgacggggctacgttattcagatgcataccctggggtggacgactccagccc 
               
               
                   
               
               
                 caggccctatggggtgaactaaacagcgctgaagtggaggcgcagtggtagcccggtgggactctgtcccacaaaactct 
               
               
                   
               
               
                 gaataacattggctgggagtcacctgacgggttggccggatacccaggtcttaccgacatagaaccggccattctcaacc 
               
               
                   
               
               
                 cttgagaagggataaaaacatgggagatttgagaaataaaccagtttatgtgcaaaatgcagatggtcaacctttgatgc 
               
               
                   
               
               
                 cgaccacgccggcccgggccaggcgaatgctggatcaaggcaaagccaggattgcgattcgatcaccctttacgatccgc 
               
               
                   
               
               
                 ttgcttcagcagattgaatcgccgcaattgcagcctgtgcgagttggtctggatacggggtccaaggcaatggggattgc 
               
               
                   
               
               
                 ggccattgccaatggcaaagccatatttgttggggaattacctttgcgacagtttcaaaaggggagtgccgtagctgaca 
               
               
                   
               
               
                 gagccatgcatcgccgggcacgcaggtcacgcttgcgctatcggaaagcgcggtttttgaatcggaccagaaaaaagtgc 
               
               
                   
               
               
                 aaggtttgtggtggcaatacgccaaagtctgatcgcaaatccggcgggcgagcggaattatgccgaaaatgtgccgctga 
               
               
                   
               
               
                 ggggcatcatgcattcgcaggaattgctaaagttccgggttggatatccccaacattaaaagcgaaaaaggacaaccatg 
               
               
                   
               
               
                 ttcatgccgtcaagaacctggctaaaatccttccggtatcagaggtggttgttgaggtagccgattgggatattcaaaag 
               
               
                   
               
               
                 atccggaatcctgatatttcgggatacgagtaccaaaatggaccattggcgcattacgagaacctgcgtgcatatgttta 
               
               
                   
               
               
                 tgcgcgtgatggatggacatgccaatactgtaaatccgaagatgggaatctgacactggatcatattatcccagaatcgc 
               
               
                   
               
               
                 ggggtggtcctacgacacccaacaacctggttgcagcgtgctataactgcaatcgagcaaaagggaatcaaactgccgag 
               
               
                   
               
               
                 gaatggggataccctgacattcaagaaagagtcaaaaagaatgagcttgcttttaagcacgcggcacacgttggcagtat 
               
               
                   
               
               
                 caaaaaccacatcatttatgagttgtcgaaggaattcccggtacgaacgacatatggtttttacacgcatatcaagcgac 
               
               
                   
               
               
                 gggatgagcttggtttagagaagcggcatgggcatgatgctgtagctattgcatgtcgatggggtgaaaaggtcgaggtt 
               
               
                   
               
               
                 gtgagccccatttaccagggccggctgaaaccttcgcgtcgccggcaaaaatatcaaatgttgatgttcccgcaatatcg 
               
               
                   
               
               
                 atataagccacgcacgaaaaaagggaaaaaagatctggacaatcaacttgctaaattgaagtacaatagtaaaaacgacc 
               
               
                   
               
               
                 cagaaaggcttaaggctattgcgcggcaattgagggccctggcgcccgagttttgggaaggtaaggggtattttgttccc 
               
               
                   
               
               
                 aaggaaagaaacaagcgcattgtcgcgaaggatggtacgatctttaaaaagagtgattatgttgaagctgtggtaagcgg 
               
               
                   
               
               
                 aaagaaatgccggggatatgttaccgctttgtattcatctggtagattgaaggttgaaacatcagagggtattaaatctg 
               
               
                   
               
               
                 caagcccagatcgttcgcgcaaattgcaatcagcgcggtcaattatgtggtgggaggaataaaatgggaatctacgtgac 
               
               
                   
               
               
                 gatcctggccggcgaccaggagctttacagcggcaaaatttcgaacggcctggcgagcgtgttgatgaacacgttcgatc 
               
               
                   
               
               
                 aggcgcggcaacataatttgttcggggaaagtttcctggcgccggcgggcaaggttgagggggcggaagatgtggaaagg 
               
               
                   
               
               
                 gtctttgtcaagctgtgccggcatcttcacaaccctcgctttttactgccgcggtgggactcgaacgacgagaactttcg 
               
               
                   
               
               
                 ggcaaagcaggcgcgggcagagttgggcaagatggttgatgagatgaaggcgctggagcttgcgctaggcagagagaggg 
               
               
                   
               
               
                 agcttgggcgggagccggcggtgaggtgggggtaatgggggaagatggaacaattggggttgccgctaaggattgaatct 
               
               
                   
               
               
                 gaaatttatagctggcatgaggggtatttctgtgattgcgatttgcagacggacgagccttcggtttgcgaaattcatgg 
               
               
                   
               
               
                 ccatcgtcgttatgagcgcaggccggcctggtggcgctgcccgctttgtgaagttgttttcgtgcctgggtcgaaagagc 
               
               
                   
               
               
                 gtccggtggtgcacaattgtcagcagaacaggtaattgcatgggaacgctttggaagggagccggcaatatgctgttgac 
               
               
                   
               
               
                 cccctgcggcactctattgtcacccggttgccggctcccctccggggcgttcccggacatggaaggcggcgatgcgcgta 
               
               
                   
               
               
                 cccgaggcatcgcgtgctggaatgagcccgggcgggcctggggaaacgctcgcccgggaaaaattgcgacggggggaggt 
               
               
                   
               
               
                 ggtaatcgaccaagagagacatcaactatgacgaactgtcctctgcatcttgggtgccggctttgcggggcatggccggc 
               
               
                   
               
               
                 acctgggaatagtgtgtaactgcagttacacgtggaaaatcgaaggatttgggagatgagcaatagtacgggtccaaaag 
               
               
                   
               
               
                 cgaa 
               
            
           
         
       
     
     In some examples, the Cas protein has the following sequence: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 17) 
               
               
                 MGDLRNKPVYVQNADGQPLMPTTPARARRMLDQGKARIAIRSPFTIRLLQ 
               
               
                   
               
               
                 QIESPQLQPVRVGLDTGSKAMGIAAIANGKAIFVGELPLRQFQKGSAVAD 
               
               
                   
               
               
                 RAMHRRARRSRLRYRKARFLNRTRKKCKVCGGNTPKSDRKSGGRAELCRK 
               
               
                   
               
               
                 CAAEGHHAFAGIAKVPGWISPTLKAKKDNHVHAVKNLAKILPVSEVVVEV 
               
               
                   
               
               
                 ADWDIQKIRNPDISGYEYQNGPLAHYENLRAYVYARDGWTCQYCKSEDGN 
               
               
                   
               
               
                 LTLDHIIPESRGGPTTPNNLVAACYNCNRAKGNQTAEEWGYPDIQERVKK 
               
               
                   
               
               
                 NELAFKHAAHVGSIKNHIIYELSKEFPVRTTYGFYTHIKRRDELGLEKRH 
               
               
                   
               
               
                 GHDAVAIACRWGEKVEVVSPIYQGRLKPSRRRQKYQMLMFPQYRYKPRTK 
               
               
                   
               
               
                 KGKKDLDNQLAKLKYNSKNDPERLKAIARQLRALAPEFWEGKGYFVPKER 
               
               
                   
               
               
                 NKRIVAKDGTIFKKSDYVEAVVSGKKCRGYVTALYSSGRLKVETSEGIKS 
               
               
                   
               
               
                 ASPDRSRKLQSARSIMWWEE 
               
            
           
         
       
     
     PAM Specificity 
     The inventors have found that at least in certain embodiments, the Cas proteins as described herein do not contain a substantial PAM specificity. In certain embodiments, the Cas protein further lacks or substantially lacks a (PAM interacting) PI domain. In certain embodiments, the Cas protein may have a PI domain or a functional fragment of a PI domain. In certain embodiments, the Cas protein may achieve a target specificity by a non-protein domain. In certain embodiments, the Cas protein may have helicase activity. In certain embodiments, the Cas protein may have reduced helicase activity compared to Cas proteins known in the art. In certain embodiments, the Cas protein may comprise additional components that contribute in mediating target recognition. In certain embodiments, targeting specificity is obtained by a central hairpin structure in a guide molecule. 
     The PAM interaction domain or PI domain as referred to herein is reported to be responsible for determining PAM specificity of Cas proteins. By means of example, the PI domain of Cas is contained in the NUC lobe and forms an elongated structure comprising seven α-helices, a three-stranded antiparallel β-sheet, a five-stranded antiparallel β-sheet, and a two-stranded antiparallel β-sheet. 
     Where the Cas protein is a protein which does have a PAM requirement, the precise sequence and length requirements for the PAM will differ depending on the Cas protein used. 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 orthologs have been identified and the skilled person will be able to identify further PAM sequences for use with a given Cas protein. 
     Further, engineering of the PAM Interacting (PI) domain may allow programing of PAM specificity, improve target site recognition fidelity, and increase the versatility of the Cas, genome engineering platform. Cas proteins may be engineered to alter their PAM specificity, for example as described 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. The skilled person will understand that other Cas proteins may be modified analogously. 
     Cas proteins are in part characterizable by the nature of the guide RNA that ensures formation of the CRISPR complex and binding to the target sequence. The guide RNA envisaged for use with a Cas protein of the present invention is capable of specifically hybridizing to a target sequence, directing binding of the CRISPR complex formed by said Cas protein and guide sequence to said target sequence. In certain embodiments, the target sequence is a coding sequence. In certain embodiments, the target sequence is a noncoding sequence. By means of example, noncoding sequences include noncoding functional RNA, cis- and trans-regulatory elements, introns, pseudogenes, repeat sequences, transposons, viral elements, and telomeres. Examples of noncoding functional RNA include ribosomal RNA, transfer RNA, piwi-interacting RNA and microRNA. In certain embodiments, the target sequence may be a regulatory DNA sequence. Non-limiting examples of regulatory DNA sequences are transcription factors, operators, enhancers, silencers, promoters, and insulators. 
     In particular embodiments, where the Cas protein is a reduced version of a naturally occurring full length Cas protein, the guide RNA envisaged for use with the Cas protein of the present invention can be the guide RNA which is known to function with the corresponding full length Cas protein. Features of the guide RNAs of Cas proteins are detailed herein below. 
     The crystal structure information (described in U.S. Provisional Patent Application Nos. 61/915,251 filed Dec. 12, 2013, 61/930,214 filed on Jan. 22, 2014, 61/980,012 filed Apr. 15, 2014; and Nishimasu et al, “Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA,” Cell 156(5):935-949, DOI: dx.doi.org/10.1016/j.cell.2014.02.001 (2014), each and all of which are incorporated herein by reference) provides structural information to truncate and create modular or multi-part CRISPR enzymes which may be incorporated into inducible CRISPR-Cas systems. In particular, structural information is provided for  S. pyogenes  Cas9 (SpCas9), and this may be extrapolated to other Cas9 orthologs or other Type II CRISPR enzymes. In some embodiments, the conformational variations in the crystal structures of the CRISPR-Cas9 system or of components of the CRISPR-Cas9 provide important and critical information about the flexibility or movement of protein structure regions relative to nucleotide (RNA or DNA) structure regions that may be important for CRISPR-Cas9 system function. The structural information provided for Cas9 (e.g.  S. pyogenes  Cas9) as the CRISPR enzyme in the present application may be used to further engineer and optimize the CRISPR-Cas system and this may be extrapolated to interrogate structure-function relationships in other CRISPR enzyme systems as well, e.g., other Type II CRISPR enzyme systems. 
     Cas Protein Modifications 
     The Cas proteins may comprise one or more modifications. In some embodiments, an unmodified nucleic acid-targeting effector protein may have cleavage activity. In some embodiments, the nucleic acid-targeting effector protein may direct cleavage of one or both nucleic acid (DNA or RNA) strands at the location of or near a target sequence, such as within the target sequence and/or within the complement of the target sequence or at sequences associated with the target sequence. In some embodiments, the nucleic acid-targeting effector protein may direct cleavage of one or both DNA or RNA strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence. In some embodiments, the cleavage may be staggered, i.e. generating sticky ends. In some embodiments, the cleavage is a staggered cut with a 5′ overhang. In some embodiments, the cleavage is a staggered cut with a 5′ overhang of 1 to 5 nucleotides, preferably of 4 or 5 nucleotides. 
     In some embodiments, the cleavage site is distant from the PAM, e.g., the cleavage occurs after the 18th nucleotide on the non-target strand and after the 23rd nucleotide on the targeted strand. In some embodiments, the cleavage site occurs after the 18th nucleotide (counted from the PAM) on the non-target strand and after the 23rd nucleotide (counted from the PAM) on the targeted strand. In some embodiments, a vector encodes a nucleic acid-targeting effector protein that may be mutated with respect to a corresponding wild-type enzyme such that the mutated nucleic acid-targeting effector protein lacks the ability to cleave one or both DNA or RNA strands of a target polynucleotide containing a target sequence. As a further example, two or more catalytic domains of a Cas protein (e.g. RuvC I, RuvC II, and RuvC III or the HNH domain of a Cas9 protein) may be mutated to produce a mutated Cas protein substantially lacking all DNA cleavage activity. As described herein, corresponding catalytic domains of a Cas effector protein may also be mutated to produce a mutated Cas effector protein lacking all DNA cleavage activity or having substantially reduced DNA cleavage activity. In some embodiments, a nucleic acid-targeting effector protein may be considered to substantially lack all polynucleotide cleavage activity when the polynucleotide 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. An effector protein may be identified with reference to the general class of enzymes that share homology to the biggest nuclease with multiple nuclease domains from the Type I, II, III, IV, V, or VI CRISPR systems. Most preferably, the effector protein is a Cas protein. In further embodiments, the effector protein is a Type II protein. By derived, Applicants mean that the derived enzyme is largely based, in the sense of having a high degree of sequence homology with, a wildtype enzyme, but that it has been mutated (modified) in some way as known in the art or as described herein. 
     In certain embodiments, the nuclease domains of the Cas protein are catalytically inactive, or modified to be catalytically inactive, or when the protein is a nickase. In certain embodiments, both nuclease domains are catalytically inactive. 
     By means of guidance and not limitation, regarding the Cas9 sequence of SEQ ID NO: 1, a Cas9 protein or functional fragment hereof may contain a D10A mutation that is causative for nickase activity and wherein no nuclease activity is associated with the protein, or a H840A mutation that is causative for nickase activity wherein no nuclease activity is associated with the protein. In some embodiments, a “nuclease-dead” Cas9 can be a D10A H840A Cas9 protein or functional fragment hereof that has neither nickase nor nuclease activity. A Cas9 protein can also be a D10A D839A H840A N863A Cas9 protein in which alanine residues are substituted for the aspartic acid residues at positions 10 and 839, the histidine residue at position 840, and the asparagine residue at position 863 in SEQ ID NO: 1. Methods to generate catalytically attenuated or catalytically inactivated are available in the art and are therefore known to a skilled person. In certain embodiments, modification of the catalytical activity of the Cas protein may be achieved by binding of a non-Cas protein. In certain embodiments, the non-Cas protein may be provided in trans. 
     Again, it will be appreciated that the terms CRISPR enzyme and CRISPR protein and Cas protein are generally used interchangeably and at all points of reference herein refer by analogy to novel CRISPR effector proteins further described in this application, unless otherwise apparent, such as by specific reference to Cas9. As mentioned above, many of the residue numberings used herein refer to the effector protein from the Types I, II, III, IV, V, and VI CRISPR loci. 
     In addition to the mutations described above, the CRISPR-Cas protein may be additionally modified. As used herein, the term “modified” with regard to a CRISPR-Cas protein generally refers to a CRISPR-Cas protein having one or more modifications or mutations (including point mutations, truncations, insertions, deletions, chimeras, fusion proteins, etc.) compared to the wild type Cas protein from which it is derived. By derived is meant that the derived enzyme is largely based, in the sense of having a high degree of sequence homology with, a wildtype enzyme, but that it has been mutated (modified) in some way as known in the art or as described herein. 
     The additional modifications of the CRISPR-Cas protein may or may not cause an altered functionality. By means of example, and in particular with reference to CRISPR-Cas protein, modifications which do not result in an altered functionality include for instance codon optimization for expression into a particular host, or providing the nuclease with a particular marker (e.g. for visualization). Modifications with may result in altered functionality may also include mutations, including point mutations, insertions, deletions, truncations (including split nucleases), etc., as well as chimeric nucleases (e.g. comprising domains from different orthologues or homologues) or fusion proteins. Fusion proteins may without limitation include, for instance, fusions with heterologous domains or functional domains (e.g. localization signals, catalytic domains, etc.). In certain embodiments, various different modifications may be combined (e.g. a mutated nuclease which is catalytically inactive and which further is fused to a functional domain, such as for instance to induce DNA methylation or another nucleic acid modification, such as including without limitation, a break (e.g. by a different nuclease (domain)), a mutation, a deletion, an insertion, a replacement, a ligation, a digestion, a break or a recombination). As used herein, “altered functionality” includes without limitation an altered specificity (e.g. altered target recognition, increased (e.g. “enhanced” Cas proteins) or decreased specificity, or altered PAM recognition), altered activity (e.g. increased or decreased catalytic activity, including catalytically inactive nucleases or nickases), and/or altered stability (e.g. fusions with destabilization domains). Examples of all these modifications are known in the art. It will be understood that a “modified” nuclease as referred to herein, and in particular a “modified” Cas or “modified” CRISPR-Cas system or complex preferably still has the capacity to interact with or bind to the polynucleic acid (e.g. in complex with the guide molecule). Such modified Cas protein can be combined with the deaminase protein or active domain thereof as described herein. 
     In certain embodiments, CRISPR-Cas protein may comprise one or more modifications resulting in enhanced activity and/or specificity, such as including mutating residues that stabilize the targeted or non-targeted strand (e.g. eCas9; “Rationally engineered Cas9 nucleases with improved specificity”, Slaymaker et al. (2016), Science, 351(6268):84-88, incorporated herewith in its entirety by reference). In certain embodiments, the altered or modified activity of the engineered CRISPR protein comprises increased targeting efficiency or decreased off-target binding. In certain embodiments, the altered activity of the engineered CRISPR protein comprises modified cleavage activity. In certain embodiments, the altered activity comprises increased cleavage activity as to the target polynucleotide loci. In certain embodiments, the altered activity comprises decreased cleavage activity as to the target polynucleotide loci. In certain embodiments, the altered activity comprises decreased cleavage activity as to off-target polynucleotide loci. In certain embodiments, the altered or modified activity of the modified nuclease comprises altered helicase kinetics. In certain embodiments, the modified nuclease comprises a modification that alters association of the protein with the nucleic acid molecule comprising RNA (in the case of a Cas protein), or a strand of the target polynucleotide loci, or a strand of off-target polynucleotide loci. In an aspect of the invention, the engineered CRISPR protein comprises a modification that alters formation of the CRISPR complex. In certain embodiments, the altered activity comprises increased cleavage activity as to off-target polynucleotide loci. Accordingly, in certain embodiments, there is increased specificity for target polynucleotide loci as compared to off-target polynucleotide loci. In other embodiments, there is reduced specificity for target polynucleotide loci as compared to off-target polynucleotide loci. In certain embodiments, the mutations result in decreased off-target effects (e.g. cleavage or binding properties, activity, or kinetics), such as in case for Cas proteins for instance resulting in a lower tolerance for mismatches between target and guide RNA. Other mutations may lead to increased off-target effects (e.g. cleavage or binding properties, activity, or kinetics). Other mutations may lead to increased or decreased on-target effects (e.g. cleavage or binding properties, activity, or kinetics). In certain embodiments, the mutations result in altered (e.g. increased or decreased) helicase activity, association or formation of the functional nuclease complex (e.g. CRISPR-Cas complex). In certain embodiments, the mutations result in an altered PAM recognition, i.e. a different PAM may be (in addition or in the alternative) be recognized, compared to the unmodified Cas protein (see e.g. “Engineered CRISPR-Cas9 nucleases with altered PAM specificities”, Kleinstiver et al. (2015), Nature, 523(7561):481-485, incorporated herein by reference in its entirety). Particularly preferred mutations include positively charged residues and/or (evolutionary) conserved residues, such as conserved positively charged residues, in order to enhance specificity. In certain embodiments, such residues may be mutated to uncharged residues, such as alanine. 
     Additional Domains 
     Aspects of the invention relate to CRISPR-Cas systems or Cas proteins, as described in any of the embodiments herein wherein the Cas protein and/or guide molecule further comprise one or more additional, typically functional, domains. 
     In an embodiment, the Cas protein, or an ortholog or homolog thereof, may be used as a generic nucleic acid binding protein with fusion to or being operably linked to a functional domain. Exemplary functional domains may include 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, molecular switches (e.g., light inducible), translational initiator, translational activator, translational repressor, nucleases, in particular ribonucleases, a spliceosome, beads, a light inducible/controllable domain or a chemically inducible/controllable domain, base editing activity, nucleotide deaminase activity, methylase activity, demethylase activity, translation activation activity, translation repression activity, transcription activation activity, transcription repression activity, transcription release factor activity, chromatin modifying or remodeling 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, nucleic acid binding activity, detectable activity. In certain embodiments, the Cas protein can be linked by physical interaction to one or more functional domains wherein at least one functional domain comprises base editing activity, nucleotide deaminase activity, methylase activity, demethylase activity, translation activation activity, translation repression activity, transcription activation activity, transcription repression activity, transcription release factor activity, chromatin modifying or remodeling 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, nucleic acid binding activity, detectable activity, and exonuclease. In certain embodiments, the functional domain can be linked to the Cas protein by a specific trigger, such as a compound or light as described elsewhere herein. 
     Preferred domains are Fok1, VP64, P65, HSF1, MyoD1. In the event that FokI is provided, it is advantageous that multiple FokI functional domains are provided to allow for a functional dimer and that gRNAs are designed to provide proper spacing for functional use (Fok1) as specifically described in Tsai et al. Nature Biotechnology, Vol. 32, Number 6, June 2014). 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. In some examples, the functional domain is an exonuclease domain, e.g., Trex2. 
     Examples of domains include a nuclease, a ligase, a reverse transcriptase, deminase, a repair protein, a methyltransferase, (viral) integrase, a recombinase, a transposase, an argonaute, a cytidine deaminase, a retron, a group II intron, a phosphatase, a phosphorylase, a sulpfurylase, a kinase, a polymerase, and an exonuclease. In some example, the function domain is a deaminase. In some examples, the functional domain is a transposase. In some examples, the functional domain is a reverse transcriptase. 
     In an aspect the invention provides a composition as herein discussed wherein the one or more functional domains is attached to the Cas effector protein or adaptor protein via a linker, optionally a GlySer linker, as discussed herein. 
     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. 
     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. 
     In some embodiments, the functional domain may be a HDAC Recruiter Effector Domain. Preferred examples include 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 Histone Methyltransferase (HMT) Recruiter Effector Domain. Preferred examples include Hp1a, PHF19, and NIPP1. 
     In some embodiments, the functional domain may be a Methyltransferase (HMT) Effector Domain. Preferred examples include those in the Table 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. 
     The invention comprehends a nucleic acid-targeting complex comprising a nucleic acid-targeting effector protein and a guide RNA, wherein the nucleic acid-targeting effector protein comprises at least one mutation, such that the nucleic acid-targeting effector protein has no more than 5% of the activity of the nucleic acid-targeting effector protein not having the at least one mutation and, optional, at least one or more nuclear localization sequences; the guide RNA comprises a guide sequence capable of hybridizing to a target sequence of interest in a cell; and wherein: the nucleic acid-targeting effector protein is associated with two or more functional domains; or at least one loop of the guide RNA 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 two or more functional domains; or the nucleic acid-targeting Cas protein is associated with one or more functional domains and at least one loop of the guide RNA 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. 
     It is also envisaged that the nucleic acid-targeting effector protein-guide RNA 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 nucleic acid-targeting effector protein, or there may be two or more functional domains associated with the guide RNA (via one or more adaptor proteins), or there may be one or more functional domains associated with the nucleic acid-targeting effector protein and one or more functional domains associated with the guide RNA (via one or more adaptor proteins). 
     The term “associated with” is used here in relation to the association of the functional domain to the Cas effector protein or the adaptor protein. It is used in respect of how one molecule ‘associates’ with respect to another, for example between an adaptor protein and a functional domain, or between the Cas effector protein and a functional domain. In the case of such protein-protein interactions, this association may be viewed in terms of recognition in the way an antibody recognizes an epitope. Alternatively, one protein may be associated with another protein via a fusion of the two, for instance one subunit being fused to another subunit. Fusion typically occurs by addition of the amino acid sequence of one to that of the other, for instance via splicing together of the nucleotide sequences that encode each protein or subunit. Alternatively, this may essentially be viewed as binding between two molecules or direct linkage, such as a fusion protein. In any event, the fusion protein may include a linker between the two subunits of interest (i.e. between the enzyme and the functional domain or between the adaptor protein and the functional domain). Thus, in some embodiments, the Cas effector protein or adaptor protein is associated with a functional domain by binding thereto. In other embodiments, the Cas effector protein or adaptor protein is associated with a functional domain because the two are fused together, optionally via an intermediate linker. 
     In some embodiments, the protein domain and/or protein structure formed by the protein linker sequence may introduce affinity of the Cas protein for additional proteins or parts of proteins that have no affinity for Cas proteins without the protein linker sequence. In certain embodiments, the protein linker sequence may introduce affinity of the Cas protein for non-protein molecules. In further embodiments, the affinity may be specific for polynucleotides. 
     Attachment of a functional domain or fusion protein can be via a linker, e.g., a flexible glycine-serine (GlyGlyGlySer) (SEQ ID NO:18) or (GGGS) 3  (SEQS ID NO: 19) or a rigid alpha-helical linker such as (Ala(GluAlaAlaAlaLys)Ala) (SEQ ID NO: 20). Linkers such as (GGGGS) 3  (SEQ ID NO: 21) are preferably used herein to separate protein or peptide domains. (GGGGS) 3 (SEQ ID NO: 21) 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: 22) (GGGGS) 9  (SEQ ID NO: 23) or (GGGGS) 12  (SEQ ID NO: 24) may preferably be used as alternatives. Other preferred alternatives are (GGGGS) 1 (SEQ ID NO: 25), (GGGGS) 2  (SEQ ID NO: 26), (GGGGS) 4  (SEQ ID NO: 27), (GGGGS) 5  (SEQ ID NO: 28), (GGGGS) 7  (SEQ ID NO: 29), (GGGGS) 8  (SEQ ID NO: 30), (GGGGS) 10  (SEQ ID NO: 31), or (GGGGS) 11  (SEQ ID NO: 32). 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 protein and any functional domain and/or between the guide RNA and the functional domain (e.g. activator or repressor). Again, a (GGGGS) 3  (SEQ ID NO: 21) 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. 
     In general, the positioning of the one or more functional domains on the Cas protein is one which allows for correct spatial orientation for the functional domain to affect the target with the attributed functional effect. 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., FokI) will be advantageously positioned to cleave or partially cleave the target. This may include positions other than the N-/C-terminus of the Cas protein. 
     In certain embodiments, a functional domain is linked to the Cas protein. In particular embodiments, a functional domain may be coupled by an aptamer mediated interaction. In further embodiments, the aptamer is a MS2 sequence, or any sequence that has been described in the art that is able to bind a protein or protein fragment. In certain embodiments, the functional domain acts on a residue or group of residues of the polynucleotide sequence part of the DNA:RNA duplex. In certain embodiments, the functional domain acts on a residue that has a specific position in the DNA:RNA duplex. In certain embodiments, the CRISPR-Cas system or Cas protein as described in any of the embodiments herein further comprise more than one functional domain. In certain embodiments, all further functional domains may be genetically coupled to the Cas protein. In certain embodiments, all further functional domains may be coupled to the CRISPR-Cas system by aptamer mediated interaction. In certain embodiments wherein multiple additional functional domains are present, at least one functional domain may be genetically coupled to the CRISPR-Cas system and at least one functional domain may be coupled to the CRISPR-Cas system by an aptamer mediated interaction. In certain embodiments wherein multiple additional functional domains are present, the different functional domains act on different residues of the target polynucleotide sequence. In alternative embodiments wherein multiple additional functional domains are present, the different functional domains act on the same residue of the target polynucleotide sequence. 
     In the practice of the invention and as will be described below, loops of the gRNA may be extended, without colliding with the Cas 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). The adaptor proteins may include but are not limited to orthogonal RNA-binding protein/aptamer combinations that exist within the diversity of bacteriophage coat proteins. A list of such coat proteins includes, but is not limited to: 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. These adaptor proteins or orthogonal RNA binding proteins can further recruit effector proteins or fusions which comprise one or more functional domains as described above. 
     The use of two different aptamers (each associated with a distinct nucleic acid-targeting guide RNAs) allows an activator-adaptor protein fusion and a repressor-adaptor protein fusion to be used, with different nucleic acid-targeting guide RNAs, to activate expression of one DNA or RNA, whilst repressing another. They, along with their different guide RNAs can be administered together, or substantially together, in a multiplexed approach. A large number of such modified nucleic acid-targeting guide RNAs 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 effector protein molecules need to be delivered, as a comparatively small number of effector protein molecules 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. 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. 
     In particular embodiments the Cas protein contains additional amino acids such as amino acids contributing to a peptide or protein tag sequence, regulatory sequence, or localization signal. Non-limiting examples of commonly used peptide tag sequences are the AviTag, C-tag, calmodulin-tag, polyglutamate tag, E-tag, Flag-tag, HA-tag, His-tag, Myc-tag, NE-tag, Rho1D4-tag, S-tag, SBP-tag, Softag 1, Softag 3, Spot-tag, Strep-tag, TC tag, Ty tag, V5 tag, VSV-tag, Xpress tag, isopeptag, SpyTag, SnoopTag, DogTag, and the SdyTag In further embodiments, the sequence comprises additional amino acids contributing to the turnover time of the Cas protein or to its activity. 
     In some embodiments, a nucleic acid-targeting effector protein such as the cas effector protein or an ortholog or homolog thereof comprises 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. In some embodiments, the nucleic acid-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: 33); the NLS from nucleoplasmin (e.g., the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 34)); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 35) or RQRRNELKRSP (SEQ ID NO: 36); the hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 37); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 38) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 39) and PPKKARED (SEQ ID NO: 40) of the myoma T protein; the sequence PQPKKKPL (SEQ ID NO: 41) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO: 42) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 43) and PKQKKRK (SEQ ID NO: 44) of the influenza virus NS1; the sequence RKLKKKIKKL (SEQ ID NO: 45) of the Hepatitis virus delta antigen; the sequence REKKKFLKRR (SEQ ID NO: 46) of the mouse Mx1 protein; the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 47) of the human poly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 48) 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 protein complexes and systems the codon optimized Cas effector proteins comprise an NLS attached to the C-terminal of the protein. 
     Guide Sequences 
     The systems herein may further comprise one or more guide molecules. As used herein, the term “guide sequence” or “guide molecules” has the leaning as used herein elsewhere and 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 of the guide sequence to a given 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. In certain example embodiments, the guide molecule comprises a guide sequence that may be designed to have at least one mismatch with the target sequence, such that a RNA duplex formed between the guide sequence and the target sequence. Accordingly, the degree of complementarity is preferably less than 99%. For instance, where the guide sequence consists of 24 nucleotides, the degree of complementarity is more particularly about 96% or less. In particular embodiments, the guide sequence is designed to have a stretch of two or more adjacent mismatching nucleotides, such that the degree of complementarity over the entire guide sequence is further reduced. For instance, where the guide sequence consists of 24 nucleotides, the degree of complementarity is more particularly about 96% or less, more particularly, about 92% or less, more particularly about 88% or less, more particularly about 84% or less, more particularly about 80% or less, more particularly about 76% or less, more particularly about 72% or less, depending on whether the stretch of two or more mismatching nucleotides encompasses 2, 3, 4, 5, 6 or 7 nucleotides, etc. In some embodiments, aside from the stretch of one or more mismatching nucleotides, 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 (or a sequence in the vicinity thereof) 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 or in the vicinity of 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 RNA may be selected to target any target nucleic acid sequence. 
     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 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 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 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 certain embodiments, the guide sequence or spacer length of the guide molecules is from 15 to 50 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 30 to 35 nt, e.g., 30, 31, 32, 33, 34, or 35 nt, or 35 nt or longer. In certain example embodiment, the guide sequence is 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, or 100 nt. 
     In some embodiments, the sequence of the guide molecule (direct repeat and/or spacer) is selected to reduce the degree secondary structure within the guide molecule. 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 RNA 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 of a 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). Further algorithms may be found in U.S. application Ser. No. TBA (attorney docket 44790.11.2022; Broad Reference BI-2013/004A); incorporated herein by reference. 
     In a particular embodiment, the guide molecule comprises a guide sequence linked to a direct repeat sequence, wherein the direct repeat sequence comprises one or more stem loops or optimized secondary structures. In particular embodiments, the direct repeat has a minimum length of 16 nts and a single stem loop. In further embodiments the direct repeat has a length longer than 16 nts, preferably more than 17 nts, and has more than one stem loops or optimized secondary structures. In particular embodiments, the guide molecule comprises or consists of the guide sequence linked to all or part of the natural direct repeat sequence. In particular embodiments, certain aspects of the guide architecture can be modified, for example by addition, subtraction, or substitution of features, whereas certain other aspects of guide architecture are maintained. Preferred locations for engineered guide molecule modifications, including but not limited to insertions, deletions, and substitutions include guide termini and regions of the guide molecule that are exposed when complexed with CRISPR protein and/or target, for example the tetraloop and/or loop2. 
     In some embodiments, a loop in the guide RNA is provided. This may be a stem loop or a tetra loop. The loop is preferably GAAA, but it is not limited to this sequence or indeed to being only 4 bp in length. Indeed, preferred loop forming sequences for use in hairpin structures are four nucleotides in length, and most preferably have the sequence GAAA. However, longer or shorter loop sequences may be used, as may alternative sequences. The sequences preferably include a nucleotide triplet (for example, AAA), and an additional nucleotide (for example C or G). Examples of loop forming sequences include CAAA and AAAG. 
     In some embodiments, the guide molecule forms a stemloop with a separate non-covalently linked sequence, which can be DNA or RNA. In particular embodiments, the sequences forming the guide 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, these 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 this sequence is functionalized, a covalent chemical bond or linkage can be formed between this sequence and the direct repeat sequence. 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, these stem-loop forming 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). 
     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” (SEQ ID NO: 49) 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: 50) 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, any base composition, as long as it doesn&#39;t alter the overall structure. 
     In particular embodiments the natural hairpin or stemloop structure of the guide molecule is extended or replaced by an extended stemloop. It has been demonstrated that extension of the stem can enhance the assembly of the guide molecule with the CRISPR-Cas protein (Chen et al. Cell. (2013); 155(7): 1479-1491). In particular embodiments the stem of the stemloop is extended by at least 1, 2, 3, 4, 5 or more complementary basepairs (i.e. corresponding to the addition of 2, 4, 6, 8, 10 or more nucleotides in the guide molecule). In particular embodiments these are located at the end of the stem, adjacent to the loop of the stemloop. 
     In particular embodiments, the susceptibility of the guide molecule to RNAses or to decreased expression can be reduced by slight modifications of the sequence of the guide molecule which do not affect its function. For instance, in particular embodiments, premature termination of transcription, such as premature transcription of U6 Pol-III, can be removed by modifying a putative Pol-III terminator (4 consecutive U&#39;s) in the guide molecules sequence. Where such sequence modification is required in the stemloop of the guide molecule, it is preferably ensured by a basepair flip. 
     In certain embodiments, the guide molecule comprises non-naturally occurring nucleic acids and/or non-naturally occurring nucleotides and/or nucleotide analogs, and/or chemically modifications. Preferably, these non-naturally occurring nucleic acids and non-naturally occurring nucleotides are located outside the guide sequence. 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, a locked nucleic acid (LNA) nucleotides comprising a methylene bridge between the 2′ and 4′ carbons of the ribose ring, or bridged nucleic acids (BNA). Other examples of modified nucleotides include 2′-O-methyl analogs, 2′-deoxy analogs, or 2′-fluoro analogs. Further examples of modified bases include, but are not limited to, 2-aminopurine, 5-bromo-uridine, pseudouridine, inosine, 7-methylguanosine. Examples of guide RNA chemical modifications include, without limitation, incorporation of 2′-O-methyl (M), 2′-O-methyl 3′phosphorothioate (MS), S-constrained ethyl(cEt), or 2′-O-methyl 3′thioPACE (MSP) 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 Jun. 29, 2015 Ragdarm et al., O 215 , 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). 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 sequence and one or more deoxyribonucleotides and/or nucleotide analogs in a region that binds to the Cas protein. In an embodiment of the invention, deoxyribonucleotides and/or nucleotide analogs are incorporated in engineered guide structures, such as, without limitation, stem-loop regions, and the seed region. 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), or 2′-O-methyl 3′ thioPACE (MSP). Such modification can enhance genome editing efficiency (see Hendel et al., Nat. Biotechnol. (2015) 33(9): 985-989). 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., O 215 , 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), or Rhodamine. 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 a particular embodiment, the direct repeat may be modified to comprise one or more protein-binding RNA aptamers. In a particular embodiment, one or more aptamers may be included such as part of optimized secondary structure. Such aptamers may be capable of binding a bacteriophage coat protein as detailed further herein. 
     In some embodiments, the Cas protein of the invention requires a tracr sequence. 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 guide 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 may correspond to the tracr mate sequence, and the portion of the sequence 3′ of the loop then corresponds to the tracr sequence. In a hairpin structure the portion of the sequence 5′ of the final “N” and upstream of the loop may alternatively correspond to the tracr sequence, and the portion of the sequence 3′ of the loop corresponds to the tracr mate sequence. 
     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′-acetoxyethyl 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. WO 2011/008730. 
     In certain embodiments, the Cas protein uses of a tracrRNA, the guide sequence, tracr mate, and tracr sequence may reside in a single RNA, i.e. an sgRNA (arranged in a 5′ to 3′ orientation or alternatively arranged in a 3′ to 5′ orientation), or the tracr RNA may be a different RNA than the RNA containing the guide and tracr mate sequence. In these embodiments, the tracr hybridizes to the tracr mate sequence and directs the CRISPR-Cas9 complex to the target sequence. 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 preferred embodiments, certain aspects of guide architecture are retained, 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 particular embodiments, the guide molecule comprises, in addition the guide sequence, a sequence corresponding to a direct repeat in the CRISPR locus. In particular embodiments, this sequence comprises at least one hairpin, i.e., a region of self-complementarity. In particular embodiments, the guide sequence is 3′ of the direct repeat comprising at least one hairpin. In further embodiments, the guide sequence is 5′ of the direct repeat comprising at least one hairpin. In particular embodiments, a hairpin is located in the middle of the guide sequence, i.e. the guide sequence is in part 5′ and in part 3′ of the direct repeat. The hairpin in the middle of the guide sequence may be involved in recognition or processing of the guide molecule. In particular embodiments, the hairpin structure comprises at least 5, preferably 7-20 nucleotides. 
     Escorted Guides 
     In particular embodiments, The CRISPR-Cas systems or complexes have a guide molecule with a functional structure designed to improve guide molecule 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, in particular embodiments, the guide molecule is modified, e.g., by one or more aptamer(s) designed to improve guide molecule 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 molecule deliverable, inducible or responsive to a selected effector. The invention accordingly comprehends a guide molecule 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. 
     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/cm2. 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 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., stke.sciencemag.org/cgi/content/abstract/sigtrans;4/164/rs2), 2. FKBP-FRB based system inducible by rapamycin (or related chemicals based on rapamycin) (see, e.g., www.nature.com/nmeth/journal/v2/n6/full/nmeth763.html), 3. GID1-GAI based system inducible by Gibberellin (GA) (see, e.g., www.nature.com/nchembio/journal/v8/n5/full/nchembio.922.html). 
     A chemical inducible system can be an estrogen receptor (ER) based system inducible by 4-hydroxytamoxifen (4OHT) (see, e.g., www.pnas.org/content/104/3/1027.abstract). A mutated ligand-binding domain of the estrogen receptor called ERT2 translocates 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., 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. 
     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/cm2 to about 100 W/cm2. 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/cm2 (FDA recommendation), although energy densities of up to 750 mW/cm2 have been used. In physiotherapy, ultrasound is typically used as an energy source in a range up to about 3 to 4 W/cm2 (WHO recommendation). In other therapeutic applications, higher intensities of ultrasound may be employed, for example, HIFU at 100 W/cm up to 1 kW/cm2 (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 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. 
     In particular embodiments, the guide molecule is modified by a secondary structure to increase the specificity of the CRISPR-Cas system and the secondary structure can protect against exonuclease activity and allow for 5′ additions to the guide sequence also referred to herein as a protected guide molecule. 
     In one aspect, the invention provides for hybridizing a “protector RNA” to a sequence of the guide molecule, wherein the “protector RNA” is an RNA strand complementary to the 3′ end of the guide molecule to thereby generate a partially double-stranded guide RNA. In an embodiment of the invention, protecting mismatched bases (i.e., the bases of the guide molecule which do not form part of the guide sequence) with a perfectly complementary protector sequence decreases the likelihood of target DNA binding to the mismatched basepairs at the 3′ end. In particular embodiments of the invention, additional sequences comprising an extended length may also be present within the guide molecule such that the guide comprises a protector sequence within the guide molecule. This “protector sequence” ensures that the guide molecule comprises a “protected sequence” in addition to an “exposed sequence” (comprising the part of the guide sequence hybridizing to the target sequence). In particular embodiments, the guide molecule is modified by the presence of the protector guide to comprise a secondary structure such as a hairpin. Advantageously there are three or four to thirty or more, e.g., about 10 or more, contiguous base pairs having complementarity to the protected sequence, the guide sequence or both. It is advantageous that the protected portion does not impede thermodynamics of the CRISPR-Cas system interacting with its target. By providing such an extension including a partially double stranded guide molecule, the guide molecule is considered protected and results in improved specific binding of the CRISPR-Cas complex, while maintaining specific activity. 
     In particular embodiments, use is made of a truncated guide (tru-guide), i.e. a guide molecule which comprises a guide sequence which is truncated in length with respect to the canonical guide sequence length. As described by Nowak et al. (Nucleic Acids Res (2016) 44 (20): 9555-9564), such guides may allow catalytically active CRISPR-Cas enzyme to bind its target without cleaving the target DNA. In particular embodiments, a truncated guide is used which allows the binding of the target but retains only nickase activity of the CRISPR-Cas enzyme. 
     In some embodiments, conjugation of triantennary N-acetyl galactosamine (GalNAc) to oligonucleotide components may be used to improve delivery, for example delivery to select cell types, for example hepatocytes (see International Patent Publication No. WO 2014/118272 incorporated herein by reference; Nair, J K et al., 2014, Journal of the American Chemical Society 136 (49), 16958-16961). This is considered to be a sugar-based particle and further details on other particle delivery systems and/or formulations are provided herein. GalNAc can therefore be considered to be a particle in the sense of the other particles described herein, such that general uses and other considerations, for instance delivery of said particles, apply to GalNAc particles as well. A solution-phase conjugation strategy may for example be used to attach triantennary GalNAc clusters (mol. wt. ˜2000) activated as PFP (pentafluorophenyl) esters onto 5′-hexylamino modified oligonucleotides (5′-HA ASOs, mol. wt. ˜8000 Da; Ostergaard et al., Bioconjugate Chem., 2015, 26 (8), pp 1451-1455). Similarly, poly(acrylate) polymers have been described for in vivo nucleic acid delivery (see WO2013158141 incorporated herein by reference). In further alternative embodiments, pre-mixing CRISPR nanoparticles (or protein complexes) with naturally occurring serum proteins may be used in order to improve delivery (Akinc A et al, 2010, Molecular Therapy vol. 18 no. 7, 1357-1364). 
     Screening techniques are available to identify delivery enhancers, for example by screening chemical libraries (Gilleron J. et al., 2015, Nucl. Acids Res. 43 (16): 7984-8001). Approaches have also been described for assessing the efficiency of delivery vehicles, such as lipid nanoparticles, which may be employed to identify effective delivery vehicles for CRISPR components (see Sahay G. et al., 2013, Nature Biotechnology 31, 653-658). 
     In some embodiments, delivery of protein CRISPR components may be facilitated with the addition of functional peptides to the protein, such as peptides that change protein hydrophobicity, for example so as to improve in vivo functionality. CRISPR component proteins may similarly be modified to facilitate subsequent chemical reactions. For example, amino acids may be added to a protein that have a group that undergoes click chemistry (Nikić I. et al., 2015, Nature Protocols 10,780-791). In embodiments of this kind, the click chemical group may then be used to add a wide variety of alternative structures, such as poly(ethylene glycol) for stability, cell penetrating peptides, RNA aptamers, lipids, or carbohydrates such as GalNAc. In further alternatives, a CRISPR component protein may be modified to adapt the protein for cell entry (see Svensen et al., 2012, Trends in Pharmacological Sciences, Vol. 33, No. 4), for example by adding cell penetrating peptides to the protein (see Kauffman, W. Berkeley et al., 2015, Trends in Biochemical Sciences, Volume 40, Issue 12, 749-764; Koren and Torchilin, 2012, Trends in Molecular Medicine, Vol. 18, No. 7). In further alternative embodiment, patients or subjects may be pre-treated with compounds or formulations that facilitate the later delivery of CRISPR components. Cas Development and Use In general 
     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. 8,999,641, 8,993,233, 8,945,839, 8,932,814, 8,906,616, 8,895,308, 8,889,418, 8,889,356, 8,871,445, 8,865,406, 8,795,965, 8,771,945 and 8,697,359; US patent Publications 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), US 2014-0273232 A1 (U.S. application Ser. No. 14/290,575), US 2014-0273231 (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 (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 (U.S. application Ser. No. 14/183,429); European Patents EP 2 784 162 B1 and EP 2 771 468 B1; European Patent Applications EP 2 771 468 (EP13818570.7), EP 2 764 103 (EP13824232.6), and EP 2 784 162 (EP14170383.5); and PCT Patent Publications 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). 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 Application No. 61/836,123, filed on Jun. 17, 2013. Reference is additionally made to U.S. Provisional Application Nos. 61/835,931, 61/835,936, 61/836,127, 61/836,101, 61/836,080 and 61/835,973, 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 Patent Application Nos.: PCT/US2014/041803, PCT/US2014/041800, PCT/US2014/041809, PCT/US2014/041804 and PCT/US2014/041806, each filed Jun. 10, 2014 6/10/14; PCT/US2014/041808 filed Jun. 11, 2014; and PCT/US2014/62558 filed Oct. 28, 2014, and U.S. Provisional Application Nos. 61/915,150, 61/915,301, 61/915,267 and 61/915,260, each filed Dec. 12, 2013; 61/757,972 and 61/768,959, filed on Jan. 29, 2013 and Feb. 25, 2013; 61/835,936, 61/836,127, 61/836,101, 61/836,080, 61/835,973, and 61/835,931, filed Jun. 17, 2013; 62/010,888 and 62/010,879, both filed Jun. 11, 2014; 62/010,329 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; 62/054,490, 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 also made to U.S. Provisional Application Nos. 62/055,484, 62/055,460, and 62/055,487, filed Sep. 25, 2014; U.S. Provisional Application No. 61/980,012, filed Apr. 15, 2014; and U.S. Provisional Application No. 61/939,242 filed Feb. 12, 2014. Reference is made to PCT application designating, inter alia, the United States, Application No. PCT/US14/41806, 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 U.S. Provisional Application Nos. 61/915,251; 61/915,260 and 61/915,267, each filed on Dec. 12, 2013. Reference is made to U.S. Provisional Application No. 61/980,012 filed Apr. 15, 2014. Reference is made to PCT application designating, inter alia, the United States, application No. PCT/US14/41806, 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 U.S. Provisional Application Nos. 61/915,251; 61/915,260 and 61/915,267, each filed on Dec. 12, 2013. 
     Mention is also made of 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 No. 62/091,462, filed 12 Dec. 2014, DEAD GUIDES FOR CRISPR TRANSCRIPTION FACTORS; U.S. Provisional Application No. 62/096,324, filed 23 Dec. 2014, DEAD GUIDES FOR CRISPR TRANSCRIPTION FACTORS; U.S. Provisional Application No. 62/091,456, filed 12 Dec. 2014, 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. 2014, ENGINEERING OF SYSTEMS, METHODS AND OPTIMIZED ENZYME AND GUIDE SCAFFOLDS FOR SEQUENCE MANIPULATION; U.S. Provisional Application No. 62/098,059, filed 30 Dec. 2014, RNA-TARGETING SYSTEM; U.S. Provisional Application No. 62/096,656, filed 24 Dec. 2014, 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. Provisional Application No. 62/098,158, filed 30 Dec. 2014, ENGINEERED CRISPR COMPLEX INSERTIONAL TARGETING SYSTEMS; U.S. Provisional Application No. 62/151,052, filed 22 Apr. 2015, 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. 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, 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 No. 62/054,675, filed 24 Sep. 2014, 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, filed 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, 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, 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, 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. 
     Each of these patents, patent publications, and applications, and all documents cited therein or during their prosecution (“appln cited documents”) and all documents cited or referenced in the appln cited documents, together with any instructions, descriptions, product specifications, and product sheets for any products mentioned therein or in any document therein and incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. All documents (e.g., these patents, patent publications and applications and the appln cited documents) are incorporated herein by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. 
     Also with respect to general information on CRISPR-Cas Systems, mention is made of the following (also hereby incorporated herein by reference): 
     Multiplex genome engineering using CRISPR/Cas systems. Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A., &amp; Zhang, F. Science February 15; 339(6121):819-23 (2013); 
     RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Jiang W., Bikard D., Cox D., Zhang F, Marraffini L A. Nat Biotechnol March; 31(3):233-9 (2013); 
     One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering. Wang H., Yang H., Shivalila C S., Dawlaty M M., Cheng A W., Zhang F., Jaenisch R. Cell May 9; 153(4):910-8 (2013); 
     Optical control of mammalian endogenous transcription and epigenetic states. Konermann S, Brigham M D, Trevino A E, Hsu P D, Heidenreich M, Cong L, Platt R J, Scott D A, Church G M, Zhang F. Nature. August 22; 500(7463):472-6. doi: 10.1038/Nature12466. Epub Aug. 23, 2013 (2013); 
     Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity. Ran, F A., Hsu, P D., Lin, C Y., Gootenberg, J S., Konermann, S., Trevino, A E., Scott, D A., Inoue, A., Matoba, S., Zhang, Y., &amp; Zhang, F. Cell August 28. pii: S0092-8674(13)01015-5 (2013-A); 
     DNA targeting specificity of RNA-guided Cas9 nucleases. Hsu, P., Scott, D., Weinstein, J., Ran, F A., Konermann, S., Agarwala, V., Li, Y., Fine, E., Wu, X., Shalem, O., Cradick, T J., Marraffini, L A., Bao, G., &amp; Zhang, F. Nat Biotechnol doi:10.1038/nbt.2647 (2013); 
     Genome engineering using the CRISPR-Cas9 system. Ran, F A., Hsu, P D., Wright, J., Agarwala, V., Scott, D A., Zhang, F. Nature Protocols November; 8(11):2281-308 (2013-B); 
     Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells. Shalem, O., Sanjana, N E., Hartenian, E., Shi, X., Scott, D A., Mikkelson, T., Heckl, D., Ebert, B L., Root, D E., Doench, J G., Zhang, F. Science December 12. (2013). [Epub ahead of print]; 
     Crystal structure of cas9 in complex with guide RNA and target DNA. Nishimasu, H., Ran, F A., Hsu, P D., Konermann, S., Shehata, S I., Dohmae, N., Ishitani, R., Zhang, F., Nureki, O. Cell February 27, 156(5):935-49 (2014); 
     Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Wu X., Scott D A., Kriz A J., Chiu A C., Hsu P D., Dadon D B., Cheng A W., Trevino A E., Konermann S., Chen S., Jaenisch R., Zhang F., Sharp P A. Nat Biotechnol. April 20. doi: 10.1038/nbt.2889 (2014); 
     CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling. Platt R J, Chen S, Zhou Y, Yim M J, Swiech L, Kempton H R, Dahlman J E, Parnas O, Eisenhaure T M, Jovanovic M, Graham D B, Jhunjhunwala S, Heidenreich M, Xavier R J, Langer R, Anderson D G, Hacohen N, Regev A, Feng G, Sharp P A, Zhang F. Cell 159(2): 440-455 DOI: 10.1016/j.cell.2014.09.014(2014); 
     Development and Applications of CRISPR-Cas9 for Genome Engineering, Hsu P D, Lander E S, Zhang F., Cell. June 5; 157(6):1262-78 (2014); 
     Genetic screens in human cells using the CRISPR/Cas9 system, Wang T, Wei J J, Sabatini D M, Lander E S., Science. January 3; 343(6166): 80-84. doi:10.1126/science.1246981 (2014); 
     Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation, Doench J G, Hartenian E, Graham D B, Tothova Z, Hegde M, Smith I, Sullender M, Ebert B L, Xavier R J, Root D E., (published online Sep. 3, 2014) Nat Biotechnol. December; 32(12):1262-7 (2014); 
     In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9, Swiech L, Heidenreich M, Banerjee A, Habib N, Li Y, Trombetta J, Sur M, Zhang F., (published online Oct. 19, 2014) Nat Biotechnol. January; 33(1):102-6 (2015); 
     Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex, Konermann S, Brigham M D, Trevino A E, Joung J, Abudayyeh O O, Barcena C, Hsu P D, Habib N, Gootenberg J S, Nishimasu H, Nureki O, Zhang F., Nature. January 29; 517(7536):583-8 (2015). 
     A split-Cas9 architecture for inducible genome editing and transcription modulation, Zetsche B, Volz S E, Zhang F., (published online Feb. 2, 2015) Nat Biotechnol. February; 33(2):139-42 (2015); 
     Genome-wide CRISPR Screen in a Mouse Model of Tumor Growth and Metastasis, Chen S, Sanjana N E, Zheng K, Shalem O, Lee K, Shi X, Scott D A, Song J, Pan J Q, Weissleder R, Lee H, Zhang F, Sharp P A. Cell 160, 1246-1260, Mar. 12, 2015 (multiplex screen in mouse), and 
     In vivo genome editing using  Staphylococcus aureus  Cas9, Ran F A, Cong L, Yan W X, Scott D A, Gootenberg J S, Kriz A J, Zetsche B, Shalem O, Wu X, Makarova K S, Koonin E V, Sharp P A, Zhang F., (published online 1 Apr. 2015), Nature. April 9; 520(7546):186-91 (2015); 
     Shalem et al., “High-throughput functional genomics using CRISPR-Cas9,” Nature Reviews Genetics 16, 299-311 (May 2015). 
     Xu et al., “Sequence determinants of improved CRISPR sgRNA design,” Genome Research 25, 1147-1157 (August 2015); 
     Parnas et al., “A Genome-wide CRISPR Screen in Primary Immune Cells to Dissect Regulatory Networks,” Cell 162, 675-686 (Jul. 30, 2015); 
     Ramanan et al., CRISPR/Cas9 cleavage of viral DNA efficiently suppresses hepatitis B virus,” Scientific Reports 5:10833. doi: 10.1038/srep10833 (Jun. 2, 2015); 
     Nishimasu et al., Crystal Structure of  Staphylococcus aureus  Cas9,” Cell 162, 1113-1126 (Aug. 27, 2015); 
     Zetsche et al. (2015), “Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system,” Cell 163, 759-771 (Oct. 22, 2015) doi: 10.1016/j.cell.2015.09.038. Epub Sep. 25, 2015; 
     Shmakov et al. (2015), “Discovery and Functional Characterization of Diverse Class CRISPR-Cas Systems,” Molecular Cell 60, 385-397 (Nov. 5, 2015) doi: 10.1016/j.molcel.2015.10.008. Epub Oct. 22, 2015; 
     Dahlman et al., “Orthogonal gene control with a catalytically active Cas9 nuclease,” Nature Biotechnology 33, 1159-1161 (November, 2015); 
     Gao et al, “Engineered Cpf1 Enzymes with Altered PAM Specificities,” bioRxiv 091611; doi: dx.doi.org/10.1101/091611 Epub Dec. 4, 2016; 
     Smargon et al. (2017), “Cas13b Is a Type VI-B CRISPR-Associated RNA-Guided RNase Differentially Regulated by Accessory Proteins Csx27 and Csx28,” Molecular Cell 65, 618-630 (Feb. 16, 2017) doi: 10.1016/j.molcel.2016.12.023. Epub Jan. 5, 2017; 
     each of which is incorporated herein by reference, may be considered in the practice of the instant invention, and discussed briefly below: 
     Cong et al. engineered type II CRISPR-Cas systems for use in eukaryotic cells based on both  Streptococcus thermophilus  Cas9 and also  Streptococcus pyogenes  Cas9 and demonstrated that Cas9 nucleases can be directed by short RNAs to induce precise cleavage of DNA in human and mouse cells. Their study further showed that Cas9 as converted into a nicking enzyme can be used to facilitate homology-directed repair in eukaryotic cells with minimal mutagenic activity. Additionally, their study demonstrated that multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several at endogenous genomic loci sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology. This ability to use RNA to program sequence specific DNA cleavage in cells defined a new class of genome engineering tools. These studies further showed that other CRISPR loci are likely to be transplantable into mammalian cells and can also mediate mammalian genome cleavage. Importantly, it can be envisaged that several aspects of the CRISPR-Cas system can be further improved to increase its efficiency and versatility. 
     Jiang et al. used the clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated Cas9 endonuclease complexed with dual-RNAs to introduce precise mutations in the genomes of  Streptococcus pneumoniae  and  Escherichia coli . The approach relied on dual-RNA:Cas9-directed cleavage at the targeted genomic site to kill unmutated cells and circumvents the need for selectable markers or counter-selection systems. The study reported reprogramming dual-RNA:Cas9 specificity by changing the sequence of short CRISPR RNA (crRNA) to make single- and multinucleotide changes carried on editing templates. The study showed that simultaneous use of two crRNAs enabled multiplex mutagenesis. Furthermore, when the approach was used in combination with recombineering, in  S. pneumoniae , nearly 100% of cells that were recovered using the described approach contained the desired mutation, and in  E. coli,  65% that were recovered contained the mutation. 
     Wang et al. (2013) used the CRISPR/Cas system for the one-step generation of mice carrying mutations in multiple genes which were traditionally generated in multiple steps by sequential recombination in embryonic stem cells and/or time-consuming intercrossing of mice with a single mutation. The CRISPR/Cas system will greatly accelerate the in vivo study of functionally redundant genes and of epistatic gene interactions. 
     Konermann et al. (2013) addressed the need in the art for versatile and robust technologies that enable optical and chemical modulation of DNA-binding domains based CRISPR Cas9 enzyme and also Transcriptional Activator Like Effectors 
     Ran et al. (2013-A) described an approach that combined a Cas9 nickase mutant with paired guide RNAs to introduce targeted double-strand breaks. This addresses the issue of the Cas9 nuclease from the microbial CRISPR-Cas system being targeted to specific genomic loci by a guide sequence, which can tolerate certain mismatches to the DNA target and thereby promote undesired off-target mutagenesis. Because individual nicks in the genome are repaired with high fidelity, simultaneous nicking via appropriately offset guide RNAs is required for double-stranded breaks and extends the number of specifically recognized bases for target cleavage. The authors demonstrated that using paired nicking can reduce off-target activity by 50- to 1,500-fold in cell lines and to facilitate gene knockout in mouse zygotes without sacrificing on-target cleavage efficiency. This versatile strategy enables a wide variety of genome editing applications that require high specificity. 
     Hsu et al. (2013) characterized SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. The study evaluated &gt;700 guide RNA variants and SpCas9-induced indel mutation levels at &gt;100 predicted genomic off-target loci in 293T and 293FT cells. The authors that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. The authors further showed that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. Additionally, to facilitate mammalian genome engineering applications, the authors reported providing a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses. 
     Ran et al. (2013-B) described a set of tools for Cas9-mediated genome editing via non-homologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies. To minimize off-target cleavage, the authors further described a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs. The protocol provided by the authors experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off-target activity. The studies showed that beginning with target design, gene modifications can be achieved within as little as 1-2 weeks, and modified clonal cell lines can be derived within 2-3 weeks. 
     Shalem et al. described a new way to interrogate gene function on a genome-wide scale. Their studies showed that delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeted 18,080 genes with 64,751 unique guide sequences enabled both negative and positive selection screening in human cells. First, the authors showed use of the GeCKO library to identify genes essential for cell viability in cancer and pluripotent stem cells. Next, in a melanoma model, the authors screened for genes whose loss is involved in resistance to vemurafenib, a therapeutic that inhibits mutant protein kinase BRAF. Their studies showed that the highest-ranking candidates included previously validated genes NF1 and MED12 as well as novel hits NF2, CUL3, TADA2B, and TADA1. The authors observed a high level of consistency between independent guide RNAs targeting the same gene and a high rate of hit confirmation, and thus demonstrated the promise of genome-scale screening with Cas9. 
     Nishimasu et al. reported the crystal structure of  Streptococcus pyogenes  Cas9 in complex with sgRNA and its target DNA at 2.5 A° resolution. The structure revealed a bilobed architecture composed of target recognition and nuclease lobes, accommodating the sgRNA:DNA heteroduplex in a positively charged groove at their interface. Whereas the recognition lobe is essential for binding sgRNA and DNA, the nuclease lobe contains the HNH and RuvC nuclease domains, which are properly positioned for cleavage of the complementary and non-complementary strands of the target DNA, respectively. The nuclease lobe also contains a carboxyl-terminal domain responsible for the interaction with the protospacer adjacent motif (PAM). This high-resolution structure and accompanying functional analyses have revealed the molecular mechanism of RNA-guided DNA targeting by Cas9, thus paving the way for the rational design of new, versatile genome-editing technologies. 
     Wu et al. mapped genome-wide binding sites of a catalytically inactive Cas9 (dCas9) from  Streptococcus pyogenes  loaded with single guide RNAs (sgRNAs) in mouse embryonic stem cells (mESCs). The authors showed that each of the four sgRNAs tested targets dCas9 to between tens and thousands of genomic sites, frequently characterized by a 5-nucleotide seed region in the sgRNA and an NGG protospacer adjacent motif (PAM). Chromatin inaccessibility decreases dCas9 binding to other sites with matching seed sequences; thus 70% of off-target sites are associated with genes. The authors showed that targeted sequencing of 295 dCas9 binding sites in mESCs transfected with catalytically active Cas9 identified only one site mutated above background levels. The authors proposed a two-state model for Cas9 binding and cleavage, in which a seed match triggers binding but extensive pairing with target DNA is required for cleavage. 
     Platt et al. established a Cre-dependent Cas9 knockin mouse. The authors demonstrated in vivo as well as ex vivo genome editing using adeno-associated virus (AAV)-, lentivirus-, or particle-mediated delivery of guide RNA in neurons, immune cells, and endothelial cells. 
     Hsu et al. (2014) is a review article that discusses generally CRISPR-Cas9 history from yogurt to genome editing, including genetic screening of cells. 
     Wang et al. (2014) relates to a pooled, loss-of-function genetic screening approach suitable for both positive and negative selection that uses a genome-scale lentiviral single guide RNA (sgRNA) library. 
     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 online tool for designing sgRNAs. 
     Swiech et al. demonstrate that AAV-mediated SpCas9 genome editing can enable reverse genetic studies of gene function in the brain. 
     Konermann et al. (2015) discusses the ability to attach multiple effector domains, e.g., transcriptional activator, functional and epigenomic regulators at appropriate positions on the guide such as stem or tetraloop with and without linkers. 
     Zetsche et al. demonstrates that the Cas9 enzyme can be split into two and hence the assembly of Cas9 for activation can be controlled. 
     Chen et al. relates to multiplex screening by demonstrating that a genome-wide in vivo CRISPR-Cas9 screen in mice reveals genes regulating lung metastasis. 
     Ran et al. (2015) relates to SaCas9 and its ability to edit genomes and demonstrates that one cannot extrapolate from biochemical assays. Shalem et al. (2015) described ways in which catalytically inactive Cas9 (dCas9) fusions are used to synthetically repress (CRISPRi) or activate (CRISPRa) expression, showing. advances using Cas9 for genome-scale screens, including arrayed and pooled screens, knockout approaches that inactivate genomic loci and strategies that modulate transcriptional activity. 
     Shalem et al. (2015) described ways in which catalytically inactive Cas9 (dCas9) fusions are used to synthetically repress (CRISPRi) or activate (CRISPRa) expression, showing. advances using Cas9 for genome-scale screens, including arrayed and pooled screens, knockout approaches that inactivate genomic loci and strategies that modulate transcriptional activity. 
     Xu et al. (2015) assessed the DNA sequence features that contribute to single guide RNA (sgRNA) efficiency in CRISPR-based screens. The authors explored efficiency of CRISPR/Cas9 knockout and nucleotide preference at the cleavage site. The authors also found that the sequence preference for CRISPRi/a is substantially different from that for CRISPR/Cas9 knockout. 
     Parnas et al. (2015) introduced genome-wide pooled CRISPR-Cas9 libraries into dendritic cells (DCs) to identify genes that control the induction of tumor necrosis factor (Tnf) by bacterial lipopolysaccharide (LPS). Known regulators of Tlr4 signaling and previously unknown candidates were identified and classified into three functional modules with distinct effects on the canonical responses to LPS. 
     Ramanan et al (2015) demonstrated cleavage of viral episomal DNA (cccDNA) in infected cells. The HBV genome exists in the nuclei of infected hepatocytes as a 3.2 kb double-stranded episomal DNA species called covalently closed circular DNA (cccDNA), which is a key component in the HBV life cycle whose replication is not inhibited by current therapies. The authors showed that sgRNAs specifically targeting highly conserved regions of HBV robustly suppresses viral replication and depleted cccDNA. 
     Nishimasu et al. (2015) reported the crystal structures of SaCas9 in complex with a single guide RNA (sgRNA) and its double-stranded DNA targets, containing the 5′-TTGAAT-3′ PAM and the 5′-TTGGGT-3′ PAM. A structural comparison of SaCas9 with SpCas9 highlighted both structural conservation and divergence, explaining their distinct PAM specificities and orthologous sgRNA recognition. 
     Also, “Dimeric CRISPR RNA-guided FokI 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 FokI Nucleases that recognize extended sequences and can edit endogenous genes with high efficiencies in human cells. In addition, mention is made of International Patent Application No. PCT/US14/70057, Attorney Reference 47627.99.2060 and BI-2013/107 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 US Provisional Application Nos. 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, with respect to a method of preparing an sgRNA-and-Cas9 protein containing particle comprising admixing a mixture comprising an sgRNA and Cas9 protein (and optionally HDR template) with a mixture comprising or consisting essentially of or consisting of surfactant, phospholipid, biodegradable polymer, lipoprotein and alcohol; and particles from such a process. For example, wherein Cas9 protein and sgRNA were mixed together at a suitable, e.g., 3:1 to 1:3 or 2:1 to 1:2 or 1:1 molar ratio, at a suitable temperature, e.g., 15-30C, e.g., 20-25C, e.g., room temperature, for a suitable time, e.g., 15-45, such as 30 minutes, advantageously in sterile, nuclease free buffer, e.g., 1×PBS. Separately, particle components such as or comprising: a surfactant, e.g., cationic lipid, e.g., 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP); phospholipid, e.g., dimyristoylphosphatidylcholine (DMPC); biodegradable polymer, such as an ethylene-glycol polymer or PEG, and a lipoprotein, such as a low-density lipoprotein, e.g., cholesterol were dissolved in an alcohol, advantageously a C1-6 alkyl alcohol, such as methanol, ethanol, isopropanol, e.g., 100% ethanol. The two solutions were mixed together to form particles containing the Cas9-sgRNA complexes. Accordingly, sgRNA may be pre-complexed with the Cas9 protein, before formulating the entire complex in a particle. Formulations may be made with a different molar ratio of different components known to promote delivery of nucleic acids into cells (e.g. 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC), polyethylene glycol (PEG), and cholesterol) For example DOTAP: DMPC: PEG: Cholesterol Molar Ratios may be DOTAP 100, DMPC 0, PEG 0, Cholesterol 0; or DOTAP 90, DMPC 0, PEG 10, Cholesterol 0; or DOTAP 90, DMPC 0, PEG 5, Cholesterol 5. DOTAP 100, DMPC 0, PEG 0, Cholesterol 0. That application accordingly comprehends admixing sgRNA, Cas protein and components that form a particle; as well as particles from such admixing. Aspects of the instant invention can involve particles; for example, particles using a process analogous to that of the Particle Delivery PCT, e.g., by admixing a mixture comprising crRNA and/or CRISPR-Cas as in the instant invention and components that form a particle, e.g., as in the Particle Delivery PCT, to form a particle and particles from such admixing (or, of course, other particles involving crRNA and/or CRISPR-Cas as in the instant invention). 
     Systems and Complexes 
     The systems herein may be nucleic-acid targeting systems. In some embodiments, one or more elements of a nucleic acid-targeting system. In some embodiments, one or more elements of a nucleic acid-targeting system is derived from a particular organism comprising an endogenous nucleic acid-targeting CRISPR system. In general, a nucleic acid-targeting system is characterized by elements that promote the formation of a nucleic acid-targeting complex at the site of a target sequence. In the context of formation of a nucleic acid-targeting 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 RNA promotes the formation of a DNA or RNA-targeting complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a nucleic acid-targeting complex. A target sequence may comprise RNA polynucleotides. In some embodiments, a target sequence is located in the nucleus or cytoplasm of a cell. In some embodiments, the target sequence may be within an organelle of a eukaryotic cell, for example, mitochondrion or chloroplast. A sequence or template that may be used for recombination into the targeted locus comprising the target sequences is referred to as an “editing template” or “editing sequence”. In aspects of the invention, an exogenous template may be referred to as an editing template. In an aspect of the invention the recombination is homologous recombination. 
     Typically, in the context of an endogenous nucleic acid-targeting system, formation of a nucleic acid-targeting complex (comprising a guide RNA hybridized to a target sequence and complexed with one or more nucleic acid-targeting effector proteins) results in cleavage of one or both nucleic acid 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, one or more vectors driving expression of one or more elements of a nucleic acid-targeting system are introduced into a host cell such that expression of the elements of the nucleic acid-targeting system direct formation of a nucleic acid-targeting complex at one or more target sites. For example, a nucleic acid-targeting effector protein and a guide RNA could each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements, may be combined in a single vector, with one or more additional vectors providing any components of the nucleic acid-targeting system not included in the first vector. nucleic acid-targeting system elements 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 a nucleic acid-targeting effector protein and a guide RNA 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 nucleic acid-targeting effector protein and guide RNA are operably linked to and expressed from the same promoter. 
     Aspects of the invention relate to CRISPR-Cas systems comprising a Cas protein as specified in any of the embodiments described herein and a guide molecule capable of forming a complex with the Cas protein and directing specific binding of the complex to a target sequence within a target polypeptide. 
     In one aspect, the invention provides for methods of modifying a target polynucleotide in a eukaryotic cell, which may be in vivo, ex vivo or in vitro. In some embodiments, the method comprises sampling a cell or population of cells from a human or non-human animal, and modifying the cell or cells. Culturing may occur at any stage ex vivo. The cell or cells may even be re-introduced into the non-human animal or plant. For re-introduced cells it is particularly preferred that the cells are stem cells. 
     In some embodiments, the method comprises allowing a CRISPR complex to bind to the target polynucleotide to effect cleavage of said target polynucleotide thereby modifying the target polynucleotide, wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized or hybridizable to a target sequence within said target polynucleotide. 
     In one aspect, the invention provides a method of modifying expression of a polynucleotide in a eukaryotic cell. In some embodiments, the method comprises allowing a CRISPR complex to bind to the polynucleotide such that said binding results in increased or decreased expression of said polynucleotide; wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized or hybridizable to a target sequence within said polynucleotide. Similar considerations and conditions apply as above for methods of modifying a target polynucleotide. In fact, these sampling, culturing and re-introduction options apply across the aspects of the present invention. 
     In some embodiments, the method comprises allowing a CRISPR complex to bind to the target polynucleotide to effect editing of one or more bases of said target polynucleotide thereby modifying the target polynucleotide, wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized or hybridizable to a target sequence within said target polynucleotide. 
     Indeed, in any aspect of the invention, the CRISPR complex may comprise a CRISPR enzyme complexed with a guide sequence hybridized or hybridizable to a target sequence. 
     Multiplexing 
     Enzymes according to the invention used in a multiplex (tandem) targeting approach. The inventors have shown that 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. It is noted that the terms “CRISPR-Cas system”, “CRISP-Cas complex” “CRISPR complex” and “CRISPR system” are used interchangeably. Also the terms “CRISPR enzyme”, “Cas enzyme”, or “CRISPR-Cas enzyme”, can be used interchangeably. 
     In one aspect, the invention provides a non-naturally occurring or engineered CRISPR enzyme, 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, 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 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 Cas herein, e.g., the Type II-like Cas protines such as ProCas9. 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 Cas 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. The inventors have found that the Cas proteins as described herein may enable improved and/or direct access to one or more nucleotides involved in the DNA:RNA duplex. 
     Inducible Systems 
     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 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. Further examples of inducible DNA binding proteins and methods for their use are provided in US Provisional Application Nos. 61/736,465 and U.S. 61/721,283, and International Patent Publication No. WO 2014/018423 A2 which is hereby incorporated by reference in its entirety. 
     Self-Inactivating Systems 
     Once all copies of a gene in the genome of a cell have been edited, continued CRISRP/Cas expression in that cell is no longer necessary. Indeed, sustained expression would be undesirable in 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 CRISPR system that relies on the use of a non-coding guide target sequence within the CRISPR vector itself. Thus, after expression begins, the CRISPR-Cas 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 (e.g., 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 effector protein gene, (c) within 100 bp of the ATG translational start codon in the Cas effector protein coding sequence, (d) within the inverted terminal repeat (iTR) of a viral delivery vector, e.g., in the AAV genome. 
     In some aspects, 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. In some aspects, 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 a inactivation of one or more, or in some cases all, of the CRISPR-Cas system. 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 guide RNA capable of targeting a genomic locus or loci to be edited, and a second subset of CRISPR complexes comprise at least one second guide RNA 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 various coding sequences (CRISPR enzyme and guide RNAs) 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 guide RNA on one vector, and the remaining guide RNA 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. 
     The first guide RNA can target any target sequence of interest within a genome, as described elsewhere herein. The second guide RNA targets a sequence within the vector which encodes the CRISPR Cas enzyme, and thereby inactivates the enzyme&#39;s expression from that vector. Thus the target sequence in the vector must be capable of inactivating expression. Suitable target sequences can be, for instance, near to or within the translational start codon for the Cas coding sequence, in a non-coding sequence in the promoter driving expression of the non-coding RNA elements, within the promoter driving expression of the Cas gene, within 100 bp of the ATG translational start codon in the Cas coding sequence, 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 coding sequence, 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 for the stability of the vector. For instance, if the promoter for the Cas 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 a AAV vector a useful target sequence is within the iTR. Other useful sequences to target can be promoter sequences, polyadenylation sites, 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 in order to provide regulation of the CRISPR-Cas. 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. 
     Addition of non-targeting nucleotides to the 5′ end (e.g. 1-10 nucleotides, preferably 1-5 nucleotides) of the “self-inactivating” 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 shutdown. 
     In one aspect of the self-inactivating AAV—CRISPR-Cas system, plasmids that co-express one or more guide RNA targeting genomic sequences of interest (e.g. 1-2, 1-5, 1-10, 1-15, 1-20, 1-30) may be established with “self-inactivating” guide RNAs that target an Cas 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 guide RNA. The U6-driven guide RNAs may be designed in an array format such that multiple guide RNA sequences can be simultaneously released. When first delivered into target tissue/cells (left cell) guide RNAs begin to accumulate while Cas levels rise in the nucleus. Cas complexes with all of the guide RNAs to mediate genome editing and self-inactivation of the CRISPR-Cas plasmids. 
     One aspect of a self-inactivating CRISPR-Cas system is expression of singly 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—guide RNA(s)-Pol2 promoter-Cas. 
     One aspect of a tandem array transcript is that one or more guide(s) edit the one or more target(s) while one or more self-inactivating guides inactivate the CRISPR-Cas system. Thus, for example, the described CRISPR-Cas system for repairing expansion disorders may 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 the CRISPR-Cas. Reference is made to application Ser. No. PCT/US2014/069897, entitled “Compositions And Methods Of Use Of Crispr-Cas Systems In Nucleotide Repeat Disorders,” published Dec. 12, 2014 as International Patent Publication No. WO/2015/089351. 
     The guideRNA may be a control guide. For example it may be engineered to target a nucleic acid sequence encoding the CRISPR Enzyme itself, as described in U.S. patent publication No. US2015232881A1, the disclosure of which is hereby incorporated by reference. In some embodiments, a system or composition may be provided with just the guideRNA engineered to target the nucleic acid sequence encoding the CRISPR Enzyme. In addition, the system or composition may be provided with the guideRNA engineered to target the nucleic acid sequence encoding the CRISPR Enzyme, as well as nucleic acid sequence encoding the CRISPR Enzyme and, optionally a second guide RNA and, further optionally, a repair template. The second guideRNA may be the primary target of the CRISPR system or composition (such a therapeutic, diagnostic, knock out etc. as defined herein). In this way, the system or composition is self-inactivating. This is exemplified in relation to Cas in US2015232881A1 (also published as WO2015070083 (A1) referenced elsewhere herein, and may be extrapolated to Cas. 
     Polynucleotides 
     The systems herein may comprise one or more polynucleotides. The polynucleotide(s) may comprise coding sequences of Cas protein(s), guide sequences, or any combination thereof. The present disclosure further provides vectors or vector systems comprising one or more polynucleotides herein. The vectors or vector systems include those described in the delivery sections herein. 
     The terms “polynucleotide”, “nucleotide”, “nucleotide sequence”, “nucleic acid” and “oligonucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. The term also encompasses nucleic-acid-like structures with synthetic backbones, see, e.g., Eckstein, 1991; Baserga et al., 1992; Milligan, 1993; WO 97/03211; WO 96/39154; Mata, 1997; Strauss-Soukup, 1997; and Samstag, 1996. A polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. As used herein the term “wild type” is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms. A “wild type” can be a base line. As used herein the term “variant” should be taken to mean the exhibition of qualities that have a pattern that deviates from what occurs in nature. The terms “non-naturally occurring” or “engineered” are used interchangeably and indicate the involvement of the hand of man. The terms, when referring to nucleic acid molecules or polypeptides mean that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which they are naturally associated in nature and as found in nature. “Complementarity” refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick base pairing or other non-traditional types. A percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). “Perfectly complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. “Substantially complementary” as used herein refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions. As used herein, “stringent conditions” for hybridization refer to conditions under which a nucleic acid having complementarity to a target sequence predominantly hybridizes with the target sequence, and substantially does not hybridize to non-target sequences. Stringent conditions are generally sequence-dependent, and vary depending on a number of factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence. Non-limiting examples of stringent conditions are described in detail in Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology-Hybridization With Nucleic Acid Probes Part I, Second Chapter “Overview of principles of hybridization and the strategy of nucleic acid probe assay”, Elsevier, N.Y. Where reference is made to a polynucleotide sequence, then complementary or partially complementary sequences are also envisaged. These are preferably capable of hybridizing to the reference sequence under highly stringent conditions. Generally, in order to maximize the hybridization rate, relatively low-stringency hybridization conditions are selected: about 20 to 25° C. lower than the thermal melting point (Tm). The Tm is the temperature at which 50% of specific target sequence hybridizes to a perfectly complementary probe in solution at a defined ionic strength and pH. Generally, in order to require at least about 85% nucleotide complementarity of hybridized sequences, highly stringent washing conditions are selected to be about 5 to 15° C. lower than the Tm. In order to require at least about 70% nucleotide complementarity of hybridized sequences, moderately-stringent washing conditions are selected to be about 15 to 30° C. lower than the Tm. Highly permissive (very low stringency) washing conditions may be as low as 50° C. below the Tm, allowing a high level of mis-matching between hybridized sequences. Those skilled in the art will recognize that other physical and chemical parameters in the hybridization and wash stages can also be altered to affect the outcome of a detectable hybridization signal from a specific level of homology between target and probe sequences. Preferred highly stringent conditions comprise incubation in 50% formamide, 5×SSC, and 1% SDS at 42° C., or incubation in 5×SSC and 1% SDS at 65° C., with wash in 0.2×SSC and 0.1% SDS at 65° C. “Hybridization” refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson Crick base pairing, Hoogstein binding, or in any other sequence specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi stranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of PCR, or the cleavage of a polynucleotide by an enzyme. A sequence capable of hybridizing with a given sequence is referred to as the “complement” of the given sequence. As used herein, the term “genomic locus” or “locus” (plural loci) is the specific location of a gene or DNA sequence on a chromosome. A “gene” refers to stretches of DNA or RNA that encode a polypeptide or an RNA chain that has functional role to play in an organism and hence is the molecular unit of heredity in living organisms. For the purpose of this invention, it may be considered that genes include regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and locus control regions. As used herein, “expression of a genomic locus” or “gene expression” is the process by which information from a gene is used in the synthesis of a functional gene product. The products of gene expression are often proteins, but in non-protein coding genes such as rRNA genes or tRNA genes, the product is functional RNA. The process of gene expression is used by all known life—eukaryotes (including multicellular organisms), prokaryotes (bacteria and archaea) and viruses to generate functional products to survive. As used herein “expression” of a gene or nucleic acid encompasses not only cellular gene expression, but also the transcription and translation of nucleic acid(s) in cloning systems and in any other context. As used herein, “expression” also refers to the process by which a polynucleotide is transcribed from a DNA template (such as into and mRNA or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as “gene product.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non amino acids. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. As used herein the term “amino acid” includes natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics. As used herein, the term “domain” or “protein domain” refers to a part of a protein sequence that may exist and function independently of the rest of the protein chain. As described in aspects of the invention, sequence identity is related to sequence homology. Homology comparisons may be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs may calculate percent (%) homology between two or more sequences and may also calculate the sequence identity shared by two or more amino acid or nucleic acid sequences. 
     In certain embodiments, the polynucleotide sequence is recombinant DNA. In further embodiments, the polynucleotide sequence further comprises additional sequences as described elsewhere herein. In certain embodiments, the nucleic acid sequence is synthesized in vitro. 
     Aspects of the invention relate to polynucleotide molecules that encode one or more components of the CRISPR-Cas system or Cas protein as referred to in any embodiment herein. In certain embodiments, the polynucleotide molecules may comprise further regulatory sequences. By means of guidance and not limitation, the polynucleotide sequence can be part of an expression plasmid, a minicircle, a lentiviral vector, a retroviral vector, an adenoviral or adeno-associated viral vector, a piggyback vector, or a tol2 vector. In certain embodiments, the polynucleotide sequence may be a bicistronic expression construct. In further embodiments, the isolated polynucleotide sequence may be incorporated in a cellular genome. In yet further embodiments, the isolated polynucleotide sequence may be part of a cellular genome. In further embodiments, the isolated polynucleotide sequence may be comprised in an artificial chromosome. In certain embodiments, the 5′ and/or 3′ end of the isolated polynucleotide sequence may be modified to improve the stability of the sequence of actively avoid degradation. In certain embodiments, the isolated polynucleotide sequence may be comprised in a bacteriophage. In other embodiments, the isolated polynucleotide sequence may be contained in  Agrobacterium  species. In certain embodiments, the isolated polynucleotide sequence is lyophilized. 
     Aspects of the invention relate to polynucleotide molecules that encode one or more components of one or more CRISPR-Cas systems as described in any of the embodiments herein, wherein at least one or more regions of the polynucleotide molecule may be codon optimized for expression in a eukaryotic cells. In certain embodiments, the polynucleotide molecules that encode one or more components of one or more CRISPR-Cas systems as described in any of the embodiments herein are optimized for expression in a mammalian cell or a plant cell. 
     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 human codon optimized sequence in International Patent Publication No. 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 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 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. 
     Base Editing 
     The present disclosure also provides for base editing systems. In general, such a system may comprise a deaminase (e.g., an adenosine deaminase or cytidine deaminase) fused with a nucleic acid-guided nuclease, e.g., Cas protein. The Cas protein may be a dead Cas protein or a Cas nickase protein. In certain examples, 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. 
     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. In some cases, the modifications by base editors herein may be used for targeting post-translational signaling or catalysis. In some embodiments, compositions herein comprise nucleotide sequence comprising encoding sequences for one or more components of a base editing system. A base-editing system may comprise a deaminase (e.g., an adenosine deaminase or cytidine deaminase) fused with a Cas protein or a variant thereof. 
     In some cases, the adenosine deaminase is double-stranded RNA-specific adenosine deaminase (ADAR). Examples of ADARs include those described Yiannis A Savva et al., The ADAR protein family, Genome Biol. 2012; 13(12): 252, which is incorporated by reference in its entirety. In some examples, the ADAR may be hADAR1. In certain examples, the ADAR may be hADAR2. The sequence of hADAR2 may be that described under Accession No. AF525422.1. 
     In some cases, the deaminase may be a deaminase domain, e.g., a deaminase domain of ADAR (“ADAR-D”). In one example, the deaminase may be the deaminase domain of hADAR2 (“hADAR2-D), e.g., as described in Phelps K J et al., Recognition of duplex RNA by the deaminase domain of the RNA editing enzyme ADAR2. Nucleic Acids Res. 2015 January; 43(2):1123-32, which is incorporated by reference herein in its entirety. In a particular example, the hADAR2-D has a sequence comprising amino acid 299-701 of hADAR2-D, e.g., amino acid 299-701 of the sequence under Accession No. AF525422.1. 
     In certain examples, 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. 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 dead CRISPR-Cas protein or CRISPR-Cas nickase. In some examples, 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 dead CRISPR-Cas protein or a CRISPR-Cas nickase. In some examples, 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, S661T, and S375N fused with a dead CRISPR-Cas protein or a CRISPR-Cas nickase. 
     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. 
     In some examples, the base editing systems may comprise an intein-mediated trans-splicing system that enables in vivo delivery of a base editor, e.g., a split-intein cytidine base editors (CBE) or adenine base editor (ABE) engineered to trans-splice. Examples of the such base editing systems include those described in Colin K. W. Lim et al., Treatment of a Mouse Model of ALS by In Vivo Base Editing, Mol Ther. Jan. 14, 2020. pii: S1525-0016(20)30011-3. doi: 10.1016/j.ymthe.2020.01.005; and Jonathan M. Levy et al., Cytosine and adenine base editing of the brain, liver, retina, heart and skeletal muscle of mice via adeno-associated viruses, Nature Biomedical Engineering volume 4, pages 97-110(2020), which are incorporated by reference herein in their entireties. 
     Examples of base editing systems include those described in International Patent Publication Nos. WO 2019/071048 (e.g. paragraphs [0933]-[0938]), WO 2019/084063 (e.g., paragraphs [0173]-[0186], [0323]-[0475], [0893]-[1094]), WO 2019/126716 (e.g., paragraphs [O 290 ]-[0425], [1077]-[1084]), WO 2019/126709 (e.g., paragraphs [O 294 ]-[0453]), WO 2019/126762 (e.g., paragraphs [0309]-[0438]), WO 2019/126774 (e.g., paragraphs [0511]-[0670]), Cox D B T, et al., RNA editing with CRISPR-Cas13, Science. Nov. 24, 2017; 358(6366):1019-1027; Abudayyeh 00, et al., A cytosine deaminase for programmable single-base RNA editing, Science 26 Jul. 2019: Vol. 365, Issue 6451, pp. 382-386; Gaudelli N M et al., Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage, Nature volume 551, pages 464-471 (23 Nov. 2017); Komor A C, et al., Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature. May 19, 2016; 533(7603):420-4; Jordan L. Doman et al., Evaluation and minimization of Cas9-independent off-target DNA editing by cytosine base editors, Nat Biotechnol (2020). doi.org/10.1038/s41587-020-0414-6; and Richter M F et al., Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity, Nat Biotechnol (2020). doi.org/10.1038/s41587-020-0453-z, which are incorporated by reference herein in their entireties. 
     Prime Editing 
     In some embodiments, the Cas proteins herein may be used for prime editing. In some cases, the Cas protein may be a nickase, e.g., a DNA nickase. The Cas protein may be a dCas. In some cases, the Cas has one or more mutations. In some examples, the Cas is a homolog or ortholog of Cas9 is from or derived from  Streptococcus pyogenes  and comprises mutations corresponding to the H840A mutation of SpCas9. In some examples, the Cas comprises mutations corresponding to D10A of SpCas9. In some examples, the Cas has mutation(s) corresponding to D10A or H840A of SpCas9. 
     The Cas protein may be associated with a reverse transcriptase. The reverse transcriptase may be fused to the C-terminus of a Cas protein. Alternatively or additionally, the reverse transcriptase may be fused to the N-terminus of a Cas protein. The fusion may be via a linker and/or an adaptor protein. In some examples, the reverse transcriptase may be an M-MLV reverse transcriptase or variant thereof. The M-MLV reverse transcriptase variant may comprise one or more mutations. For the examples, the M-MLV reverse transcriptase may comprise D200N, L603W, and T330P. In another example, the M-MLV reverse transcriptase may comprise D200N, L603W, T330P, T306K, and W313F. In a particular example, the fusion of Cas and reverse transcriptase is Cas (with a mutation corresponding to H840A of SpCas9) fused with M-MLV reverse transcriptase (D200N+L603W+T330P+T306K+W313F). 
     In some embodiments, the Cas protein herein may target DNA using a guide RNA containing a binding sequence that hybridizes to the target sequence on the DNA. The guide RNA may further comprise an editing sequence that contains new genetic information that replaces target DNA nucleotides. The small sizes of the Cas protein herein may allow easier packaging and delivery of the prime editing system, e.g., with a viral vector, e.g., AAV or lentiviral vector. 
     A single-strand break (a nick) may be generated on the target DNA by the Cas protein at the target site to expose a 3′-hydroxyl group, thus priming the reverse transcription of an edit-encoding extension on the guide directly into the target site. These steps may result in a branched intermediate with two redundant single-stranded DNA flaps: a 5′ flap that contains the unedited DNA sequence, and a 3′ flap that contains the edited sequence copied from the guide RNA. The 5′ flaps may be removed by a structure-specific endonuclease, e.g., FEN122, which excises 5′ flaps generated during lagging-strand DNA synthesis and long-patch base excision repair. The non-edited DNA strand may be nicked to induce bias DNA repair to preferentially replace the non-edited strand. Examples of prime editing systems and methods include those described in Anzalone A V et al., Search-and-replace genome editing without double-strand breaks or donor DNA, Nature. Oct. 21, 2019. doi: 10.1038/s41586-019-1711-4, which is incorporated by reference herein in its entirety. 
     The Cas protein may be used to prime-edit a single nucleotide on a target DNA. Alternatively or additionally, the Cas protein may be used to prime-edit at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 10000 nucleotides on a target DNA. 
     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. Dec. 1, 1993; 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 systems and compositions herein. A cargo may comprise one or more of the following: i) a plasmid encoding one or more Cas proteins; ii) a plasmid encoding one or more guide RNAs, iii) mRNA of one or more Cas proteins; iv) one or more guide RNAs; v) one or more Cas proteins; vi) 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 cases, the plasmid may also encode a recombination template (e.g., for HDR). In some embodiments, a cargo may comprise mRNA encoding one or more Cas proteins and one or more guide RNAs. 
     In some examples, a cargo may comprise one or more Cas proteins 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. 
     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, 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. 
     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 have 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 International Patent Publication No. WO 2008042156, US Publication Application No. US 20130185823, and International Patent Publication No WO 2015/089419. 
     Vectors 
     The systems, compositions, and/or delivery systems may comprise one or more vectors. The present disclosure also include vector systems. A vector system may comprise one or more vectors. In some embodiments, a vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors include 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. A vector may be a plasmid, e.g., a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Certain vectors may be 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). Some 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. In certain examples, vectors may be expression vectors, e.g., capable of directing the expression of genes to which they are operatively-linked. In some cases, the expression vectors may be for expression in eukaryotic cells. Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. 
     Examples of vectors include pGEX, pMAL, pRIT5,  E. coli  expression vectors (e.g., pTrc, pET 11d, yeast expression vectors (e.g., pYepSec1, pMFa, pJRY88, pYES2, and picZ, Baculovirus vectors (e.g., for expression in insect cells such as SF9 cells) (e.g., pAc series and the pVL series), mammalian expression vectors (e.g., pCDM8 and pMT2PC. 
     A vector may comprise i) Cas encoding sequence(s), and/or ii) a single, or at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 32, at least 48, at least 50 guide RNA(s) encoding sequences. In a single vector there can be a promoter for each RNA coding sequence. Alternatively or additionally, in a single vector, there may be a promoter controlling (e.g., driving transcription and/or expression) multiple RNA encoding sequences. 
     Furthermore, that compositions or systems may be delivered via a vector, e.g., a separate vector or the same vector that is encoding the CRISPR complex. When provided by a separate vector, the CRISPR RNA that targets Cas expression can be administered sequentially or simultaneously. When administered sequentially, the CRISPR RNA that targets Cas 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 enzyme may then associate with the second gRNA capable of hybridizing to the sequence comprising at least part of the Cas or CRISPR cassette. Where the guide RNA targets the sequences encoding expression of the Cas protein, the enzyme becomes impeded and the system becomes self-inactivating. In the same manner, CRISPR RNA that targets Cas expression applied via, for example liposome, lipofection, particles, microvesicles as explained herein, may be administered sequentially or simultaneously. Similarly, self-inactivation may be used for inactivation of one or more guide RNA used to target one or more targets. 
     Regulatory Elements 
     A vector may comprise one or more regulatory elements. The regulatory element(s) may be operably linked to coding sequences of Cas proteins, accessary proteins, guide RNAs (e.g., a single guide RNA, crRNA, and/or tracrRNA), or combination thereof. 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). In certain examples, a vector may comprise: a first regulatory element operably linked to a nucleotide sequence encoding a Cas protein, and a second regulatory element operably linked to a nucleotide sequence encoding a guide RNA. 
     Examples of regulatory elements include promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences). 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 may 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. 
     Examples of promoters include 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), the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1α promoter. 
     Viral Vectors 
     The cargos may be delivered by viruses. In some embodiments, viral vectors are used. A viral vector may comprise virally-derived DNA or RNA sequences for packaging into a virus (e.g., retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses). Viral vectors also include polynucleotides carried by a virus for transfection into a host cell. Viruses and viral vectors may be used for in vitro, ex vivo, and/or in vivo deliveries. 
     Adeno Associated Virus (AAV) 
     The systems and compositions herein may be delivered by adeno associated virus (AAV). AAV vectors may be used for such delivery. AAV, of the Dependovirus genus and Parvoviridae family, is a single stranded DNA virus. In some embodiments, AAV may provide a persistent source of the provided DNA, as AAV delivered genomic material can exist indefinitely in cells, e.g., either as exogenous DNA or, with some modification, be directly integrated into the host DNA. In some embodiments, AAV do not cause or relate with any diseases in humans. The virus itself is able to efficiently infect cells while provoking little to no innate or adaptive immune response or associated toxicity. 
     Examples of AAV that can be used herein include AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-8, and AAV-9. The type of AAV may be selected with regard to the cells to be targeted; e.g., one can select AAV serotypes 1, 2, 5 or a hybrid capsid AAV1, AAV2, AAV5 or any combination thereof for targeting brain or neuronal cells; and one can select AAV4 for targeting cardiac tissue. AAV8 is useful for delivery to the liver. AAV-2-based vectors were originally proposed for CFTR delivery to CF airways, other serotypes such as AAV-1, AAV-5, AAV-6, and AAV-9 exhibit improved gene transfer efficiency in a variety of models of the lung epithelium. Examples of cell types targeted by AAV are described in Grimm, D. et al, J. Virol. 82: 5887-5911 (2008)), and shown as follows: 
     
       
         
           
               
               
               
               
               
               
               
               
               
             
               
                 TABLE 1 
               
               
                   
               
               
                 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 
               
               
                   
               
            
           
         
       
     
     CRISPR-Cas AAV particles may be created in HEK 293 T cells. Once particles with specific tropism have been created, they are used to infect the target cell line much in the same way that native viral particles do. This may allow for persistent presence of CRISPR-Cas components in the infected cell type, and what makes this version of delivery particularly suited to cases where long-term expression is desirable. Examples of doses and formulations for AAV that can be used include those describe in U.S. Pat. Nos. 8,454,972 and 8,404,658. 
     Various strategies may be used for delivery the systems and compositions herein with AAVs. In some examples, coding sequences of Cas and gRNA may be packaged directly onto one DNA plasmid vector and delivered via one AAV particle. In some examples, AAVs may be used to deliver gRNAs into cells that have been previously engineered to express Cas. In some examples, coding sequences of Cas and gRNA may be made into two separate AAV particles, which are used for co-transfection of target cells. In some examples, markers, tags, and other sequences may be packaged in the same AAV particles as coding sequences of Cas and/or gRNAs. 
     Lentiviruses 
     The systems and compositions herein may be delivered by lentiviruses. Lentiviral vectors may be used for such delivery. Lentiviruses are complex retroviruses that have the ability to infect and express their genes in both mitotic and post-mitotic cells. 
     Examples of lentiviruses include human immunodeficiency virus (HIV), which may use its envelope glycoproteins of other viruses to target a broad range of cell types; minimal non-primate lentiviral vectors based on the equine infectious anemia virus (EIAV), which may be used for ocular therapies. In certain embodiments, 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) may be used/and or adapted to the nucleic acid-targeting system herein. 
     Lentiviruses may be pseudo-typed with other viral proteins, such as the G protein of vesicular stomatitis virus. In doing so, the cellular tropism of the lentiviruses can be altered to be as broad or narrow as desired. In some cases, to improve safety, second- and third-generation lentiviral systems may split essential genes across three plasmids, which may reduce the likelihood of accidental reconstitution of viable viral particles within cells. 
     In some examples, leveraging the integration ability, lentiviruses may be used to create libraries of cells comprising various genetic modifications, e.g., for screening and/or studying genes and signaling pathways. 
     Adenoviruses 
     The systems and compositions herein may be delivered by adenoviruses. Adenoviral vectors may be used for such delivery. Adenoviruses include nonenveloped viruses with an icosahedral nucleocapsid containing a double stranded DNA genome. Adenoviruses may infect dividing and non-dividing cells. In some embodiments, adenoviruses do not integrate into the genome of host cells, which may be used for limiting off-target effects of CRISPR-Cas systems in gene editing applications. 
     Viral Vehicles 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. 
     Non-Viral 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, gold 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. 
     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-dimyristyloxypropyl-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). 
     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-distearoyl-sn-glycero-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. 
     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 (DLinDMA) 
     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. 
     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), Ca2ρ (e.g., forming DNA/Ca 2+  microcomplexes), polyethenimine (PEI) (e.g., branched PEI), and poly(L-lysine) (PLL). 
     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 (33 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. Oct. 22, 2014; 136(42):14722-5; and Sun W et al, Angew Chem Int Ed Engl. Oct. 5, 2015; 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. 
     Gold 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. 
     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. 
     Streptolysin O (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; El-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. Apr. 28, 2020. doi: 10.1039/d0bm00427h. 
     Applications and Uses in General 
     The systems, the vector systems, the vectors and the compositions described herein may be used in various nucleic acids-targeting applications, altering or modifying synthesis of a gene product, such as a protein, nucleic acids cleavage, nucleic acids editing, nucleic acids splicing; trafficking of target nucleic acids, tracing of target nucleic acids, isolation of target nucleic acids, visualization of target nucleic acids, etc. 
     Aspects of the invention thus also encompass methods and uses of the compositions and systems described herein in genome engineering, e.g. for altering or manipulating the expression of one or more genes or the one or more gene products, in prokaryotic or eukaryotic cells, in vitro, in vivo or ex vivo. 
     Typically, in the context of a nucleic acid-targeting system, formation of a nucleic acid-targeting complex (comprising a guide RNA hybridized to a target sequence and complexed with one or more nucleic acid-targeting effector proteins) results in cleavage of one or both DNA or RNA 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. As used herein the term “sequence(s) associated with a target locus of interest” refers to sequences near the vicinity of the target sequence (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from the target sequence, wherein the target sequence is comprised within a target locus of interest). 
     Examples of target polynucleotides include a sequence associated with a signaling biochemical pathway, e.g., a signaling biochemical pathway-associated gene or polynucleotide. Examples of target polynucleotides include a disease associated gene or polynucleotide. A “disease-associated” gene or polynucleotide refers to any gene or polynucleotide which is yielding transcription or translation products at an abnormal level or in an abnormal form in cells derived from a disease-affected tissues compared with tissues or cells of a non-disease control. It may be a gene that becomes expressed at an abnormally high level; it may be a gene that becomes expressed at an abnormally low level, where the altered expression correlates with the occurrence and/or progression of the disease. A disease-associated gene also refers to a gene possessing mutation(s) or genetic variation that is directly responsible or is in linkage disequilibrium with a gene(s) that is responsible for the etiology of a disease. The transcribed or translated products may be known or unknown, and may be at a normal or abnormal level. 
     The target polynucleotide of a CRISPR complex can be any polynucleotide endogenous or exogenous to the eukaryotic cell. For example, the target polynucleotide can be a polynucleotide residing in the nucleus of the eukaryotic cell. The target polynucleotide can be a sequence coding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or a junk DNA). Without wishing to be bound by theory, it is believed that the target sequence should be associated with a PAM (protospacer adjacent motif); that is, a short sequence recognized by the CRISPR complex. The precise sequence and length requirements for the PAM differ depending on the CRISPR enzyme used, but PAMs are typically 2-5 base pair sequences adjacent the protospacer (that is, the target sequence) Examples of PAM sequences are given in the examples section below, and the skilled person will be able to identify further PAM sequences for use with a given CRISPR enzyme. Further, engineering of the PAM Interacting (PI) domain may allow programing of PAM specificity, improve target site recognition fidelity, and increase the versatility of the Cas, genome engineering platform. Cas proteins may be engineered to alter their PAM specificity, for example as described 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. 
     The target polynucleotide of a CRISPR complex may include a number of disease-associated genes and polynucleotides as well as signaling biochemical pathway-associated genes and polynucleotides as listed in U.S. provisional patent applications 61/736,527 and 61/748,427 having Broad reference BI-2011/008/WSGR Docket No. 44063-701.101 and BI-2011/008/WSGR Docket No. 44063-701.102 respectively, both entitled SYSTEMS METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION filed on Dec. 12, 2012 and Jan. 2, 2013, respectively, and PCT Application PCT/US2013/074667, entitled DELIVERY, ENGINEERING AND OPTIMIZATION OF SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION AND THERAPEUTIC APPLICATIONS, filed Dec. 12, 2013, the contents of all of which are herein incorporated by reference in their entirety. 
     Examples of target polynucleotides include a sequence associated with a signaling biochemical pathway, e.g., a signaling biochemical pathway-associated gene or polynucleotide. Examples of target polynucleotides include a disease associated gene or polynucleotide. A “disease-associated” gene or polynucleotide refers to any gene or polynucleotide which is yielding transcription or translation products at an abnormal level or in an abnormal form in cells derived from a disease-affected tissues compared with tissues or cells of a non-disease control. It may be a gene that becomes expressed at an abnormally high level; it may be a gene that becomes expressed at an abnormally low level, where the altered expression correlates with the occurrence and/or progression of the disease. A disease-associated gene also refers to a gene possessing mutation(s) or genetic variation that is directly responsible or is in linkage disequilibrium with a gene(s) that is responsible for the etiology of a disease. The transcribed or translated products may be known or unknown, and may be at a normal or abnormal level. 
     Aspects of the invention relate to a method of targeting a polynucleotide, comprising contacting a sample that comprises the polynucleotide with a CRISPR-Cas system or Cas protein as described in any embodiment herein, a delivery system comprising a CRISPR-Cas system or Cas protein as described in any embodiment herein, a polynucleotide comprising a CRISPR-Cas system or Cas protein as described in any embodiment herein, a vector comprising a CRISPR-Cas system or Cas protein as described in any embodiment herein, or a vector system comprising a CRISPR-Cas system or Cas protein as described in any embodiment herein. In certain embodiments, a target polynucleotide is contacted with at least two different CRISPR-Cas systems or Cas proteins. In further embodiments, the two different CRISPR-Cas systems or Cas proteins have different target polynucleotide specificities, or degrees of specificity. In certain embodiments, the two different CRISPR-Cas systems or Cas proteins have a different PAM specificity. 
     Further intended is the method of targeting a polynucleotide, comprising contacting a sample that comprises the polynucleotide with a CRISPR-Cas system or Cas protein as described in any embodiment herein, a polynucleotide comprising a CRISPR-Cas system or Cas protein as described in any embodiment herein, a delivery system comprising a CRISPR-Cas system or Cas protein as described in any embodiment herein, a vector comprising a CRISPR-Cas system or Cas protein as described in any embodiment herein, or a vector system comprising a CRISPR-Cas system or Cas protein as described in any embodiment herein wherein the method further comprises detection of binding of the CRISPR-Cas system or Cas protein to the polynucleotide. 
     Also envisaged are methods of targeting a polynucleotide, comprising contacting a sample that comprises the polynucleotide with a CRISPR-Cas system or Cas protein as described in any embodiment herein, a polynucleotide comprising a CRISPR-Cas system or Cas protein as described in any embodiment herein, a vector comprising a CRISPR-Cas system or Cas protein as described in any embodiment herein, or a vector system comprising a CRISPR-Cas system or Cas protein as described in any embodiment herein wherein contacting results in modification of a gene product or modification of the amount or expression of a gene product. In certain embodiments, the expression of the targeted gene product is increased by the method. In certain embodiments, the expression of the targeted gene product is increased by at least 10%, preferably by at least 15%, preferably by at least 20%, preferably by at least 25%, preferably by at least 30%, preferably by at least 35%, preferably by at least 40%, preferably by at least 45%, preferably by at least 50%, preferably by at least 55%, preferably by at least 60%, preferably by at least 65%, preferably by at least 70%, preferably by at least 75%, preferably by at least 80%, preferably by at least 85%, preferably by at least 90%, preferably by at least 95%, preferably by at least 100%. In certain embodiments, the expression of the targeted gene product is increased at least 1.5-fold, preferably at least 2-fold, preferably at least 2.5-fold, preferably at least 3-fold, preferably at least 3.5-fold, preferably at least 3.5-fold, preferably at least 4-fold, preferably at least 4.5-fold, preferably at least 5-fold, preferably at least 10-fold, preferably at least 10-fold, preferably at least 15-fold, preferably at least 20-fold, preferably at least 25-fold, preferably at least 50-fold, preferably at least 100-fold. In certain embodiments, the expression of the targeted gene product is reduced by at least 10%, preferably by at least 15%, preferably by at least 20%, preferably by at least 25%, preferably by at least 30%, preferably by at least 35%, preferably by at least 40%, preferably by at least 45%, preferably by at least 50%, preferably by at least 55%, preferably by at least 60%, preferably by at least 65%, preferably by at least 70%, preferably by at least 75%, preferably by at least 80%, preferably by at least 85%, preferably by at least 90%, preferably by at least 95%, preferably by at least 100%. In certain embodiments, the expression of the targeted gene product is reduced at least 1.5-fold, preferably at least 2-fold, preferably at least 2.5-fold, preferably at least 3-fold, preferably at least 3.5-fold, preferably at least 3.5-fold, preferably at least 4-fold, preferably at least 4.5-fold, preferably at least 5-fold, preferably at least 10-fold, preferably at least 10-fold, preferably at least 15-fold, preferably at least 20-fold, preferably at least 25-fold, preferably at least 50-fold, preferably at least 100-fold. In alternative embodiments, the expression of the targeted gene product is reduced by the method. In further embodiments, expression of the targeted gene may be completely eliminated, or may be considered eliminated as remnant expression levels of the targeted gene fall below the detection limit of methods known in the art that are used to quantify, detect, or monitor expression levels of genes. 
     In some embodiments, one or more polynucleotide molecules, vectors, or vector systems driving expression of one or more elements of a nucleic acid-targeting system or delivery systems comprising one or more elements of the nucleic acid-targeting system are introduced into a host cell such that expression of the elements of the nucleic acid-targeting system direct formation of a nucleic acid-targeting complex at one or more target sites. In certain embodiments of the invention the host cell may be a eukaryotic cell, a prokaryotic cell, or a plant cell. 
     In particular embodiments, the host cell is a cell of a cell line. 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. 
     Further intended are isolated human cells or tissues, plants or non-human animals comprising one or more of the polynucleotide molecules, vectors, vector systems, or cells described in any of the embodiments herein. In an aspect, host cells and cell lines modified by or comprising the compositions, systems or modified enzymes of present invention are provided, including (isolated) stem cells, and progeny thereof. 
     In certain embodiments, the plants or non-human animals comprise at least one of the CRISPR system components, polynucleotide molecules, vectors, vector systems, or cells described in any of the embodiments herein at least one tissue type of the plant or non-human animal. In certain embodiment, non-human animals comprise at least one of the CRISPR system components, polynucleotide molecules, vectors, vector systems, or cells described in any of the embodiments herein in at least one tissue type. In certain embodiments, the presence of the CRISPR system components is transient, in that they are degraded over time. In certain embodiments, expression of the CRISPR-Cas systems or Cas proteins described in any of the embodiments comprised in polynucleotide molecules, vectors, vector systems, or cells is limited to certain tissue types or regions in the plant or non-human animal. In certain embodiments, the expression of the CRISPR-Cas systems or Cas proteins described in any of the embodiments comprised in polynucleotide molecules, vectors, vector systems, or cells is dependent of a physiological cue. In certain embodiments, expression of the CRISPR-Cas systems or Cas proteins described in any of the embodiments comprised in polynucleotide molecules, vectors, vector systems, or cells may be triggered by an exogenous molecule. In certain embodiments, expression of the CRISPR-Cas systems or Cas proteins described in any of the embodiments comprised in polynucleotide molecules, vectors, vector systems, or cells is dependent on the expression of a non-cas molecule in the plant or non-human animal. 
     In one aspect, the invention provides methods for using one or more elements of a nucleic acid-targeting system. The nucleic acid-targeting complex of the invention provides an effective means for modifying a target DNA or RNA (single or double stranded, linear or super-coiled). The nucleic acid-targeting complex of the invention has a wide variety of utility including modifying (e.g., deleting, inserting, translocating, inactivating, activating) a target DNA or RNA in a multiplicity of cell types. As such, the nucleic acid-targeting complex of the invention has a broad spectrum of applications in, e.g., gene therapy, drug screening, disease diagnosis, and prognosis. An exemplary nucleic acid-targeting complex comprises a DNA or RNA-targeting effector protein complexed with a guide RNA hybridized to a target sequence within the target locus of interest. 
     In one embodiment, this invention provides a method of cleaving a target polynucleotide. The method may comprise modifying a target polynucleotide using a nucleic acid-targeting complex that binds to the target polynucleotide and effect cleavage of said target polynucleotide. In an embodiment, the nucleic acid-targeting complex of the invention, when introduced into a cell, may create a break (e.g., a single or a double strand break) in the polynucleotide sequence. For example, the method can be used to cleave a disease polynucleotide in a cell. For example, an exogenous template comprising a sequence to be integrated flanked by an upstream sequence and a downstream sequence may be introduced into a cell. The upstream and downstream sequences share sequence similarity with either side of the site of integration in the polynucleotide. The exogenous template comprises a sequence to be integrated (e.g., a mutated RNA). The sequence for integration may be a sequence endogenous or exogenous to the cell. Examples of a sequence to be integrated include polynucleotide 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. The upstream and downstream sequences in the recombination template are selected to promote recombination between the RNA sequence of interest and the recombination. The upstream sequence is a polynucleotide sequence that shares sequence similarity with the sequence upstream of the targeted site for integration. Similarly, the downstream sequence is a polynucleotide sequence that shares sequence similarity with the polynucleotide sequence downstream of the targeted site of integration. The upstream and downstream sequences in the recombination template can have 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with the targeted sequence. Preferably, the upstream and downstream sequences in the recombination template have about 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the targeted sequence. In some methods, the upstream and downstream sequences in the recombination template have about 99% or 100% sequence identity with the targeted sequence. 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 bp. In some methods, the recombination 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 recombination template of the invention can be constructed using recombinant techniques (see, for example, Sambrook et al., 2001 and Ausubel et al., 1996). In a method for modifying a target sequence by integrating an recombination template, a break (e.g., double or single stranded break in double or single stranded DNA or RNA) is introduced into the DNA or RNA sequence by the nucleic acid-targeting complex, the break is repaired via homologous recombination with an recombination template such that the template is integrated into the target. The presence of a double-stranded break facilitates integration of the template. In other embodiments, this invention provides a method of modifying expression of a RNA in a eukaryotic cell. The method comprises increasing or decreasing expression of a target polynucleotide by using a nucleic acid-targeting complex that binds to the DNA or RNA (e.g., mRNA or pre-mRNA). In some methods, a target can be inactivated to affect the modification of the expression in a cell. For example, upon the binding of a nucleic acid-targeting complex to a target sequence in a cell, the target is inactivated such that the sequence is not translated, the coded protein is not produced, or the sequence does not function as the wild-type sequence does. For example, a protein or microRNA coding sequence may be inactivated such that the protein or microRNA or pre-microRNA transcript is not produced. The target of a nucleic acid-targeting complex can be any polynucleotide endogenous or exogenous to the eukaryotic cell. For example, the target polynucleotide can be a polynucleotide residing in the nucleus of the eukaryotic cell. The target polynucleotide can be a sequence coding a gene product (e.g., a protein) or a non-coding sequence (e.g., ncRNA, lncRNA, tRNA, or rRNA). Examples of target RNA include a sequence associated with a signaling biochemical pathway, e.g., a signaling biochemical pathway-associated polynucleotide. Examples of target polynucleotide include a disease associated polynucleotide. A “disease-associated” polynucleotide refers to any polynucleotide which is yielding translation products at an abnormal level or in an abnormal form in cells derived from a disease-affected tissues compared with tissues or cells of a non-disease control. It may be a a gene that becomes expressed at an abnormally high level; it may be a a gene that becomes expressed at an abnormally low level, where the altered expression correlates with the occurrence and/or progression of the disease. A disease-associated polynucleotide also refers to a a gene possessing mutation(s) or genetic variation that is directly responsible or is in linkage disequilibrium with a gene(s) that is responsible for the etiology of a disease. The translated products may be known or unknown, and may be at a normal or abnormal level. The target RNA of a nucleic acid-targeting complex can be any polynucleotide endogenous or exogenous to the eukaryotic cell. For example, the target RNA can be a RNA residing in the nucleus of the eukaryotic cell. The target polynucleotide can be a sequence coding a gene product (e.g., a protein) or a non-coding sequence (e.g., ncRNA, lncRNA, tRNA, or rRNA). 
     In some embodiments, the method may comprise allowing a nucleic acid-targeting complex to bind to the target DNA or RNA to effect cleavage of said target DNA or RNA thereby modifying the target DNA or RNA, wherein the nucleic acid-targeting complex comprises a nucleic acid-targeting effector protein complexed with a guide RNA hybridized to a target sequence within said target DNA or RNA. In one aspect, the invention provides a method of modifying expression of DNA or RNA in a eukaryotic cell. In some embodiments, the method comprises allowing a nucleic acid-targeting complex to bind to the DNA or RNA such that said binding results in increased or decreased expression of said DNA or RNA; wherein the nucleic acid-targeting complex comprises a nucleic acid-targeting effector protein complexed with a guide RNA. Similar considerations and conditions apply as above for methods of modifying a target DNA or RNA. In fact, these sampling, culturing and re-introduction options apply across the aspects of the present invention. In one aspect, the invention provides for methods of modifying a target DNA or RNA in a eukaryotic cell, which may be in vivo, ex vivo or in vitro. In some embodiments, the method comprises sampling a cell or population of cells from a human or non-human animal, and modifying the cell or cells. Culturing may occur at any stage ex vivo. The cell or cells may even be re-introduced into the non-human animal or plant. For re-introduced cells it is particularly preferred that the cells are stem cells. The Cas proteins as described in any embodiment herein may be used to detect nucleic acid identifiers. Nucleic acid identifiers are non-coding nucleic acids that may be used to identify a particular article. Example nucleic acid identifiers, such as DNA watermarks, are described in Heider and Barnekow. “DNA watermarks: A proof of concept” BMC Molecular Biology 9:40 (2008). The nucleic acid identifiers may also be a nucleic acid barcode. A nucleic-acid based barcode is a short sequence of nucleotides (for example, DNA, RNA, or combinations thereof) that is used as an identifier for an associated molecule, such as a target molecule and/or target nucleic acid. A nucleic acid barcode can have a length of at least, for example, 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, 60, 70, 80, 90, or 100 nucleotides, and can be in single- or double-stranded form. One or more nucleic acid barcodes can be attached, or “tagged,” to a target molecule and/or target nucleic acid. This attachment can be direct (for example, covalent or non-covalent binding of the barcode to the target molecule) or indirect (for example, via an additional molecule, for example, a specific binding agent, such as an antibody (or other protein) or a barcode receiving adaptor (or other nucleic acid molecule). Target molecule and/or target nucleic acids can be labeled with multiple nucleic acid barcodes in combinatorial fashion, such as a nucleic acid barcode concatemer. Typically, a nucleic acid barcode is used to identify target molecules and/or target nucleic acids as being from a particular compartment (for example a discrete volume), having a particular physical property (for example, affinity, length, sequence, etc.), or having been subject to certain treatment conditions. Target molecule and/or target nucleic acid can be associated with multiple nucleic acid barcodes to provide information about all of these features (and more). Methods of generating nucleic acid-barcodes are disclosed, for example, in International Patent Application Publication No. WO/2014/047561. 
     In an embodiment, a guide RNA and a Cas and a Cas nuclease induce a double strand break for the purpose of inducing HDR-mediated correction. In a further embodiment, two or more guide RNAs complexing with Cas or an ortholog or homolog thereof, may be used to induce multiplexed breaks for purpose of inducing HDR-mediated correction. 
     A recombination template nucleic acid, as that term is used herein, refers to a nucleic acid sequence which can be used in conjunction with CRISPR-Cas systems discloser herein to alter the structure of a target position. In an embodiment, the target nucleic acid is modified to have some or all of the sequence of the recombination template nucleic acid, typically at or near cleavage site(s). In an embodiment, the recombination template nucleic acid is single stranded. In an alternate embodiment, the recombination template nucleic acid is double stranded. In an embodiment, the recombination template nucleic acid is DNA, e.g., double stranded DNA. In an alternate embodiment, the recombination template nucleic acid is single stranded DNA. 
     In some embodiments, a recombination template is provided 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. 
     A recombination template may be a component of another vector as described herein, contained in a separate vector, or provided as a separate polynucleotide. A recombination 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 some embodiments, the recombination template polynucleotide is complementary to a portion of a polynucleotide comprising the target sequence. When optimally aligned, a recombination 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 recombination template sequence and a polynucleotide comprising a target sequence are optimally aligned, the nearest nucleotide of the recombination 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 an embodiment, the recombination template nucleic acid alters the structure of the target position by participating in homologous recombination. In an embodiment, the recombination template nucleic acid alters the sequence of the target position. In an embodiment, the recombination template nucleic acid results in the incorporation of a modified, or non-naturally occurring base into the target nucleic acid. 
     The recombination template sequence may undergo a breakage mediated or catalyzed recombination with the target sequence. In an embodiment, the recombination template nucleic acid may include sequence that corresponds to a site on the target sequence that is cleaved by an Cas mediated cleavage event. In an embodiment, the recombination template nucleic acid may include sequence that corresponds to both, a first site on the target sequence that is cleaved in a first Cas mediated event and a second site on the target sequence that is cleaved in a second Cas mediated event. 
     In certain embodiments, the recombination template nucleic acid can include 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 recombination 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 recombination 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 recombination template sequence may be used to alter an unwanted structure, e.g., an unwanted or mutant nucleotide. The recombination 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 recombination 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 recombination template nucleic acid may be 20+/−10, 30+/−10, 40+/−10, 50+/−10, 60+/−10, 70+/−10, 80+/−10, 90+/−10, 100+/−10, 110+/−10, 120+/−10, 130+/−10, 140+/−10, 150+/−10, 160+/−10, 170+/−10, 180+/−10, 190+/−10, 200+/−10, 210+/−10, of 220+/−10 nucleotides in length. In an embodiment, the recombination 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, 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 recombination 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 recombination 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 recombination 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. 
     Unlike CRISPR-Cas-mediated gene knockout, which permanently eliminates expression by mutating the gene at the DNA level, CRISPR-Cas knockdown allows for temporary reduction of gene expression through the use of artificial transcription factors. Mutating key residues in both DNA cleavage domains of the Cas protein, results in the generation of a catalytically inactive Cas. A catalytically inactive Cas complexes with a guide RNA and localizes to the DNA sequence specified by that guide RNA&#39;s targeting domain, however, it does not cleave the target DNA. Fusion of the inactive Cas protein (e.g. the D10A and H840A mutations) to an effector domain, e.g., a transcription repression domain, enables recruitment of the effector to any DNA site specified by the guide RNA. In certain embodiments, Cas may be fused to a transcriptional repression domain and recruited to the promoter region of a gene. Especially for gene repression, it is contemplated herein that blocking the binding site of an endogenous transcription factor would aid in downregulating gene expression. In another embodiment, an inactive Cas can be fused to a chromatin modifying protein. Altering chromatin status can result in decreased expression of the target gene. 
     In an embodiment, a guide RNA molecule can be targeted to a known transcription response elements (e.g., promoters, enhancers, etc.), a known upstream activating sequences, and/or sequences of unknown or known function that are suspected of being able to control expression of the target DNA. 
     In some methods, a target polynucleotide can be inactivated to affect the modification of the expression in a cell. For example, upon the binding of a CRISPR complex to a target sequence in a cell, the target polynucleotide is inactivated such that the sequence is not transcribed, the coded protein is not produced, or the sequence does not function as the wild-type sequence does. For example, a protein or microRNA coding sequence may be inactivated such that the protein is not produced. 
     Cas Effector Protein Complex System Promoted Non-Homologous End-Joining 
     In certain embodiments, nuclease-induced non-homologous end-joining (NHEJ) can be used to target gene-specific knockouts. 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. Two-thirds of these mutations typically alter the reading frame and, therefore, produce a non-functional protein. Additionally, mutations that maintain the reading frame, but which insert or delete a significant amount of sequence, can destroy functionality of the protein. This is locus dependent as mutations in critical functional domains are likely less tolerable than mutations in non-critical regions of the protein. The indel mutations generated by NHEJ are unpredictable in nature; however, at a given break site certain indel sequences are favored and are over represented in the population, likely due to small regions of microhomology. The lengths of deletions can vary widely; most commonly in the 1-50 bp range, but they can easily be greater than 50 bp, e.g., they can easily reach greater than about 100-200 bp. Insertions tend to be shorter and often include short duplications of the sequence immediately surrounding the break site. However, it is possible to obtain large insertions, and in these cases, the inserted sequence has often been traced to other regions of the genome or to plasmid DNA present in the cells. 
     Because NHEJ is a mutagenic process, it may also be used to delete small sequence motifs as long as the generation of a specific final sequence is not required. 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; however, the error-prone nature of NHEJ may still produce indel mutations at the site of repair. 
     Both double strand cleaving Cas proteins, or an ortholog or homolog thereof, and single strand, or nickase, Cas proteins, or an ortholog or homolog thereof, molecules can be used in the methods and compositions described herein to generate NHEJ-mediated indels. NHEJ-mediated indels 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 protein, or an ortholog or homolog thereof, 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 Cas proteins, or an ortholog or homolog thereof, preferably 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. In some examples, the systems herein may introduce one or more indels via NHEJ pathway and insert sequence from a combination template via HDR. 
     Exemplary Applications 
     The invention provides a non-naturally occurring or engineered composition, or one or more polynucleotides encoding components of said composition, or vector or delivery systems comprising one or more polynucleotides encoding components of said composition for use in a modifying a target cell in vivo, ex vivo or in vitro and, may be conducted in a manner alters the cell such that once modified the progeny or cell line of the CRISPR modified cell retains the altered phenotype. The modified cells and progeny may be part of a multicellular organism such as a plant or animal with ex vivo or in vivo application of CRISPR system to desired cell types. The CRISPR invention may be a therapeutic method of treatment. The therapeutic method of treatment may comprise gene or genome editing, or gene therapy. 
     In some embodiments, one or more vectors described herein are used to produce a non-human transgenic animal or transgenic plant. In some embodiments, the transgenic animal is a mammal, such as a mouse, rat, or rabbit. Methods for producing transgenic animals and plants are known in the art, and generally begin with a method of cell transfection, such as described herein. 
     Use of Orthogonal Catalytically Inactive CRISPR-Cas Proteins 
     In particular embodiments, the Cas nickase is used in combination with an orthogonal catalytically inactive CRISPR-Cas protein to increase efficiency of said Cas nickase (as described in Chen et al. 2017, Nature Communications 8:14958; doi:10.1038/ncomms14958). More particularly, the orthogonal catalytically inactive CRISPR-Cas protein is characterized by a different PAM recognition site than the Cα9 nickase used in the AD-functionalized CRISPR system and the corresponding guide sequence is selected to bind to a target sequence proximal to that of the Cas nickase of the functionalized CRISPR system. The orthogonal catalytically inactive CRISPR-Cas protein as used in the context of the present invention does not form part of the functionalized CRISPR system but merely functions to increase the efficiency of said Cas nickase and is used in combination with a standard guide molecule as described in the art for said CRISPR-Cas protein. In particular embodiments, said orthogonal catalytically inactive CRISPR-Cas protein is a dead CRISPR-Cas protein, i.e. comprising one or more mutations which abolishes the nuclease activity of said CRISPR-Cas protein. In particular embodiments, the catalytically inactive orthogonal CRISPR-Cas protein is provided with two or more guide molecules which are capable of hybridizing to target sequences which are proximal to the target sequence of the Cas nickase. In particular embodiments, at least two guide molecules are used to target said catalytically inactive CRISPR-Cas protein, of which at least one guide molecule is capable of hybridizing to a target sequence 5″ of the target sequence of the Cas nickase and at least one guide molecule is capable of hybridizing to a target sequence 3′ of the target sequence of the Cas nickase of the functionalized CRISPR system, whereby said one or more target sequences may be on the same or the opposite DNA strand as the target sequence of the Cas nickase. In particular embodiments, the guide sequences for the one or more guide molecules of the orthogonal catalytically inactive CRISPR-Cas protein are selected such that the target sequences are proximal to that of the guide molecule for the targeting of the functionalized CRISPR, i.e. for the targeting of the Cas nickase. In particular embodiments, the one or more target sequences of the orthogonal catalytically inactive CRISPR-Cas enzyme are each separated from the target sequence of the Cas nickase by more than 5 but less than 450 basepairs. Optimal distances between the target sequences of the guides for use with the orthogonal catalytically inactive CRISPR-Cas protein and the target sequence of the functionalized CRISPR system can be determined by the skilled person. In particular embodiments, the orthogonal CRISPR-Cas protein is a Class II, type II CRISPR protein. In particular embodiments, the orthogonal CRISPR-Cas protein is a Class II, type V CRISPR protein. In particular embodiments, the catalytically inactive orthogonal CRISPR-Cas protein. In particular embodiments, the catalytically inactive orthogonal CRISPR-Cas protein has been modified to alter its PAM specificity as described elsewhere herein. In particular embodiments, the Cas protein nickase is a nickase which, by itself has limited activity in human cells, but which, in combination with an inactive orthogonal CRISPR-Cas protein and one or more corresponding proximal guides ensures the required nickase activity. 
     Use of Inactivated CRISPR Cas Enzyme for Detection Methods Such as FISH 
     In one aspect, the invention provides an engineered, non-naturally occurring CRISPR-Cas system comprising a catalytically inactivate Cas protein described herein, and use this system in detection methods such as fluorescence in situ hybridization (FISH). A dCas protein 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 teleomeric repeats in vivo. The dCas system can be used to visualize both repetitive sequences and individual genes in the human genome. Such new applications of labelled dCas CRISPR-cas systems may be important in imaging cells and studying the functional nuclear architecture, especially in cases with a small nucleus volume or complex 3-D structures. (Chen B, Gilbert L A, Cimini B A, Schnitzbauer J, Zhang W, Li G W, Park J, Blackburn E H, Weissman J S, Qi L S, Huang B. 2013. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155(7):1479-91. doi: 10.1016/j.cell.2013.12.001) 
     Patient-Specific Screening Methods 
     A nucleic acid-targeting system that targets DNA, e.g., trinucleotide repeats can be used to screen patients or patent samples for the presence of such repeats. The repeats can be the target of the RNA of the nucleic acid-targeting system, and if there is binding thereto by the nucleic acid-targeting system, that binding can be detected, to thereby indicate that such a repeat is present. Thus, a nucleic acid-targeting system can be used to screen patients or patient samples for the presence of the repeat. The patient can then be administered suitable compound(s) to address the condition; or, can be administered a nucleic acid-targeting system to bind to and cause insertion, deletion or mutation and alleviate the condition. 
     Models of Genetic and Epigenetic Conditions 
     A method of the invention may be used to create a plant, an animal or cell that may be used to model and/or study genetic or epigenetic conditions of interest, such as a through a model of mutations of interest or a disease model. As used herein, “disease” refers to a disease, disorder, or indication in a subject. For example, a method of the invention may be used to create an animal or cell that comprises a modification in one or more nucleic acid sequences associated with a disease, or a plant, animal or cell in which the expression of one or more nucleic acid sequences associated with a disease are altered. Such a nucleic acid sequence may encode a disease associated protein sequence or may be a disease associated control sequence. Accordingly, it is understood that in embodiments of the invention, a plant, subject, patient, organism or cell can be a non-human subject, patient, organism or cell. Thus, the invention provides a plant, animal or cell, produced by the present methods, 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. In the instance where the cell is in cultured, a cell line may be established if appropriate culturing conditions are met and preferably if the cell is suitably adapted for this purpose (for instance a stem cell). Bacterial cell lines produced by the invention are also envisaged. Hence, cell lines are also envisaged. 
     In some methods, the disease model can be used to study the effects of mutations on the animal or cell and development and/or progression of the disease using measures commonly used in the study of the disease. Alternatively, such a disease model is useful for studying the effect of a pharmaceutically active compound on the disease. 
     In some methods, the disease model can be used to assess the efficacy of a potential gene therapy strategy. That is, a disease-associated gene or polynucleotide can be modified such that the disease development and/or progression is inhibited or reduced. In particular, the method comprises modifying a disease-associated gene or polynucleotide such that an altered protein is produced and, as a result, the animal or cell has an altered response. Accordingly, in some methods, a genetically modified animal may be compared with an animal predisposed to development of the disease such that the effect of the gene therapy event may be assessed. 
     In another embodiment, this invention provides a method of developing a biologically active agent that modulates a cell signaling event associated with a disease gene. The method comprises contacting a test compound with a cell comprising one or more vectors that drive expression of one or more of a CRISPR enzyme, and a direct repeat sequence linked to a guide sequence; and detecting a change in a readout that is indicative of a reduction or an augmentation of a cell signaling event associated with, e.g., a mutation in a disease gene contained in the cell. 
     A cell model or animal model can be constructed in combination with the method of the invention for screening a cellular function change. Such a model may be used to study the effects of a genome sequence modified by the CRISPR complex of the invention on a cellular function of interest. For example, a cellular function model may be used to study the effect of a modified genome sequence on intracellular signaling or extracellular signaling. Alternatively, a cellular function model may be used to study the effects of a modified genome sequence on sensory perception. In some such models, one or more genome sequences associated with a signaling biochemical pathway in the model are modified. 
     Several disease models have been specifically investigated. These include de novo autism risk genes CHD8, KATNAL2, and SCN2A; and the syndromic autism (Angelman Syndrome) gene UBE3A. These genes and resulting autism models are of course preferred, but serve to show the broad applicability of the invention across genes and corresponding models. An altered expression of one or more genome sequences associated with a signaling biochemical pathway can be determined by assaying for a difference in the mRNA levels of the corresponding genes between the test model cell and a control cell, when they are contacted with a candidate agent. Alternatively, the differential expression of the sequences associated with a signaling biochemical pathway is determined by detecting a difference in the level of the encoded polypeptide or gene product. 
     To assay for an agent-induced alteration in the level of mRNA transcripts or corresponding polynucleotides, nucleic acid contained in a sample is first extracted according to standard methods in the art. For instance, mRNA can be isolated using various lytic enzymes or chemical solutions according to the procedures set forth in Sambrook et al. (1989), or extracted by nucleic-acid-binding resins following the accompanying instructions provided by the manufacturers. The mRNA contained in the extracted nucleic acid sample is then detected by amplification procedures or conventional hybridization assays (e.g. Northern blot analysis) according to methods widely known in the art or based on the methods exemplified herein. 
     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 particular, the isolated RNA can be subjected to a reverse transcription assay that is coupled with a quantitative polymerase chain reaction (RT-PCR) in order to quantify the expression level of a sequence associated with a signaling biochemical pathway. 
     Detection of the gene expression level can be conducted in real time in an amplification assay. In one aspect, the amplified products can be directly visualized with fluorescent DNA-binding agents including but not limited to DNA intercalators and DNA groove binders. Because the amount of the intercalators incorporated into the double-stranded DNA molecules is typically proportional to the amount of the amplified DNA products, one can conveniently determine the amount of the amplified products by quantifying the fluorescence of the intercalated dye using conventional optical systems in the art. DNA-binding dye suitable for this application include SYBR green, SYBR blue, DAPI, propidium iodine, Hoeste, SYBR gold, ethidium bromide, acridines, proflavine, acridine orange, acriflavine, fluorcoumanin, ellipticine, daunomycin, chloroquine, distamycin D, chromomycin, homidium, mithramycin, ruthenium polypyridyls, anthramycin, and the like. 
     In another aspect, other fluorescent labels such as sequence specific probes can be employed in the amplification reaction to facilitate the detection and quantification of the amplified products. Probe-based quantitative amplification relies on the sequence-specific detection of a desired amplified product. It utilizes fluorescent, target-specific probes (e.g., TaqMan® probes) resulting in increased specificity and sensitivity. Methods for performing probe-based quantitative amplification are well established in the art and are taught in U.S. Pat. No. 5,210,015. 
     In yet another aspect, conventional hybridization assays using hybridization probes that share sequence homology with sequences associated with a signaling biochemical pathway can be performed. Typically, probes are allowed to form stable complexes with the sequences associated with a signaling biochemical pathway contained within the biological sample derived from the test subject in a hybridization reaction. It will be appreciated by one of skill in the art that where antisense is used as the probe nucleic acid, the target polynucleotides provided in the sample are chosen to be complementary to sequences of the antisense nucleic acids. Conversely, where the nucleotide probe is a sense nucleic acid, the target polynucleotide is selected to be complementary to sequences of the sense nucleic acid. 
     Hybridization can be performed under conditions of various stringency. Suitable hybridization conditions for the practice of the present invention are such that the recognition interaction between the probe and sequences associated with a signaling biochemical pathway is both sufficiently specific and sufficiently stable. Conditions that increase the stringency of a hybridization reaction are widely known and published in the art. See, for example, (Sambrook, et al., (1989); Nonradioactive In Situ Hybridization Application Manual, Boehringer Mannheim, second edition). The hybridization assay can be formed using probes immobilized on any solid support, including but are not limited to nitrocellulose, glass, silicon, and a variety of gene arrays. A preferred hybridization assay is conducted on high-density gene chips as described in U.S. Pat. No. 5,445,934. 
     For a convenient detection of the probe-target complexes formed during the hybridization assay, the nucleotide probes are conjugated to a detectable label. Detectable labels suitable for use in the present invention include any composition detectable by photochemical, biochemical, spectroscopic, immunochemical, electrical, optical or chemical means. A wide variety of appropriate detectable labels are known in the art, which include fluorescent or chemiluminescent labels, radioactive isotope labels, enzymatic or other ligands. In preferred embodiments, one will likely desire to employ a fluorescent label or an enzyme tag, such as digoxigenin, ß-galactosidase, urease, alkaline phosphatase or peroxidase, avidin/biotin complex. 
     The detection methods used to detect or quantify the hybridization intensity will typically depend upon the label selected above. For example, radiolabels may be detected using photographic film or a phosphoimager. Fluorescent markers may be detected and quantified using a photodetector to detect emitted light. Enzymatic labels are typically detected by providing the enzyme with a substrate and measuring the reaction product produced by the action of the enzyme on the substrate; and finally colorimetric labels are detected by simply visualizing the colored label. 
     An agent-induced change in expression of sequences associated with a signaling biochemical pathway can also be determined by examining the corresponding gene products. Determining the protein level typically involves a) contacting the protein contained in a biological sample with an agent that specifically bind to a protein associated with a signaling biochemical pathway; and (b) identifying any agent:protein complex so formed. In one aspect of this embodiment, the agent that specifically binds a protein associated with a signaling biochemical pathway is an antibody, preferably a monoclonal antibody. 
     The reaction is performed by contacting the agent with a sample of the proteins associated with a signaling biochemical pathway derived from the test samples under conditions that will allow a complex to form between the agent and the proteins associated with a signaling biochemical pathway. The formation of the complex can be detected directly or indirectly according to standard procedures in the art. In the direct detection method, the agents are supplied with a detectable label and unreacted agents may be removed from the complex; the amount of remaining label thereby indicating the amount of complex formed. For such method, it is preferable to select labels that remain attached to the agents even during stringent washing conditions. It is preferable that the label does not interfere with the binding reaction. In the alternative, an indirect detection procedure may use an agent that contains a label introduced either chemically or enzymatically. A desirable label generally does not interfere with binding or the stability of the resulting agent:polypeptide complex. However, the label is typically designed to be accessible to an antibody for an effective binding and hence generating a detectable signal. 
     A wide variety of labels suitable for detecting protein levels are known in the art. Non-limiting examples include radioisotopes, enzymes, colloidal metals, fluorescent compounds, bioluminescent compounds, and chemiluminescent compounds. 
     The amount of agent:polypeptide complexes formed during the binding reaction can be quantified by standard quantitative assays. As illustrated above, the formation of agent:polypeptide complex can be measured directly by the amount of label remained at the site of binding. In an alternative, the protein associated with a signaling biochemical pathway is tested for its ability to compete with a labeled analog for binding sites on the specific agent. In this competitive assay, the amount of label captured is inversely proportional to the amount of protein sequences associated with a signaling biochemical pathway present in a test sample. 
     A number of techniques for protein analysis based on the general principles outlined above are available in the art. They include but are not limited to radioimmunoassays, ELISA (enzyme linked immunoradiometric assays), “sandwich” immunoassays, immunoradiometric assays, in situ immunoassays (using e.g., colloidal gold, enzyme or radioisotope labels), western blot analysis, immunoprecipitation assays, immunofluorescent assays, and SDS-PAGE. 
     Antibodies that specifically recognize or bind to proteins associated with a signaling biochemical pathway are preferable for conducting the aforementioned protein analyses. Where desired, antibodies that recognize a specific type of post-translational modifications (e.g., signaling biochemical pathway inducible modifications) can be used. Post-translational modifications include but are not limited to glycosylation, lipidation, acetylation, and phosphorylation. These antibodies may be purchased from commercial vendors. For example, anti-phosphotyrosine antibodies that specifically recognize tyrosine-phosphorylated proteins are available from a number of vendors including Invitrogen and Perkin Elmer. Anti-phosphotyrosine antibodies are particularly useful in detecting proteins that are differentially phosphorylated on their tyrosine residues in response to an ER stress. Such proteins include but are not limited to eukaryotic translation initiation factor 2 alpha (eIF-2α). Alternatively, these antibodies can be generated using conventional polyclonal or monoclonal antibody technologies by immunizing a host animal or an antibody-producing cell with a target protein that exhibits the desired post-translational modification. 
     Genome Wide Knock-Out Screening 
     The CRISPR proteins and systems described herein can be used to perform efficient and cost effective functional genomic screens. Such screens can utilize CRISPR effector protein based genome wide libraries. Such screens and libraries can provide for determining the function of genes, cellular pathways genes are involved in, and how any alteration in gene expression can result in a particular biological process. An advantage of the present invention is that the CRISPR system avoids off-target binding and its resulting side effects. This is achieved using systems arranged to have a high degree of sequence specificity for the target DNA. In preferred embodiments of the invention, the CRISPR effector protein complexes are Cas effector protein complexes. 
     In embodiments of the invention, a genome wide library may comprise a plurality of Cas protein guide RNAs, as described herein, comprising guide sequences that are capable of targeting a plurality of target sequences in a plurality of genomic loci in a population of eukaryotic cells. The population of cells may be a population of embryonic stem (ES) cells. The target sequence in the genomic locus may be a non-coding sequence. The non-coding sequence may be an intron, regulatory sequence, splice site, 3′ UTR, 5′ UTR, or polyadenylation signal. Gene function of one or more gene products may be altered by said targeting. The targeting may result in a knockout of gene function. The targeting of a gene product may comprise more than one guide RNA. A gene product may be targeted by 2, 3, 4, 5, 6, 7, 8, 9, or 10 guide RNAs, preferably 3 to 4 per gene. Off-target modifications may be minimized by exploiting the staggered double strand breaks generated by Cas effector protein complexes or by utilizing methods analogous to those used in CRISPR-Cas systems (See, e.g., DNA targeting specificity of RNA-guided Cas nucleases. Hsu, P., Scott, D., Weinstein, J., Ran, F A., Konermann, S., Agarwala, V., Li, Y., Fine, E., Wu, X., Shalem, O., Cradick, T J., Marraffini, L A., Bao, G., &amp; Zhang, F. Nat Biotechnol doi:10.1038/nbt.2647 (2013)), incorporated herein by reference. The targeting may be of about 100 or more sequences. The targeting may be of about 1000 or more sequences. The targeting may be of about 20,000 or more sequences. The targeting may be of the entire genome. The targeting may be of a panel of target sequences focused on a relevant or desirable pathway. The pathway may be an immune pathway. The pathway may be a cell division pathway. 
     One aspect of the invention comprehends a genome wide library that may comprise a plurality of Cas guide RNAs that may comprise guide sequences that are capable of targeting a plurality of target sequences in a plurality of genomic loci, wherein said targeting results in a knockout of gene function. This library may potentially comprise guide RNAs that target each and every gene in the genome of an organism. 
     In some embodiments of the invention, the organism or subject is a eukaryote (including mammal including human) or a non-human eukaryote or a non-human animal or a non-human mammal. In some embodiments, the organism or subject is a non-human animal, and may be an arthropod, for example, an insect, or may be a nematode. In some methods of the invention the organism or subject is a plant. In some methods of the invention, the organism or subject is a mammal or a non-human mammal. A non-human mammal may be for example a rodent (preferably a mouse or a rat), an ungulate, or a primate. In some methods of the invention the organism or subject is algae, including microalgae, or is a fungus. 
     The knockout of gene function may comprise introducing into each cell in the population of cells a vector system of one or more vectors comprising an engineered, non-naturally occurring Cas effector protein system comprising I. a Cas effector protein, and II. one or more guide RNAs, wherein components I and II may be same or on different vectors of the system, integrating components I and II into each cell, wherein the guide sequence targets a unique gene in each cell, wherein the Cas effector protein is operably linked to a regulatory element, wherein when transcribed, the guide RNA comprising the guide sequence directs sequence-specific binding of the Cas effector protein system to a target sequence in the genomic loci of the unique gene, inducing cleavage of the genomic loci by the Cas effector protein, and confirming different knockout mutations in a plurality of unique genes in each cell of the population of cells thereby generating a gene knockout cell library. The invention comprehends that the population of cells is a population of eukaryotic cells, and in a preferred embodiment, the population of cells is a population of embryonic stem (ES) cells. 
     The one or more vectors may be plasmid vectors. The vector may be a single vector comprising a Cas effector protein, a gRNA, and optionally, a selection marker into target cells. Not being bound by a theory, the ability to simultaneously deliver a Cas effector protein and gRNA through a single vector enables application to any cell type of interest, without the need to first generate cell lines that express the Cas effector protein. The regulatory element may be an inducible promoter. The inducible promoter may be a doxycycline inducible promoter. In some methods of the invention the expression of the guide sequence is under the control of the T7 promoter and is driven by the expression of T7 polymerase. The confirming of different knockout mutations may be by whole exome sequencing. The knockout mutation may be achieved in 100 or more unique genes. The knockout mutation may be achieved in 1000 or more unique genes. The knockout mutation may be achieved in 20,000 or more unique genes. The knockout mutation may be achieved in the entire genome. The knockout of gene function may be achieved in a plurality of unique genes which function in a particular physiological pathway or condition. The pathway or condition may be an immune pathway or condition. The pathway or condition may be a cell division pathway or condition. 
     Useful in the practice of the instant invention utilizing Cas effector protein complexes are methods used in CRISPR-Cas systems and reference is made to: Genome-Scale CRISPR-Cas Knockout Screening in Human Cells. Shalem, O., Sanjana, N E., Hartenian, E., Shi, X., Scott, D A., Mikkelson, T., Heckl, D., Ebert, B L., Root, D E., Doench, J G., Zhang, F. Science December 12. (2013). [Epub ahead of print]; Published in final edited form as: Science. Jan. 3, 2014; 343(6166): 84-87. Shalem et al. involves a new way to interrogate gene function on a genome-wide scale. Their studies showed that delivery of a genome-scale CRISPR-Cas knockout (GeCKO) library targeted 18,080 genes with 64,751 unique guide sequences enabled both negative and positive selection screening in human cells. First, the authors showed use of the GeCKO library to identify genes essential for cell viability in cancer and pluripotent stem cells. Next, in a melanoma model, the authors screened for genes whose loss is involved in resistance to vemurafenib, a therapeutic that inhibits mutant protein kinase BRAF. Their studies showed that the highest-ranking candidates included previously validated genes NF1 and MED12 as well as novel hitsNF2, CUL3, TADA2B, and TADA1. The authors observed a high level of consistency between independent guide RNAs targeting the same gene and a high rate of hit confirmation, and thus demonstrated the promise of genome-scale screening with Cas. 
     Reference is also made to US patent publication number US20140357530; and PCT Patent Publication WO2014093701, hereby incorporated herein by reference. Reference is also made to NIH Press Release of Oct. 22, 2015 entitled, “Researchers identify potential alternative to CRISPR-Cas genome editing tools: New Cas enzymes shed light on evolution of CRISPR-Cas systems, which is incorporated by reference. 
     Functional Alteration and Screening 
     In another aspect, the present invention provides for a method of functional evaluation and screening of genes. The use of the CRISPR system of the present invention to precisely deliver functional domains, to activate or repress genes or to alter epigenetic state by precisely altering the methylation site on a specific locus of interest, can be with one or more guide RNAs applied to a single cell or population of cells or with a library applied to genome in a pool of cells ex vivo or in vivo comprising the administration or expression of a library comprising a plurality of guide RNAs (gRNAs) and wherein the screening further comprises use of a Cas effector protein, wherein the CRISPR complex comprising the Cas effector protein is modified to comprise a heterologous functional domain. In an aspect the invention provides a method for screening a genome comprising the administration to a host or expression in a host in vivo of a library. In an aspect the invention provides a method as herein discussed further comprising an activator administered to the host or expressed in the host. In an aspect the invention provides a method as herein discussed wherein the activator is attached to a Cas effector protein. In an aspect the invention provides a method as herein discussed wherein the activator is attached to the N terminus or the C terminus of the Cas effector protein. In an aspect the invention provides a method as herein discussed wherein the activator is attached to a gRNA loop. In an aspect the invention provides a method as herein discussed further comprising a repressor administered to the host or expressed in the host. In an aspect the invention provides a method as herein discussed, wherein the screening comprises affecting and detecting gene activation, gene inhibition, or cleavage in the locus. 
     It is also preferred to target endogenous (regulatory) control elements (such as enhancers and silencers) e.g. 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. 
     Saturating Mutagenesis 
     The Cas effector protein system(s) described herein can be used to perform saturating or deep scanning mutagenesis of genomic loci in conjunction with a cellular phenotype—for instance, for determining critical minimal features and discrete vulnerabilities of functional elements required for gene expression, drug resistance, and reversal of disease. By saturating or deep scanning mutagenesis is meant that every or essentially every DNA base is cut within the genomic loci. A library of Cas1 effector protein guide RNAs may be introduced into a population of cells. The library may be introduced, such that each cell receives a single guide RNA (gRNA). In the case where the library is introduced by transduction of a viral vector, as described herein, a low multiplicity of infection (MOI) is used. The library may include gRNAs targeting every sequence upstream of a (protospacer adjacent motif) (PAM) sequence in a genomic locus. The library may include at least 100 non-overlapplng genomic sequences upstream of a PAM sequence for every 1000 base pairs within the genomic locus. The library may include gRNAs targeting sequences upstream of at least one different PAM sequence. The Cas effector protein systems may include more than one Cas protein. Any Cas effector protein as described herein, including orthologues or engineered Cas effector proteins that recognize different PAM sequences may be used. The frequency of off target sites for a gRNA may be less than 500. Off target scores may be generated to select gRNAs with the lowest off target sites. Any phenotype determined to be associated with cutting at a gRNA target site may be confirmed by using gRNAs targeting the same site in a single experiment. Validation of a target site may also be performed by using a modified Cas effector protein, as described herein, and two gRNAs targeting the genomic site of interest. Not being bound by a theory, a target site is a true hit if the change in phenotype is observed in validation experiments. 
     The genomic loci may include at least one continuous genomic region. The at least one continuous genomic region may comprise up to the entire genome. The at least one continuous genomic region may comprise a functional element of the genome. The functional element may be within a non-coding region, coding gene, intronic region, promoter, or enhancer. The at least one continuous genomic region may comprise at least 1 kb, preferably at least 50 kb of genomic DNA. The at least one continuous genomic region may comprise a transcription factor binding site. The at least one continuous genomic region may comprise a region of DNase I hypersensitivity. The at least one continuous genomic region may comprise a transcription enhancer or repressor element. The at least one continuous genomic region may comprise a site enriched for an epigenetic signature. The at least one continuous genomic DNA region may comprise an epigenetic insulator. The at least one continuous genomic region may comprise two or more continuous genomic regions that physically interact. Genomic regions that interact may be determined by ‘4C technology’. 4C technology allows the screening of the entire genome in an unbiased manner for DNA segments that physically interact with a DNA fragment of choice, as is described in Zhao et al. ((2006) Nat Genet 38, 1341-7) and in U.S. Pat. No. 8,642,295, both incorporated herein by reference in its entirety. The epigenetic signature may be histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, DNA methylation, or a lack thereof. 
     The Cas effector protein system(s) for saturating or deep scanning mutagenesis can be used in a population of cells. The Cas effector protein system(s) can be used in eukaryotic cells, including but not limited to mammalian and plant cells. The population of cells may be prokaryotic cells. The population of eukaryotic cells may be a population of embryonic stem (ES) cells, neuronal cells, epithelial cells, immune cells, endocrine cells, muscle cells, erythrocytes, lymphocytes, plant cells, or yeast cells. 
     In one aspect, the present invention provides for a method of screening for functional elements associated with a change in a phenotype. The library may be introduced into a population of cells that are adapted to contain a Cas effector protein. The cells may be sorted into at least two groups based on the phenotype. The phenotype may be expression of a gene, cell growth, or cell viability. The relative representation of the guide RNAs present in each group are determined, whereby genomic sites associated with the change in phenotype are determined by the representation of guide RNAs present in each group. The change in phenotype may be a change in expression of a gene of interest. The gene of interest may be upregulated, downregulated, or knocked out. The cells may be sorted into a high expression group and a low expression group. The population of cells may include a reporter construct that is used to determine the phenotype. The reporter construct may include a detectable marker. Cells may be sorted by use of the detectable marker. 
     In another aspect, the present invention provides for a method of screening for genomic sites associated with resistance to a chemical compound. The chemical compound may be a drug or pesticide. The library may be introduced into a population of cells that are adapted to contain a Cas effector protein, wherein each cell of the population contains no more than one guide RNA; the population of cells are treated with the chemical compound; and the representation of guide RNAs are determined after treatment with the chemical compound at a later time point as compared to an early time point, whereby genomic sites associated with resistance to the chemical compound are determined by enrichment of guide RNAs. Representation of gRNAs may be determined by deep sequencing methods. 
     Useful in the practice of the instant invention utilizing Cas effector protein complexes are methods used in CRISPR-Cas systems and reference is made to the article entitled BCL11A enhancer dissection by Cas-mediated in situ saturating mutagenesis. Canver, M. C., Smith, E. C., Sher, F., Pinello, L., Sanjana, N. E., Shalem, O., Chen, D. D., Schupp, P. G., Vinjamur, D. S., Garcia, S. P., Luc, S., Kurita, R., Nakamura, Y., Fujiwara, Y., Maeda, T., Yuan, G., Zhang, F., Orkin, S. H., &amp; Bauer, D. E. DOI:10.1038/nature15521, published online Sep. 16, 2015, the article is herein incorporated by reference and discussed briefly below: 
     Canver et al. involves novel pooled CRISPR-Cas guide RNA libraries to perform in situ saturating mutagenesis of the human and mouse BCL11A erythroid enhancers previously identified as an enhancer associated with fetal hemoglobin (HbF) level and whose mouse ortholog is necessary for erythroid BCL11A expression. This approach revealed critical minimal features and discrete vulnerabilities of these enhancers. Through editing of primary human progenitors and mouse transgenesis, the authors validated the BCL11A erythroid enhancer as a target for HbF reinduction. The authors generated a detailed enhancer map that informs therapeutic genome editing. 
     Modification of a Cell or Organism 
     The present disclosure further provides cells comprising one or more components of the systems herein, e.g., the Cas protein and/or guide molecule(s). Also provided include cells modified by the systems and methods herein, and cell cultures, tissues, organs, organism comprising such cells or progeny thereof. The invention in some embodiments comprehends a method of modifying an cell or organism. The cell may be a prokaryotic cell or a eukaryotic cell. The cell may be a mammalian cell. The mammalian cell many be a non-human primate, bovine, porcine, rodent or mouse cell. The cell may be a non-mammalian eukaryotic cell such as poultry, fish or shrimp. The cell may also be a plant cell. The plant cell may be of a crop plant such as cassava, corn, sorghum, wheat, or rice. The plant cell may also be of an algae, tree or vegetable. The modification introduced to the cell by the present invention may be such that the cell and progeny of the cell are altered for improved production of biologic products such as an antibody, starch, alcohol or other desired cellular output. The modification introduced to the cell by the present invention may be such that the cell and progeny of the cell include an alteration that changes the biologic product produced. 
     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 composition, system, or component thereof described herein and/or include detecting a diseased or healthy polynucleotide in a subject or cell thereof using a composition, system, or component thereof described herein. In some embodiments, the method of treatment or prevention can include using a composition, 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 composition, system, or component thereof to modify a polynucleotide of an infectious organism or symbiotic organism within a subject. The composition, system, and components thereof can be used to develop models of diseases, states, or conditions. The composition, system, 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 composition, system, and components thereof can be used to screen and select cells that can be used, for example, as treatments or preventions described herein. The composition, system, 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 composition, 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. 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 composition, system, 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 composition, system, 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 composition, system, 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 composition, system, 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 composition, system, 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 composition, 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. The repair template may be a recombination template herein. 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 composition, 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 composition, 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 composition, 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 composition and 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 at least 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 at least 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 at least 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 at least 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 at least 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 at least 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 composition, 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 composition, system, or technique. 
     In some embodiments, the composition, system, or component thereof can promote Non-Homologous End-Joining (NHEJ). In some embodiments, modification of a polynucleotide by a composition, system, or a component thereof, such as a diseased polynucleotide, can include NHEJ. In some embodiments, promotion of this repair pathway by the composition, 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 composition, 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. 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, composition, system, mediated NHEJ can be used in the method to delete small sequence motifs. In some embodiments, composition, 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 International Patent Publication No. WO 2014/093622 (PCT/US2013/074667); or, via mutation. Others are as described elsewhere herein. 
     Typically, in the context of an endogenous CRISPR or system, formation of a CRISPR or 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 composition, system, or component thereof to bind to the target polynucleotide, e.g., to effect cleavage, nicking, or other modification as the composition, system, is capable of said target polynucleotide, thereby modifying the target polynucleotide, wherein the composition, 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 composition, 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 composition, system, or component thereof. 
     The cleavage, nicking, or other modification capable of being performed by the composition, 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 recombination 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 composition, 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 composition, 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 composition, 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 composition and system, according to the invention as described herein, such as the composition and system, for use in the methods according to the invention as described herein, may be suitably used for any type of application known for composition, system, preferably in eukaryotes. In certain aspects, 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. Alternatively, or in addition, in certain aspects, 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 composition, 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 2 and 3. 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 composition, 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 composition, 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 composition, system, 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 composition, system, herein or a component thereof, wherein the composition, 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-thalassemia”, Nature 467, 318-322 (Sep. 16, 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) 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 composition, system, herein in view of the description herein). In some embodiments, iPSCs can be modified using a composition, system, described herein to correct a disease polynucleotide associated with a circulatory disease. In this regard, the teachings of Xu et al. (Sci Rep. Jul. 9, 2015; 5:12065. doi: 10.1038/srep12065) and Song et al. (Stem Cells Dev. May 1, 2015; 24(9):1053-65. doi: 10.1089/scd.2014.0347. Epub Feb. 5, 2015) with respect to modifying iPSCs can be adapted for use in view of the description herein with the composition, system, 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, CD5, 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 protein and the gRNA being admixed. The gRNA and Cas effector 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 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. May 16, 2013; 121(20):4082-9. doi: 10.1182/blood-2012-09-455204. Epub Mar. 21, 2013. 
     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 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. 
     Treating Neurological Diseases 
     In some embodiments, the compositions, 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 compositions, 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 compositions, systems, herein. 
     Treating Hearing Diseases 
     In some embodiments the composition and 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 composition, 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 Publication No. 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. Patent 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. Patent Publication No. 2007/0093878, which provides an exemplary cochlear implant suitable for delivery of the compositions, 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 Publication No. 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. Patent Publication No. 2005/0287127) and Li et al., (U.S. patent 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 composition and system may be delivered to the ear by direct application of pharmaceutical composition to the outer ear, with compositions modified from US Patent Publication No. 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 compositions, 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 Mukherjea et al. (Antioxidants &amp; Redox Signaling, Volume 13, Number 5, 2010) can be adapted for transtympanic administration of the composition, 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 composition, 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-Cas-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 compositions, 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 composition, system, or component thereof described herein is delivered to one or both eyes. 
     The composition, 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 composition, 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 composition, 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, 189rα76 (2013)), which can be adapted for use with the composition, 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 composition, 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 may be used or adapted. 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 composition, 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 compositions, 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 compositions, 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 composition, 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 composition, 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 compositions, systems, described herein. 
     For example, US Patent Publication No. 20110023139, the teachings of which can be adapted for and/or applied to the compositions, 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 2. 
     The compositions, systems, herein can be used for treating diseases of the muscular system. The present invention also contemplates delivering the composition, system, described herein, 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 composition, 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 composition, system, described herein capable of RNA modification. In some embodiments, exon skipping can be achieved in dystrophin mRNA. In some embodiments, the composition, 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 composition, 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 compositions, 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 compositions, 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 compositions, systems, herein and injected at a dose of about 15 to about 50 mg into the great saphenous vein of a human. 
     In some embodiments, the method comprise treating a sickle cell related disease, e.g., sickle cell trait, sickle cell disease such as sickle cell anemia, β-thalassaemia. For example, the method and system may be used to modify the genome of the sickle cell, e.g., by correcting one or more mutations of the β-globin gene. In the case of β-thalassaemia, sickle cell anemia can be corrected by modifying HSCs with the systems. The system allows the specific editing of the cell&#39;s genome by cutting its DNA and then letting it repair itself. The Cas protein is inserted and directed by a RNA guide to the mutated point and then it cuts the DNA at that point. Simultaneously, a healthy version of the sequence is inserted. This sequence is used by the cell&#39;s own repair system to fix the induced cut. In this way, the CRISPR-Cas allows the correction of the mutation in the previously obtained stem cells. The methods and systems may be used to correct HSCs as to sickle cell anemia using a systems that targets and corrects the mutation (e.g., with a suitable HDR template that delivers a coding sequence for β-globin, advantageously non-sickling β-globin); specifically, the guide RNA can target mutation that give rise to sickle cell anemia, and the HDR can provide coding for proper expression of β-globin. An guide RNA that targets the mutation-and-Cas protein containing particle is contacted with HSCs carrying the mutation. The particle also can contain a suitable HDR template to correct the mutation for proper expression of β-globin; or the HSC can be contacted with a second particle or a vector that contains or delivers the HDR template. The so contacted cells can be administered; and optionally treated/expanded; cf. Cartier. The HDR template can provide for the HSC to express an engineered β-globin gene (e.g., βA-T87Q), or β-globin. 
     Treating Diseases of the Liver and Kidney 
     In some embodiments, the composition, 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 composition, 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 composition and system 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 recombination 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 ash.confex.com/ash/2015/webprogram/Paper86495.html and presented on Dec. 6th, 2015) which can be adapted for use with the compositions, 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 composition and system described herein can be a lung or epithelial disease. The compositions and systems described herein can be used for treating epithelial and/or lung diseases. The present invention also contemplates delivering the composition, system, described herein, to one or both lungs. 
     In some embodiments, as viral vector can be used to deliver the composition, 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, 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 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 compositions and systems described herein can be used for the treatment of skin diseases. The present invention also contemplates delivering the composition and system, described herein, to the skin. 
     In some embodiments, delivery to the skin (intradermal delivery) of the composition, 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 composition, system, described herein, for example, at a dosage of up to 300 μl of 0.1 mg/ml CRISPR-Cas 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 compositions, systems, described herein can be used for the treatment of cancer. The present invention also contemplates delivering the composition, system, described herein, to a cancer cell. Also, as is described elsewhere herein the compositions, 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 International Patent Publication No. WO 2015/161276, 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 2 and 3. In some embodiments, target genes for cancer treatment and prevention can also include those described in International Patent Publication No. WO 2015/048577 the disclosure of which is hereby incorporated by reference and can be adapted for and/or applied to the composition, system, described herein. 
     Adoptive Cell Therapy 
     The compositions, systems, and components thereof described herein can be used to modify cells for an adoptive cell therapy. In an aspect of the invention, methods and compositions which involve editing a target nucleic acid sequence, or modulating expression of a target nucleic acid sequence, and applications thereof in connection with cancer immunotherapy are comprehended by adapting the composition, system, of the present invention. In some examples, the compositions, systems, and methods may be used to modify a stem cell (e.g., induced pluripotent cell) to derive modified natural killer cells, gamma delta T cells, and alpha beta T cells, which can be used for the adoptive cell therapy. In certain examples, the compositions, systems, and methods may be used to modify modified natural killer cells, gamma delta T cells, and alpha beta T cells. 
     As used herein, “ACT”, “adoptive cell therapy” and “adoptive cell transfer” may be used interchangeably. In certain embodiments, Adoptive cell therapy (ACT) can refer to the transfer of cells to a patient with the goal of transferring the functionality and characteristics into the new host by engraftment of the cells (see, e.g., Mettananda et al., Editing an α-globin enhancer in primary human hematopoietic stem cells as a treatment for β-thalassemia, Nat Commun. Sep. 4, 2017; 8(1):424). As used herein, the term “engraft” or “engraftment” refers to the process of cell incorporation into a tissue of interest in vivo through contact with existing cells of the tissue. Adoptive cell therapy (ACT) can refer to the transfer of cells, most commonly immune-derived cells, back into the same patient or into a new recipient host with the goal of transferring the immunologic functionality and characteristics into the new host. If possible, use of autologous cells helps the recipient by minimizing GVHD issues. The adoptive transfer of autologous tumor infiltrating lymphocytes (TIL) (Zacharakis et al., (2018) Nat Med. 2018 June; 24(6):724-730; Besser et al., (2010) Clin. Cancer Res 16 (9) 2646-55; Dudley et al., (2002) Science 298 (5594): 850-4; and Dudley et al., (2005) Journal of Clinical Oncology 23 (10): 2346-57) or genetically re-directed peripheral blood mononuclear cells (Johnson et al., (2009) Blood 114 (3): 535-46; and Morgan et al., (2006) Science 314(5796) 126-9) has been used to successfully treat patients with advanced solid tumors, including melanoma, metastatic breast cancer and colorectal carcinoma, as well as patients with CD19-expressing hematologic malignancies (Kalos et al., (2011) Science Translational Medicine 3 (95): 95rα73). In certain embodiments, allogenic cells immune cells are transferred (see, e.g., Ren et al., (2017) Clin Cancer Res 23 (9) 2255-2266). As described further herein, allogenic cells can be edited to reduce alloreactivity and prevent graft-versus-host disease. Thus, use of allogenic cells allows for cells to be obtained from healthy donors and prepared for use in patients as opposed to preparing autologous cells from a patient after diagnosis. 
     Aspects of the invention involve the adoptive transfer of immune system cells, such as T cells, specific for selected antigens, such as tumor associated antigens or tumor specific neoantigens (see, e.g., 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; 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; and Rajasagi et al., 2014, Systematic identification of personal tumor-specific neoantigens in chronic lymphocytic leukemia. Blood. Jul. 17, 2014; 124(3):453-62). 
     In certain embodiments, an antigen (such as a tumor antigen) to be targeted in adoptive cell therapy (such as particularly CAR or TCR T-cell therapy) of a disease (such as particularly of tumor or cancer) may be selected from a group consisting of: MR1 (see, e.g., Crowther, et al., 2020, Genome-wide CRISPR-Cas9 screening reveals ubiquitous T cell cancer targeting via the monomorphic MHC class I-related protein MR1, Nature Immunology volume 21, pages 178-185), B cell maturation antigen (BCMA) (see, e.g., Friedman et al., Effective Targeting of Multiple BCMA-Expressing Hematological Malignancies by Anti-BCMA CAR T Cells, Hum Gene Ther. Mar. 8, 2018; Berdeja J G, et al. Durable clinical responses in heavily pretreated patients with relapsed/refractory multiple myeloma: updated results from a multicenter study of bb2121 anti-Bcma CAR T cell therapy. Blood. 2017; 130:740; and Mouhieddine and Ghobrial, Immunotherapy in Multiple Myeloma: The Era of CAR T Cell Therapy, Hematologist, May-June 2018, Volume 15, issue 3); PSA (prostate-specific antigen); prostate-specific membrane antigen (PSMA); PSCA (Prostate stem cell antigen); Tyrosine-protein kinase transmembrane receptor ROR1; fibroblast activation protein (FAP); Tumor-associated glycoprotein 72 (TAG72); Carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule (EPCAM); Mesothelin; Human Epidermal growth factor Receptor 2 (ERBB2 (Her2/neu)); Prostate; Prostatic acid phosphatase (PAP); elongation factor 2 mutant (ELF2M); Insulin-like growth factor 1 receptor (IGF-1R); gp1OO; BCR-ABL (breakpoint cluster region-Abelson); tyrosinase; New York esophageal squamous cell carcinoma 1 (NY-ESO-1); κ-light chain, LAGE (L antigen); MAGE (melanoma antigen); Melanoma-associated antigen 1 (MAGE-A1); MAGE A3; MAGE A6; legumain; Human papillomavirus (HPV) E6; HPV E7; prostein; survivin; PCTA1 (Galectin 8); Melan-A/MART-1; Ras mutant; TRP-1 (tyrosinase related protein 1, or gp75); Tyrosinase-related Protein 2 (TRP2); TRP-2/INT2 (TRP-2/intron 2); RAGE (renal antigen); receptor for advanced glycation end products 1 (RAGE1); Renal ubiquitous 1, 2 (RU1, RU2); intestinal carboxyl esterase (iCE); Heat shock protein 70-2 (HSP70-2) mutant; thyroid stimulating hormone receptor (TSHR); CD123; CD171; CD19; CD20; CD22; CD26; CD30; CD33; CD44v7/8 (cluster of differentiation 44, exons 7/8); CD53; CD92; CD100; CD148; CD150; CD200; CD261; CD262; CD362; CS-1 (CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); Tn antigen (Tn Ag); Fms-Like Tyrosine Kinase 3 (FLT3); CD38; CD138; CD44v6; B7H3 (CD276); KIT (CD117); Interleukin-13 receptor subunit alpha-2 (IL-13Rα2); Interleukin 11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); Protease Serine 21 (PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis(Y) antigen; CD24; Platelet-derived growth factor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4 (SSEA-4); Mucin 1, cell surface associated (MUC1); mucin 16 (MUC16); epidermal growth factor receptor (EGFR); epidermal growth factor receptor variant III (EGFRvIII); neural cell adhesion molecule (NCAM); carbonic anhydrase IX (CAIX); Proteasome (Prosome, Macropain) Subunit, Beta Type, 9 (LMP2); ephrin type-A receptor 2 (EphA2); Ephrin B2; Fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); TGS5; high molecular weight-melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); Folate receptor alpha; Folate receptor beta; tumor endothelial marker 1 (TEM1/CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); G protein-coupled receptor class C group 5, member D (GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); Polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide portion of globoH glycoceramide (GloboH); mammary gland differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1 (HAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K 9 (LY6K); Olfactory receptor 51E2 (OR51E2); TCR Gamma Alternate Reading Frame Protein (TARP); Wilms tumor protein (WT1); ETS translocation-variant gene 6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X Antigen Family, Member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); CT (cancer/testis (antigen)); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; p53; p53 mutant; human Telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-Acetyl glucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3); Androgen receptor; Cyclin B1; Cyclin D1; v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN); Ras Homolog Family Member C (RhoC); Cytochrome P450 1B1 (CYP1B1); CCCTC-Binding Factor (Zinc Finger Protein)-Like (BORIS); Squamous Cell Carcinoma Antigen Recognized By T Cells-1 or 3 (SART1, SART3); Paired box protein Pax-5 (PAX5); proacrosin binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint-1, -2, -3 or -4 (SSX1, SSX2, SSX3, SSX4); CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR); Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); mouse double minute 2 homolog (MDM2); livin; alphafetoprotein (AFP); transmembrane activator and CAML Interactor (TACI); B-cell activating factor receptor (BAFF-R); V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS); immunoglobulin lambda-like polypeptide 1 (IGLL1); 707-AP (707 alanine proline); ART-4 (adenocarcinoma antigen recognized by T4 cells); BAGE (B antigen; b-catenin/m, b-catenin/mutated); CAMEL (CTL-recognized antigen on melanoma); CAP1 (carcinoembryonic antigen peptide 1); CASP-8 (caspase-8); CDC27m (cell-division cycle 27 mutated); CDK4/m (cycline-dependent kinase 4 mutated); Cyp-B (cyclophilin B); DAM (differentiation antigen melanoma); EGP-2 (epithelial glycoprotein 2); EGP-40 (epithelial glycoprotein 40); Erbb2, 3, 4 (erythroblastic leukemia viral oncogene homolog-2, -3, 4); FBP (folate binding protein); fAchR (Fetal acetylcholine receptor); G250 (glycoprotein 250); GAGE (G antigen); GnT-V (N-acetylglucosaminyltransferase V); HAGE (helicose antigen); ULA-A (human leukocyte antigen-A); HST2 (human signet ring tumor 2); KIAA0205; KDR (kinase insert domain receptor); LDLR/FUT (low density lipid receptor/GDP L-fucose: b-D-galactosidase 2-a-L fucosyltransferase); L1 CAM (L1 cell adhesion molecule); MC1R (melanocortin 1 receptor); Myosin/m (myosin mutated); MUM-1, -2, -3 (melanoma ubiquitous mutated 1, 2, 3); NA88-A (NA cDNA clone of patient M88); KG2D (Natural killer group 2, member D) ligands; oncofetal antigen (h5T4); p190 minor bcr-abl (protein of 190KD bcr-abl); Pml/RARa (promyelocytic leukemia/retinoic acid receptor a); PRAME (preferentially expressed antigen of melanoma); SAGE (sarcoma antigen); TEL/AML1 (translocation Ets-family leukemia/acute myeloid leukemia 1); TPI/m (triosephosphate isomerase mutated); CD70; and any combination thereof. 
     In certain embodiments, an antigen to be targeted in adoptive cell therapy (such as particularly CAR or TCR T-cell therapy) of a disease (such as particularly of tumor or cancer) is a tumor-specific antigen (TSA). 
     In certain embodiments, an antigen to be targeted in adoptive cell therapy (such as particularly CAR or TCR T-cell therapy) of a disease (such as particularly of tumor or cancer) is a neoantigen. 
     In certain embodiments, an antigen to be targeted in adoptive cell therapy (such as particularly CAR or TCR T-cell therapy) of a disease (such as particularly of tumor or cancer) is a tumor-associated antigen (TAA). 
     In certain embodiments, an antigen to be targeted in adoptive cell therapy (such as particularly CAR or TCR T-cell therapy) of a disease (such as particularly of tumor or cancer) is a universal tumor antigen. In certain preferred embodiments, the universal tumor antigen is selected from the group consisting of: a human telomerase reverse transcriptase (hTERT), survivin, mouse double minute 2 homolog (MDM2), cytochrome P450 1B1 (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, cyclin (Dl), and any combinations thereof. 
     In certain embodiments, an antigen (such as a tumor antigen) to be targeted in adoptive cell therapy (such as particularly CAR or TCR T-cell therapy) of a disease (such as particularly of tumor or cancer) may be selected from a group consisting of: CD19, BCMA, CD70, CLL-1, MAGE A3, MAGE A6, HPV E6, HPV E7, WT1, CD22, CD171, ROR1, MUC16, and SSX2. In certain preferred embodiments, the antigen may be CD19. For example, CD19 may be targeted in hematologic malignancies, such as in lymphomas, more particularly in B-cell lymphomas, such as without limitation in diffuse large B-cell lymphoma, primary mediastinal b-cell lymphoma, transformed follicular lymphoma, marginal zone lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia including adult and pediatric ALL, non-Hodgkin lymphoma, indolent non-Hodgkin lymphoma, or chronic lymphocytic leukemia. For example, BCMA may be targeted in multiple myeloma or plasma cell leukemia (see, e.g., 2018 American Association for Cancer Research (AACR) Annual meeting Poster: Allogeneic Chimeric Antigen Receptor T Cells Targeting B Cell Maturation Antigen). For example, CLL1 may be targeted in acute myeloid leukemia. For example, MAGE A3, MAGE A6, SSX2, and/or KRAS may be targeted in solid tumors. For example, HPV E6 and/or HPV E7 may be targeted in cervical cancer or head and neck cancer. For example, WT1 may be targeted in acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), chronic myeloid leukemia (CIVIL), non-small cell lung cancer, breast, pancreatic, ovarian or colorectal cancers, or mesothelioma. For example, CD22 may be targeted in B cell malignancies, including non-Hodgkin lymphoma, diffuse large B-cell lymphoma, or acute lymphoblastic leukemia. For example, CD171 may be targeted in neuroblastoma, glioblastoma, or lung, pancreatic, or ovarian cancers. For example, ROR1 may be targeted in ROR1+ malignancies, including non-small cell lung cancer, triple negative breast cancer, pancreatic cancer, prostate cancer, ALL, chronic lymphocytic leukemia, or mantle cell lymphoma. For example, MUC16 may be targeted in MUC16ecto+ epithelial ovarian, fallopian tube or primary peritoneal cancer. For example, CD70 may be targeted in both hematologic malignancies as well as in solid cancers such as renal cell carcinoma (RCC), gliomas (e.g., GBM), and head and neck cancers (HNSCC). CD70 is expressed in both hematologic malignancies as well as in solid cancers, while its expression in normal tissues is restricted to a subset of lymphoid cell types (see, e.g., 2018 American Association for Cancer Research (AACR) Annual meeting Poster: Allogeneic CRISPR Engineered Anti-CD70 CAR-T Cells Demonstrate Potent Preclinical Activity Against Both Solid and Hematological Cancer Cells). 
     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 a 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 WO 9215322). 
     In general, CARs are comprised of an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises an antigen-binding domain that is specific for a predetermined target. While the antigen-binding domain of a CAR is often an antibody or antibody fragment (e.g., a single chain variable fragment, scFv), the binding domain is not particularly limited so long as it results in specific recognition of a target. For example, in some embodiments, the antigen-binding domain may comprise a receptor, such that the CAR is capable of binding to the ligand of the receptor. Alternatively, the antigen-binding domain may comprise a ligand, such that the CAR is capable of binding the endogenous receptor of that ligand. 
     The antigen-binding domain of a CAR is generally separated from the transmembrane domain by a hinge or spacer. The spacer is also not particularly limited, and it is designed to provide the CAR with flexibility. For example, a spacer domain may comprise a portion of a human Fc domain, including a portion of the CH3 domain, or the hinge region of any immunoglobulin, such as IgA, IgD, IgE, IgG, or IgM, or variants thereof. Furthermore, the hinge region may be modified so as to prevent off-target binding by FcRs or other potential interfering objects. For example, the hinge may comprise an IgG4 Fc domain with or without a S228P, L235E, and/or N297Q mutation (according to Kabat numbering) in order to decrease binding to FcRs. Additional spacers/hinges include, but are not limited to, CD4, CD8, and CD28 hinge regions. 
     The transmembrane domain of a CAR may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane bound or transmembrane protein. Transmembrane regions of particular use in this disclosure may be derived from CD8, CD28, CD3, CD45, CD4, CD5, CDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, TCR. Alternatively, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. Preferably a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine doublet provides a particularly suitable linker. 
     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/OX40/4-1BB-CD3; 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 CD3-chain, CD97, GDI la-CD18, CD2, ICOS, CD27, CD154, CDS, OX40, 4-1BB, CD2, CD7, LIGHT, LFA-1, NKG2C, B7-H3, CD30, CD40, PD-1, or CD28 signaling domains (for example scFv-CD28-4-1BB-CD3ζ or scFv-CD28-OX40-CD3; see U.S. Pat. Nos. 8,906,682; 8,399,645; 5,686,281; PCT Publication No. WO 2014/134165; PCT Publication No. WO 2012/079000). In certain embodiments, the primary signaling domain comprises a functional signaling domain of a protein selected from the group consisting of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, common FcR gamma (FCERIG), FcR beta (Fc Epsilon R1b), CD79a, CD79b, Fc gamma RIIa, DAP10, and DAP12. In certain preferred embodiments, the primary signaling domain comprises a functional signaling domain of CD3ζ or FcRγ. In certain embodiments, the one or more costimulatory signaling domains comprise a functional signaling domain of a protein selected, each independently, from the group consisting of: CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, and NKG2D. In certain embodiments, the one or more costimulatory signaling domains comprise a functional signaling domain of a protein selected, each independently, from the group consisting of: 4-1BB, CD27, and CD28. In certain embodiments, a chimeric antigen receptor may have the design as described in U.S. Pat. No. 7,446,190, comprising an intracellular domain of CD3 chain (such as amino acid residues 52-163 of the human CD3 zeta chain, as shown in SEQ ID NO: 14 of U.S. Pat. No. 7,446,190), a signaling region from CD28 and an antigen-binding element (or portion or domain; such as scFv). The CD28 portion, when between the zeta chain portion and the antigen-binding element, may suitably include the transmembrane and signaling domains of CD28 (such as amino acid residues 114-220 of SEQ ID NO: 10, full sequence shown in SEQ ID NO: 6 of U.S. Pat. No. 7,446,190; these can include the following portion of CD28 as set forth in Genbank identifier NM 006139. Alternatively, when the zeta sequence lies between the CD28 sequence and the antigen-binding element, intracellular domain of CD28 can be used alone (such as amino sequence set forth in SEQ ID NO: 9 of U.S. Pat. No. 7,446,190). Hence, certain embodiments employ a CAR comprising (a) a zeta chain portion comprising the intracellular domain of human CD3ζ chain, (b) a costimulatory signaling region, and (c) an antigen-binding element (or portion or domain), wherein the costimulatory signaling region comprises the amino acid sequence encoded by SEQ ID NO: 6 of U.S. Pat. No. 7,446,190. 
     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 
     By means of an example and without limitation, Kochenderfer et al., (2009) J Immunother. 32 (7): 689-702 described anti-CD19 chimeric antigen receptors (CAR). FMC63-28Z CAR contained a single chain variable region moiety (scFv) recognizing CD19 derived from the FMC63 mouse hybridoma (described in Nicholson et al., (1997) Molecular Immunology 34: 1157-1165), a portion of the human CD28 molecule, and the intracellular component of the human TCR-molecule. FMC63-CD828BBZ CAR contained the FMC63 scFv, the hinge and transmembrane regions of the CD8 molecule, the cytoplasmic portions of CD28 and 4-1BB, and the cytoplasmic component of the TCR-t molecule. The exact sequence of the CD28 molecule included in the FMC63-28Z CAR corresponded to Genbank identifier NM_006139; the sequence included all amino acids starting with the amino acid sequence IEVMYPPPY (SEQ. I.D. No. 2) and continuing all the way to the carboxy-terminus of the protein. To encode the anti-CD19 scFv component of the vector, the authors designed a DNA sequence which was based on a portion of a previously published CAR (Cooper et al., (2003) Blood 101: 1637-1644). This sequence encoded the following components in frame from the 5′ end to the 3′ end: an XhoI site, the human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor α-chain signal sequence, the FMC63 light chain variable region (as in Nicholson et al., supra), a linker peptide (as in Cooper et al., supra), the FMC63 heavy chain variable region (as in Nicholson et al., supra), and a NotI site. A plasmid encoding this sequence was digested with XhoI and NotI. To form the MSGV-FMC63-28Z retroviral vector, the XhoI and NotI-digested fragment encoding the FMC63 scFv was ligated into a second XhoI and NotI-digested fragment that encoded the MSGV retroviral backbone (as in Hughes et al., (2005) Human Gene Therapy 16: 457-472) as well as part of the extracellular portion of human CD28, the entire transmembrane and cytoplasmic portion of human CD28, and the cytoplasmic portion of the human TCR-molecule (as in Maher et al., 2002) Nature Biotechnology 20: 70-75). The FMC63-28Z CAR is included in the KTE-C19 (axicabtagene ciloleucel) anti-CD19 CAR-T therapy product in development by Kite Pharma, Inc. for the treatment of inter alia patients with relapsed/refractory aggressive B-cell non-Hodgkin lymphoma (NHL). Accordingly, in certain embodiments, cells intended for adoptive cell therapies, more particularly immunoresponsive cells such as T cells, may express the FMC63-28Z CAR as described by Kochenderfer et al. (supra). Hence, in certain embodiments, cells intended for adoptive cell therapies, more particularly immunoresponsive cells such as T cells, may comprise a CAR comprising an extracellular antigen-binding element (or portion or domain; such as scFv) that specifically binds to an antigen, an intracellular signaling domain comprising an intracellular domain of a CD3ζ chain, and a costimulatory signaling region comprising a signaling domain of CD28. Preferably, the CD28 amino acid sequence is as set forth in Genbank identifier NM_006139 (sequence version 1,2 or 3) starting with the amino acid sequence IEVMYPPPY and continuing all the way to the carboxy-terminus of the protein. Preferably, the antigen is CD19, more preferably the antigen-binding element is an anti-CD19 scFv, even more preferably the anti-CD19 scFv as described by Kochenderfer et al. (supra). 
     Additional anti-CD19 CARs are further described in International Patent Publication No. WO 2015/187528. More particularly Example 1 and Table 1 of WO2015187528, incorporated by reference herein, demonstrate the generation of anti-CD19 CARs based on a fully human anti-CD19 monoclonal antibody (47G4, as described in US20100104509) and murine anti-CD19 monoclonal antibody (as described in Nicholson et al. and explained above). Various combinations of a signal sequence (human CD8-alpha or GM-CSF receptor), extracellular and transmembrane regions (human CD8-alpha) and intracellular T-cell signaling domains (CD28-CD3ζ; 4-1BB-CD3ζ; CD27-CD3ζ; CD28-CD27-CD3ζ, 4-1BB-CD27-CD3ζ; CD27-4-1BB-CD3ζ; CD28-CD27-FcεRI gamma chain; or CD28-FcεRI gamma chain) were disclosed. Hence, in certain embodiments, cells intended for adoptive cell therapies, more particularly immunoresponsive cells such as T cells, may comprise a CAR comprising an extracellular antigen-binding element that specifically binds to an antigen, an extracellular and transmembrane region as set forth in Table 1 of WO2015187528 and an intracellular T-cell signaling domain as set forth in Table 1 of No. WO 2015/187528. Preferably, the antigen is CD19, more preferably the antigen-binding element is an anti-CD19 scFv, even more preferably the mouse or human anti-CD19 scFv as described in Example 1 of. WO 2015/187528. In certain embodiments, the CAR comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13 as set forth in Table 1 of WO2015187528. 
     By means of an example and without limitation, chimeric antigen receptor that recognizes the CD70 antigen is described in WO2012058460A2 (see also, Park et al., CD70 as a target for chimeric antigen receptor T cells in head and neck squamous cell carcinoma, Oral Oncol. 2018 March; 78:145-150; and Jin et al., CD70, a novel target of CAR T-cell therapy for gliomas, Neuro Oncol. Jan. 10, 2018; 20(1):55-65). CD70 is expressed by diffuse large B-cell and follicular lymphoma and also by the malignant cells of Hodgkins lymphoma, Waldenstrom&#39;s macroglobulinemia and multiple myeloma, and by HTLV-1- and EBV-associated malignancies. (Agathanggelou et al. Am.J.Pathol. 1995; 147: 1152-1160; Hunter et al., Blood 2004; 104:4881. 26; Lens et al., J Immunol. 2005; 174:6212-6219; Baba et al., J Virol. 2008; 82:3843-3852.) In addition, CD70 is expressed by non-hematological malignancies such as renal cell carcinoma and glioblastoma. (Junker et al., J Urol. 2005; 173:2150-2153; Chahlavi et al., Cancer Res 2005; 65:5428-5438) Physiologically, CD70 expression is transient and restricted to a subset of highly activated T, B, and dendritic cells. 
     By means of an example and without limitation, chimeric antigen receptor that recognizes BCMA has been described (see, e.g., US20160046724A1; WO2016014789A2; WO2017211900A1; WO2015158671A1; US20180085444A1; WO2018028647A1; US20170283504A1; and WO2013154760A1). 
     In certain embodiments, the immune cell may, in addition to a CAR or exogenous TCR as described herein, further comprise a chimeric inhibitory receptor (inhibitory CAR) that specifically binds to a second target antigen and is capable of inducing an inhibitory or immunosuppressive or repressive signal to the cell upon recognition of the second target antigen. In certain embodiments, the chimeric inhibitory receptor comprises an extracellular antigen-binding element (or portion or domain) configured to specifically bind to a target antigen, a transmembrane domain, and an intracellular immunosuppressive or repressive signaling domain. In certain embodiments, the second target antigen is an antigen that is not expressed on the surface of a cancer cell or infected cell or the expression of which is downregulated on a cancer cell or an infected cell. In certain embodiments, the second target antigen is an MHC-class I molecule. In certain embodiments, the intracellular signaling domain comprises a functional signaling portion of an immune checkpoint molecule, such as for example PD-1 or CTLA4. Advantageously, the inclusion of such inhibitory CAR reduces the chance of the engineered immune cells attacking non-target (e.g., non-cancer) tissues. 
     Alternatively, T-cells expressing CARs may be further modified to reduce or eliminate expression of endogenous TCRs in order to reduce off-target effects. Reduction or elimination of endogenous TCRs can reduce off-target effects and increase the effectiveness of the T cells (U.S. Pat. No. 9,181,527). T cells stably lacking expression of a functional TCR may be produced using a variety of approaches. T cells internalize, sort, and degrade the entire T cell receptor as a complex, with a half-life of about 10 hours in resting T cells and 3 hours in stimulated T cells (von Essen, M. et al. 2004. J. Immunol. 173:384-393). Proper functioning of the TCR complex requires the proper stoichiometric ratio of the proteins that compose the TCR complex. TCR function also requires two functioning TCR zeta proteins with ITAM motifs. The activation of the TCR upon engagement of its MHC-peptide ligand requires the engagement of several TCRs on the same T cell, which all must signal properly. Thus, if a TCR complex is destabilized with proteins that do not associate properly or cannot signal optimally, the T cell will not become activated sufficiently to begin a cellular response. 
     Accordingly, in some embodiments, TCR expression may eliminated using RNA interference (e.g., shRNA, siRNA, miRNA, etc.), CRISPR, or other methods that target the nucleic acids encoding specific TCRs (e.g., TCR-α and TCR-β) and/or CD3 chains in primary T cells. By blocking expression of one or more of these proteins, the T cell will no longer produce one or more of the key components of the TCR complex, thereby destabilizing the TCR complex and preventing cell surface expression of a functional TCR. 
     In some instances, CAR may also comprise a switch mechanism for controlling expression and/or activation of the CAR. For example, a CAR may comprise an extracellular, transmembrane, and intracellular domain, in which the extracellular domain comprises a target-specific binding element that comprises a label, binding domain, or tag that is specific for a molecule other than the target antigen that is expressed on or by a target cell. In such embodiments, the specificity of the CAR is provided by a second construct that comprises a target antigen binding domain (e.g., an scFv or a bispecific antibody that is specific for both the target antigen and the label or tag on the CAR) and a domain that is recognized by or binds to the label, binding domain, or tag on the CAR. See, e.g., WO 2013/044225, WO 2016/000304, WO 2015/057834, WO 2015/057852, WO 2016/070061, U.S. Pat. No. 9,233,125, US 2016/0129109. In this way, a T-cell that expresses the CAR can be administered to a subject, but the CAR cannot bind its target antigen until the second composition comprising an antigen-specific binding domain is administered. 
     Alternative switch mechanisms include CARs that require multimerization in order to activate their signaling function (see, e.g., US patent Publication Nos. US 2015/0368342, US 2016/0175359, US 2015/0368360) and/or an exogenous signal, such as a small molecule drug (US 2016/0166613, Yung et al., Science, 2015), in order to elicit a T-cell response. Some CARs may also comprise a “suicide switch” to induce cell death of the CAR T-cells following treatment (Buddee et al., PLoS One, 2013) or to downregulate expression of the CAR following binding to the target antigen (International Patent Publication No. WO 2016/011210). 
     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). CAR T cells of this kind may for example be used in animal models, for example to treat tumor xenografts. 
     In certain embodiments, ACT includes co-transferring CD4+ Th1 cells and CD8+ CTLs to induce a synergistic antitumor response (see, e.g., Li et al., Adoptive cell therapy with CD4+ T helper 1 cells and CD8+ cytotoxic T cells enhances complete rejection of an established tumor, leading to generation of endogenous memory responses to non-targeted tumor epitopes. Clin Transl Immunology. 2017 October; 6(10): e160). 
     In certain embodiments, Th17 cells are transferred to a subject in need thereof. Th17 cells have been reported to directly eradicate melanoma tumors in mice to a greater extent than Th1 cells (Muranski P, et al., Tumor-specific Th17-polarized cells eradicate large established melanoma. Blood. Jul. 15, 2008; 112(2):362-73; and Martin-Orozco N, et al., T helper 17 cells promote cytotoxic T cell activation in tumor immunity. Immunity. Nov. 20, 2009; 31(5):787-98). Those studies involved an adoptive T cell transfer (ACT) therapy approach, which takes advantage of CD4+ T cells that express a TCR recognizing tyrosinase tumor antigen. Exploitation of the TCR leads to rapid expansion of Th17 populations to large numbers ex vivo for reinfusion into the autologous tumor-bearing hosts. 
     In certain embodiments, ACT may include autologous iPSC-based vaccines, such as irradiated iPSCs in autologous anti-tumor vaccines (see e.g., Kooreman, Nigel G. et al., Autologous iPSC-Based Vaccines Elicit Anti-tumor Responses In Vivo, Cell Stem Cell 22, 1-13, 2018, doi.org/10.1016/j.stem.2018.01.016). 
     Unlike T-cell receptors (TCRs) that are MHC restricted, CARs can potentially bind any cell surface-expressed antigen and can thus be more universally used to treat patients (see Irving et al., Engineering Chimeric Antigen Receptor T-Cells for Racing in Solid Tumors: Don&#39;t Forget the Fuel, Front. Immunol., 3 Apr. 2017, doi.org/10.3389/fimmu.2017.00267). In certain embodiments, in the absence of endogenous T-cell infiltrate (e.g., due to aberrant antigen processing and presentation), which precludes the use of TIL therapy and immune checkpoint blockade, the transfer of CAR T-cells may be used to treat patients (see, e.g., Hinrichs C S, Rosenberg S A. Exploiting the curative potential of adoptive T-cell therapy for cancer. Immunol Rev (2014) 257(1):56-71. doi:10.1111/imr.12132). 
     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). 
     In certain embodiments, the treatment can be administered after lymphodepleting pretreatment in the form of chemotherapy (typically a combination of cyclophosphamide and fludarabine) or radiation therapy. Initial studies in ACT had short lived responses and the transferred cells did not persist in vivo for very long (Houot et al., T-cell-based immunotherapy: adoptive cell transfer and checkpoint inhibition. Cancer Immunol Res (2015) 3(10):1115-22; and Kamta et al., Advancing Cancer Therapy with Present and Emerging Immuno-Oncology Approaches. Front. Oncol. (2017) 7:64). Immune suppressor cells like Tregs and MDSCs may attenuate the activity of transferred cells by outcompeting them for the necessary cytokines. Not being bound by a theory lymphodepleting pretreatment may eliminate the suppressor cells allowing the TILs to persist. 
     In one embodiment, the treatment can be administrated into patients undergoing an immunosuppressive treatment (e.g., glucocorticoid 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. In certain embodiments, the immunosuppressive treatment provides for the selection and expansion of the immunoresponsive T cells within the patient. 
     In certain embodiments, the treatment can be administered before primary treatment (e.g., surgery or radiation therapy) to shrink a tumor before the primary treatment. In another embodiment, the treatment can be administered after primary treatment to remove any remaining cancer cells. 
     In certain embodiments, immunometabolic barriers can be targeted therapeutically prior to and/or during ACT to enhance responses to ACT or CAR T-cell therapy and to support endogenous immunity (see, e.g., Irving et al., Engineering Chimeric Antigen Receptor T-Cells for Racing in Solid Tumors: Don&#39;t Forget the Fuel, Front. Immunol., Apr. 3, 2017, doi.org/10.3389/fimmu.2017.00267). 
     The administration of cells or population of cells, such as immune system cells or cell populations, such as more particularly immunoresponsive cells or cell populations, as disclosed herein 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, intrathecally, by intravenous or intralymphatic injection, or intraperitoneally. In some embodiments, the disclosed CARs may be delivered or administered into a cavity formed by the resection of tumor tissue (i.e. intracavity delivery) or directly into a tumor prior to resection (i.e. intratumoral delivery). 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 CART 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; International Patent Publication WO 2011/146862; International Patent Publication WO 2014/011987; International Patent Publication WO 2013/040371; 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 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; Ren et al., 2017, Multiplex genome editing to generate universal CAR T cells resistant to PD1 inhibition, Clin Cancer Res. May 1, 2017; 23(9):2255-2266. doi: 10.1158/1078-0432.CCR-16-1300. Epub Nov. 4, 2016; Qasim et al., 2017, Molecular remission of infant B-ALL after infusion of universal TALEN gene-edited CAR T cells, Sci Transl Med. Jan. 25, 2017; 9(374); Legut, et al., 2018, CRISPR-mediated TCR replacement generates superior anticancer transgenic T cells. Blood, 131(3), 311-322; and Georgiadis et al., Long Terminal Repeat CRISPR-CAR-Coupled “Universal” T Cells Mediate Potent Anti-leukemic Effects, Molecular Therapy, In Press, Corrected Proof, Available online Mar. 6, 2018). Cells may be edited using any CRISPR system and method of use thereof as described herein. The composition and 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 for example to insert or knock-in an exogenous gene, such as an exogenous gene encoding a CAR or a TCR, at a preselected locus in a cell (e.g. TRAC locus); to eliminate potential alloreactive T-cell receptors (TCR) or to prevent inappropriate pairing between endogenous and exogenous TCR chains, such as to knock-out or knock-down expression of an endogenous TCR in a cell; to disrupt the target of a chemotherapeutic agent in a cell; to block an immune checkpoint, such as to knock-out or knock-down expression of an immune checkpoint protein or receptor in a cell; to knock-out or knock-down expression of other gene or genes in a cell, the reduced expression or lack of expression of which can enhance the efficacy of adoptive therapies using the cell; to knock-out or knock-down expression of an endogenous gene in a cell, said endogenous gene encoding an antigen targeted by an exogenous CAR or TCR; to knock-out or knock-down expression of one or more MHC constituent proteins in a cell; to activate a T cell; to modulate cells such that the cells are resistant to exhaustion or dysfunction; and/or increase the differentiation and/or proliferation of functionally exhausted or dysfunctional CD8+ T-cells (see International Patent Publication Nos. WO 2013/176915, WO 2014/059173, WO 2014/172606, WO 2014/184744, and WO 2014/191128). 
     In certain embodiments, editing may result in inactivation of a gene. By inactivating a gene, it is intended that the gene of interest is not expressed in a functional protein form. In a particular embodiment, the system specifically catalyzes cleavage in one targeted gene thereby inactivating said targeted gene. The nucleic acid strand breaks caused are commonly repaired through the distinct mechanisms of homologous recombination or non-homologous end joining (NHEJ). However, NHEJ is an imperfect repair process that often results in changes to the DNA sequence at the site of the cleavage. Repair via non-homologous end joining (NHEJ) often results in small insertions or deletions (Indel) and can be used for the creation of specific gene knockouts. Cells in which a cleavage induced mutagenesis event has occurred can be identified and/or selected by well-known methods in the art. In certain embodiments, homology directed repair (HDR) is used to concurrently inactivate a gene (e.g., TRAC) and insert an endogenous TCR or CAR into the inactivated locus. 
     Hence, in certain embodiments, editing of cells, particularly cells intended for adoptive cell therapies, more particularly immunoresponsive cells such as T cells, may be performed to insert or knock-in an exogenous gene, such as an exogenous gene encoding a CAR or a TCR, at a preselected locus in a cell. Conventionally, nucleic acid molecules encoding CARs or TCRs are transfected or transduced to cells using randomly integrating vectors, which, depending on the site of integration, may lead to clonal expansion, oncogenic transformation, variegated transgene expression and/or transcriptional silencing of the transgene. Directing of transgene(s) to a specific locus in a cell can minimize or avoid such risks and advantageously provide for uniform expression of the transgene(s) by the cells. Without limitation, suitable ‘safe harbor’ loci for directed transgene integration include CCR5 or AAVS1. Homology-directed repair (HDR) strategies are known and described elsewhere in this specification allowing to insert transgenes into desired loci (e.g., TRAC locus). 
     Further suitable loci for insertion of transgenes, in particular CAR or exogenous TCR transgenes, include without limitation loci comprising genes coding for constituents of endogenous T-cell receptor, such as T-cell receptor alpha locus (TRA) or T-cell receptor beta locus (TRB), for example T-cell receptor alpha constant (TRAC) locus, T-cell receptor beta constant 1 (TRBC1) locus or T-cell receptor beta constant 2 (TRBC1) locus. Advantageously, insertion of a transgene into such locus can simultaneously achieve expression of the transgene, potentially controlled by the endogenous promoter, and knock-out expression of the endogenous TCR. This approach has been exemplified in Eyquem et al., (2017) Nature 543: 113-117, wherein the authors used CRISPR/Cas9 gene editing to knock-in a DNA molecule encoding a CD19-specific CAR into the TRAC locus downstream of the endogenous promoter; the CAR-T cells obtained by CRISPR were significantly superior in terms of reduced tonic CAR signaling and exhaustion. 
     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 (3, 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. 
     Hence, in certain embodiments, editing of cells, particularly cells intended for adoptive cell therapies, more particularly immunoresponsive cells such as T cells, may be performed to knock-out or knock-down expression of an endogenous TCR in a cell. For example, NHEJ-based or HDR-based gene editing approaches can be employed to disrupt the endogenous TCR alpha and/or beta chain genes. For example, gene editing system or systems, such as CRISPR/Cas system or systems, can be designed to target a sequence found within the TCR beta chain conserved between the beta 1 and beta 2 constant region genes (TRBC1 and TRBC2) and/or to target the constant region of the TCR alpha chain (TRAC) gene. 
     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. 
     In certain embodiments, editing of cells, particularly cells intended for adoptive cell therapies, more particularly immunoresponsive cells such as T cells, may be performed to block an immune checkpoint, such as to knock-out or knock-down expression of an immune checkpoint protein or receptor in a cell. 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. Apr. 15, 2016; 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). 
     International Patent Publication No. WO 2014/172606 relates to the use of MT1 and/or MT2 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, TIM-3, CEACAM-1, CEACAM-3, or CEACAM-5. 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. 
     By means of an example and without limitation, International Patent Publication No. WO 2016/196388 concerns an engineered T cell comprising (a) a genetically engineered antigen receptor that specifically binds to an antigen, which receptor may be a CAR; and (b) a disrupted gene encoding a PD-L1, an agent for disruption of a gene encoding a PD-L1, and/or disruption of a gene encoding PD-L1, wherein the disruption of the gene may be mediated by a gene editing nuclease, a zinc finger nuclease (ZFN), CRISPR/Cas9 and/or TALEN. WO2015142675 relates to immune effector cells comprising a CAR in combination with an agent (such as the composition or system herein) that increases the efficacy of the immune effector cells in the treatment of cancer, wherein the agent may inhibit an immune inhibitory molecule, such as PD1, PD-L1, CTLA-4, TIM-3, LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, TGFR beta, CEACAM-1, CEACAM-3, or CEACAM-5. Ren et al., (2017) Clin Cancer Res 23 (9) 2255-2266 performed lentiviral delivery of CAR and electro-transfer of Cas9 mRNA and gRNAs targeting endogenous TCR, β-2 microglobulin (B2M) and PD1 simultaneously, to generate gene-disrupted allogeneic CART cells deficient of TCR, HLA class I molecule and PD1. 
     In certain embodiments, cells may be engineered to express a CAR, wherein expression and/or function of methylcytosine dioxygenase genes (TET1, TET2 and/or TET3) in the cells has been reduced or eliminated, (such as the composition or system herein) (for example, as described in WO201704916). 
     In certain embodiments, editing of cells, particularly cells intended for adoptive cell therapies, more particularly immunoresponsive cells such as T cells, may be performed to knock-out or knock-down expression of an endogenous gene in a cell, said endogenous gene encoding an antigen targeted by an exogenous CAR or TCR, thereby reducing the likelihood of targeting of the engineered cells. In certain embodiments, the targeted antigen may be one or more antigen selected from the group consisting of CD38, CD138, CS-1, CD33, CD26, CD30, CD53, CD92, CD100, CD148, CD150, CD200, CD261, CD262, CD362, human telomerase reverse transcriptase (hTERT), survivin, mouse double minute 2 homolog (MDM2), cytochrome P450 1B1 (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, cyclin (D1), B cell maturation antigen (BCMA), transmembrane activator and CAML Interactor (TACI), and B-cell activating factor receptor (BAFF-R) (for example, as described in International Patent Publication Nos. WO 2016/011210 and WO 2017/011804). 
     In certain embodiments, editing of cells, particularly cells intended for adoptive cell therapies, more particularly immunoresponsive cells such as T cells, may be performed to knock-out or knock-down expression of one or more MHC constituent proteins, such as one or more HLA proteins and/or beta-2 microglobulin (B2M), in a cell, whereby rejection of non-autologous (e.g., allogeneic) cells by the recipient&#39;s immune system can be reduced or avoided. In preferred embodiments, one or more HLA class I proteins, such as HLA-A, B and/or C, and/or B2M may be knocked-out or knocked-down. Preferably, B2M may be knocked-out or knocked-down. By means of an example, Ren et al., (2017) Clin Cancer Res 23 (9) 2255-2266 performed lentiviral delivery of CAR and electro-transfer of Cas mRNA and gRNAs targeting endogenous TCR, β-2 microglobulin (B2M) and PD1 simultaneously, to generate gene-disrupted allogeneic CART cells deficient of TCR, HLA class I molecule and PD1. 
     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, B2M and TCRα, B2M and TCRβ. 
     In certain embodiments, a cell may be multiplied edited (multiplex genome editing) as taught herein to (1) knock-out or knock-down expression of an endogenous TCR (for example, TRBC1, TRBC2 and/or TRAC), (2) knock-out or knock-down expression of an immune checkpoint protein or receptor (for example PD1, PD-L 1 and/or CTLA4); and (3) knock-out or knock-down expression of one or more MHC constituent proteins (for example, HLA-A, B and/or C, and/or B2M, preferably B2M). 
     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. 
     Immune cells may be obtained using any method known in the art. In one embodiment, allogenic T cells may be obtained from healthy subjects. In one embodiment T cells that have infiltrated a tumor are isolated. T cells may be removed during surgery. T cells may be isolated after removal of tumor tissue by biopsy. T cells may be isolated by any means known in the art. In one embodiment, T cells are obtained by apheresis. In one embodiment, the method may comprise obtaining a bulk population of T cells from a tumor sample by any suitable method known in the art. For example, a bulk population of T cells can be obtained from a tumor sample by dissociating the tumor sample into a cell suspension from which specific cell populations can be selected. Suitable methods of obtaining a bulk population of T cells may include, but are not limited to, any one or more of mechanically dissociating (e.g., mincing) the tumor, enzymatically dissociating (e.g., digesting) the tumor, and aspiration (e.g., as with a needle). 
     The bulk population of T cells obtained from a tumor sample may comprise any suitable type of T cell. Preferably, the bulk population of T cells obtained from a tumor sample comprises tumor infiltrating lymphocytes (TILs). 
     The tumor sample may be obtained from any mammal. Unless stated otherwise, as used herein, the term “mammal” refers to any mammal including, but not limited to, mammals of the order Logomorpha, such as rabbits; the order Carnivora, including Felines (cats) and Canines (dogs); the order Artiodactyla, including Bovines (cows) and Swines (pigs); or of the order Perssodactyla, including Equines (horses). The mammals may be non-human primates, e.g., of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). In some embodiments, the mammal may be a mammal of the order Rodentia, such as mice and hamsters. Preferably, the mammal is a non-human primate or a human. An especially preferred mammal is the human. 
     T cells can be obtained from a number of sources, including peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, spleen tissue, and tumors. In certain embodiments of the present invention, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll separation. In one preferred embodiment, cells from the circulating blood of an individual are obtained by apheresis or leukapheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In one embodiment of the invention, the cells are washed with phosphate buffered saline (PBS). In an alternative embodiment, the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. Initial activation steps in the absence of calcium lead to magnified activation. As those of ordinary skill in the art would readily appreciate a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor) according to the manufacturer&#39;s instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS. Alternatively, the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media. 
     In another embodiment, T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient. A specific subpopulation of T cells, such as CD28+, CD4+, CDC, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques. For example, in one preferred embodiment, T cells are isolated by incubation with anti-CD3/anti-CD28 (i.e., 3×28)-conjugated beads, such as DYNABEADS® M-450 CD3/CD28 T, or XCYTE DYNABEADS™ for a time period sufficient for positive selection of the desired T cells. In one embodiment, the time period is about 30 minutes. In a further embodiment, the time period ranges from 30 minutes to 36 hours or longer and all integer values there between. In a further embodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred embodiment, the time period is 10 to 24 hours. In one preferred embodiment, the incubation time period is 24 hours. For isolation of T cells from patients with leukemia, use of longer incubation times, such as 24 hours, can increase cell yield. Longer incubation times may be used to isolate T cells in any situation where there are few T cells as compared to other cell types, such in isolating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immunocompromised individuals. Further, use of longer incubation times can increase the efficiency of capture of CD8+ T cells. 
     Enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. A preferred method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. 
     Further, monocyte populations (e.g., CD14+ cells) may be depleted from blood preparations by a variety of methodologies, including anti-CD14 coated beads or columns, or utilization of the phagocytotic activity of these cells to facilitate removal. Accordingly, in one embodiment, the invention uses paramagnetic particles of a size sufficient to be engulfed by phagocytotic monocytes. In certain embodiments, the paramagnetic particles are commercially available beads, for example, those produced by Life Technologies under the trade name Dynabeads™. In one embodiment, other non-specific cells are removed by coating the paramagnetic particles with “irrelevant” proteins (e.g., serum proteins or antibodies). Irrelevant proteins and antibodies include those proteins and antibodies or fragments thereof that do not specifically target the T cells to be isolated. In certain embodiments, the irrelevant beads include beads coated with sheep anti-mouse antibodies, goat anti-mouse antibodies, and human serum albumin. 
     In brief, such depletion of monocytes is performed by preincubating T cells isolated from whole blood, apheresed peripheral blood, or tumors with one or more varieties of irrelevant or non-antibody coupled paramagnetic particles at any amount that allows for removal of monocytes (approximately a 20:1 bead:cell ratio) for about 30 minutes to 2 hours at 22 to 37 degrees C., followed by magnetic removal of cells which have attached to or engulfed the paramagnetic particles. Such separation can be performed using standard methods available in the art. For example, any magnetic separation methodology may be used including a variety of which are commercially available, (e.g., DYNAL® Magnetic Particle Concentrator (DYNAL MPC®)). Assurance of requisite depletion can be monitored by a variety of methodologies known to those of ordinary skill in the art, including flow cytometric analysis of CD14 positive cells, before and after depletion. 
     For isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and beads. For example, in one embodiment, a concentration of 2 billion cells/ml is used. In one embodiment, a concentration of 1 billion cells/ml is used. In a further embodiment, greater than 100 million cells/ml is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used. In yet another embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/ml is used. In further embodiments, concentrations of 125 or 150 million cells/ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells, or from samples where there are many tumor cells present (i.e., leukemic blood, tumor tissue, etc). Such populations of cells may have therapeutic value and would be desirable to obtain. For example, using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression. 
     In a related embodiment, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and surface (e.g., particles such as beads), interactions between the particles and cells is minimized. This selects for cells that express high amounts of desired antigens to be bound to the particles. For example, CD4+ T cells express higher levels of CD28 and are more efficiently captured than CD8+ T cells in dilute concentrations. In one embodiment, the concentration of cells used is 5×106/ml. In other embodiments, the concentration used can be from about 1×105/ml to 1×106/ml, and any integer value in between. 
     T cells can also be frozen. Wishing not to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After a washing step to remove plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or other suitable cell freezing media, the cells then are frozen to −80° C. at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at −20° C. or in liquid nitrogen. 
     T cells for use in the present invention may also be antigen-specific T cells. For example, tumor-specific T cells can be used. In certain embodiments, antigen-specific T cells can be isolated from a patient of interest, such as a patient afflicted with a cancer or an infectious disease. In one embodiment, neoepitopes are determined for a subject and T cells specific to these antigens are isolated. Antigen-specific cells for use in expansion may also be generated in vitro using any number of methods known in the art, for example, as described in U.S. patent Publication No. US 20040224402 entitled, Generation and Isolation of Antigen-Specific T Cells, or in U.S. Pat. No. 6,040,177. Antigen-specific cells for use in the present invention may also be generated using any number of methods known in the art, for example, as described in Current Protocols in Immunology, or Current Protocols in Cell Biology, both published by John Wiley &amp; Sons, Inc., Boston, Mass. 
     In a related embodiment, it may be desirable to sort or otherwise positively select (e.g. via magnetic selection) the antigen specific cells prior to or following one or two rounds of expansion. Sorting or positively selecting antigen-specific cells can be carried out using peptide-WIC tetramers (Altman, et al., Science. Oct. 4, 1996; 274(5284):94-6). In another embodiment, the adaptable tetramer technology approach is used (Andersen et al., 2012 Nat Protoc. 7:891-902). Tetramers are limited by the need to utilize predicted binding peptides based on prior hypotheses, and the restriction to specific HLAs. Peptide-MHC tetramers can be generated using techniques known in the art and can be made with any WIC molecule of interest and any antigen of interest as described herein. Specific epitopes to be used in this context can be identified using numerous assays known in the art. For example, the ability of a polypeptide to bind to WIC class I may be evaluated indirectly by monitoring the ability to promote incorporation of 1251 labeled O 2 -microglobulin (β2m) into MHC class I/β2m/peptide heterotrimeric complexes (see Parker et al., J. Immunol. 152:163, 1994). 
     In one embodiment cells are directly labeled with an epitope-specific reagent for isolation by flow cytometry followed by characterization of phenotype and TCRs. In one embodiment, T cells are isolated by contacting with T cell specific antibodies. Sorting of antigen-specific T cells, or generally any cells of the present invention, can be carried out using any of a variety of commercially available cell sorters, including, but not limited to, MoFlo sorter (DakoCytomation, Fort Collins, Colo.), FACSAria™, FACSArray™, FACSVantage™, BD™ LSR II, and FACSCalibur™ (BD Biosciences, San Jose, Calif.). 
     In a preferred embodiment, the method comprises selecting cells that also express CD3. The method may comprise specifically selecting the cells in any suitable manner. Preferably, the selecting is carried out using flow cytometry. The flow cytometry may be carried out using any suitable method known in the art. The flow cytometry may employ any suitable antibodies and stains. Preferably, the antibody is chosen such that it specifically recognizes and binds to the particular biomarker being selected. For example, the specific selection of CD3, CD8, TIM-3, LAG-3, 4-1BB, or PD-1 may be carried out using anti-CD3, anti-CD8, anti-TIM-3, anti-LAG-3, anti-4-1BB, or anti-PD-1 antibodies, respectively. The antibody or antibodies may be conjugated to a bead (e.g., a magnetic bead) or to a fluorochrome. Preferably, the flow cytometry is fluorescence-activated cell sorting (FACS). TCRs expressed on T cells can be selected based on reactivity to autologous tumors. Additionally, T cells that are reactive to tumors can be selected for based on markers using the methods described in patent publication Nos. WO2014133567 and WO2014133568, herein incorporated by reference in their entirety. Additionally, activated T cells can be selected for based on surface expression of CD107a. 
     In one embodiment of the invention, the method further comprises expanding the numbers of T cells in the enriched cell population. Such methods are described in U.S. Pat. No. 8,637,307 and is herein incorporated by reference in its entirety. The numbers of T cells may be increased at least about 3-fold (or 4-, 5-, 6-, 7-, 8-, or 9-fold), more preferably at least about 10-fold (or 20-, 30-, 40-, 50-, 60-, 70-, 80-, or 90-fold), more preferably at least about 100-fold, more preferably at least about 1,000 fold, or most preferably at least about 100,000-fold. The numbers of T cells may be expanded using any suitable method known in the art. Exemplary methods of expanding the numbers of cells are described in patent publication No. WO 2003/057171, U.S. Pat. No. 8,034,334, and U.S. Patent Publication No. 2012/0244133, each of which is incorporated herein by reference. 
     In one embodiment, ex vivo T cell expansion can be performed by isolation of T cells and subsequent stimulation or activation followed by further expansion. In one embodiment of the invention, the T cells may be stimulated or activated by a single agent. In another embodiment, T cells are stimulated or activated with two agents, one that induces a primary signal and a second that is a co-stimulatory signal. Ligands useful for stimulating a single signal or stimulating a primary signal and an accessory molecule that stimulates a second signal may be used in soluble form. Ligands may be attached to the surface of a cell, to an Engineered Multivalent Signaling Platform (EMSP), or immobilized on a surface. In a preferred embodiment both primary and secondary agents are co-immobilized on a surface, for example a bead or a cell. In one embodiment, the molecule providing the primary activation signal may be a CD3 ligand, and the co-stimulatory molecule may be a CD28 ligand or 4-1BB ligand. 
     In certain embodiments, T cells comprising a CAR or an exogenous TCR, may be manufactured as described in International Patent Publication No. WO 2015/120096, by a method comprising enriching a population of lymphocytes obtained from a donor subject; stimulating the population of lymphocytes with one or more T-cell stimulating agents to produce a population of activated T cells, wherein the stimulation is performed in a closed system using serum-free culture medium; transducing the population of activated T cells with a viral vector comprising a nucleic acid molecule which encodes the CAR or TCR, using a single cycle transduction to produce a population of transduced T cells, wherein the transduction is performed in a closed system using serum-free culture medium; and expanding the population of transduced T cells for a predetermined time to produce a population of engineered T cells, wherein the expansion is performed in a closed system using serum-free culture medium. In certain embodiments, T cells comprising a CAR or an exogenous TCR, may be manufactured as described in WO 2015/120096, by a method comprising: obtaining a population of lymphocytes; stimulating the population of lymphocytes with one or more stimulating agents to produce a population of activated T cells, wherein the stimulation is performed in a closed system using serum-free culture medium; transducing the population of activated T cells with a viral vector comprising a nucleic acid molecule which encodes the CAR or TCR, using at least one cycle transduction to produce a population of transduced T cells, wherein the transduction is performed in a closed system using serum-free culture medium; and expanding the population of transduced T cells to produce a population of engineered T cells, wherein the expansion is performed in a closed system using serum-free culture medium. The predetermined time for expanding the population of transduced T cells may be 3 days. The time from enriching the population of lymphocytes to producing the engineered T cells may be 6 days. The closed system may be a closed bag system. Further provided is population of T cells comprising a CAR or an exogenous TCR obtainable or obtained by said method, and a pharmaceutical composition comprising such cells. 
     In certain embodiments, T cell maturation or differentiation in vitro may be delayed or inhibited by the method as described in International Patent Publication No. WO 2017/070395, comprising contacting one or more T cells from a subject in need of a T cell therapy with an AKT inhibitor (such as, e.g., one or a combination of two or more AKT inhibitors disclosed in claim 8 of WO2017070395) and at least one of exogenous Interleukin-7 (IL-7) and exogenous Interleukin-15 (IL-15), wherein the resulting T cells exhibit delayed maturation or differentiation, and/or wherein the resulting T cells exhibit improved T cell function (such as, e.g., increased T cell proliferation; increased cytokine production; and/or increased cytolytic activity) relative to a T cell function of a T cell cultured in the absence of an AKT inhibitor. 
     In certain embodiments, a patient in need of a T cell therapy may be conditioned by a method as described in International Patent Publication No. WO 2016/191756 comprising administering to the patient a dose of cyclophosphamide between 200 mg/m2/day and 2000 mg/m2/day and a dose of fludarabine between 20 mg/m2/day and 900 mg/m 2 /day. 
     Diseases 
     Genetic Diseases and Diseases with a Genetic and/or Epigenetic Aspect 
     The compositions, 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 composition, system, and/or one or more components thereof to a subject, where the composition, 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 compositions, 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 2. 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 2 
               
             
            
               
                   
               
               
                 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, HIRA1, 
               
               
                   
                   
                   
                 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 compositions, 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 3. In some embodiments, the disease is a genetic disease or disorder. In some of embodiments, the composition, 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 3. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 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 
               
               
                   
               
            
           
         
       
     
     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 composition, 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 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 composition, 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 compositions, systems, and components thereof described herein. 
     In some embodiments, the composition, 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 composition, 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 composition, 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 composition, 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), Herpeesviridae (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 composition, 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 composition, 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 composition, 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 composition, 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 composition, 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 composition, 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  busk/),  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 boa  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 composition, system, and/or component thereof to a pathogenic organism described herein, allowing the composition, 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 composition, 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 composition, 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 composition, 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 composition, 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 composition, 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 composition, 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 composition, 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 composition, 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 composition, system, or component thereof can be capable of correcting an mtDNA mutation, or a combination thereof. 
     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 composition, system, and/or a component thereof to a cell, and more specifically one or more mitochondria in a cell, allowing the composition, 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 compositions, 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 compositions, 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 composition, system, and/or component thereof described herein. In some embodiments, the composition, 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 composition, 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 compositions and 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. 
     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 composition, system, and/or component thereof and/or a vector or vector system that is capable of driving expression of a composition, 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 composition, system, or 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 composition, system, or 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 composition and 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 recombination 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 2 and 3 herein. 
     In Situ Disease Detection 
     The compositions, 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 compositions, systems, and components thereof described herein in view of the description herein. 
     In some embodiments, the composition, system, or component thereof can be used in a detection method, such as an in situ detection method described herein. In some embodiments, the composition, system, or component thereof can include a catalytically inactivate Cas effector described herein 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 compositions, 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 compositions, systems, and/or components thereof described herein can be used in a method to screen and/or select cells. In some embodiments, composition, 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 composition, system(s) and/or components thereof, and/or vectors or vector systems into the cell(s), wherein the composition, system(s) and/or components thereof, and/or 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 recombination template; wherein, for example that which is being expressed is within and expressed in vivo by the composition, system, vector or vector system and/or the recombination template comprises the one or more mutations that abolish Cas effector cleavage; allowing homologous recombination of the recombination template with the target polynucleotide in the cell(s) to be selected; allowing a composition, system, or complex to bind to a target polynucleotide to effect cleavage of the target polynucleotide within said gene, wherein the AAV-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 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 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 compositions, 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. 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 composition, 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 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 composition, 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 complex with the components of the composition and system herein, and wherein said complex is operable in the cell. In some embodiments, the 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 composition, system, optionally a composition, 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 composition, system, optionally a composition, 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 composition, system, optionally a composition, 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 composition, system, optionally a composition, 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 composition, system, such as a composition, system, based therapy or therapeutic, optionally in a population; or for developing or designing a gRNA for use in a composition, system, optionally a composition, 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 compositions, 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 composition, 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 composition, 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 composition, system, selected from the set of compositions, systems, wherein the composition, 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 composition and system, in the set of compositions, 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 the Systems 
     The methods of the present invention can involve optimization of selected parameters or variables associated with the composition, system, and/or its functionality, as described herein further elsewhere. Optimization of the composition, 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 composition, system, modulation, such as composition, system, based therapeutic target(s) modulation, modification, or manipulation, as well as the delivery of the composition, 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. 
     The activity of the composition and/or system, such as CRISPR-Cas system-based therapy or therapeutics may involve target disruption, such as target mutation, such as leading to gene knockout. The activity of the composition and/or system, 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. The activity of the composition and/or system, 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 functionality of the composition and/or system, 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, the functionality of the composition and/or system comprises genomic mutation. In certain embodiments, the functionality of the composition and/or system comprises single genomic mutation. In certain embodiments, the functionality of the composition and/or system functionality comprises multiple genomic mutation. In certain embodiments, the functionality of the composition and/or system comprises gene knockout. In certain embodiments, the functionality of the composition and/or system comprises single gene knockout. In certain embodiments, the functionality of the composition and/or system comprises multiple gene knockout. In certain embodiments, the functionality of the composition and/or system comprises gene correction. In certain embodiments, the functionality of the composition and/or system comprises single gene correction. In certain embodiments, the functionality of the composition and/or system comprises multiple gene correction. In certain embodiments, the functionality of the composition and/or system comprises genomic region correction. In certain embodiments, the functionality of the composition and/or system comprises single genomic region correction. In certain embodiments, the functionality of the composition and/or system comprises multiple genomic region correction. In certain embodiments, the functionality of the composition and/or system comprises gene deletion. In certain embodiments, the functionality of the composition and/or system comprises single gene deletion. In certain embodiments, the functionality of the composition and/or system comprises multiple gene deletion. In certain embodiments, the functionality of the composition and/or system comprises genomic region deletion. In certain embodiments, the functionality of the composition and/or system comprises single genomic region deletion. In certain embodiments, the functionality of the composition and/or system comprises multiple genomic region deletion. In certain embodiments, the functionality of the composition and/or system comprises modulation of gene or genomic region functionality. In certain embodiments, the functionality of the composition and/or system comprises modulation of single gene or genomic region functionality. In certain embodiments, the functionality of the composition and/or system comprises modulation of multiple gene or genomic region functionality. In certain embodiments, the functionality of the composition and/or system comprises gene or genomic region functionality, such as gene or genomic region activity. In certain embodiments, the functionality of the composition and/or system comprises single gene or genomic region functionality, such as gene or genomic region activity. In certain embodiments, the functionality of the composition and/or system comprises multiple gene or genomic region functionality, such as gene or genomic region activity. In certain embodiments, the functionality of the composition and/or system 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, the functionality of the composition and/or system 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, the functionality of the composition and/or system 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 the system, such as CISPR-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 be optimized, for instance, 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 compositions, 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 the functionality of the composition and/or system, 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 the functionality of the composition and/or system, selecting one or more CRISPR-Cas system mode of delivery, selecting one or more 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 the functionality of the composition and/or system, optionally selecting one or more mode of delivery, optionally selecting one or more delivery vehicle or expression system, and optimization of selected parameters or variables associated with the 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 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 functionality of the composition and/or system, the 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 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 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 compositions, 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 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 compositions, 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 compositions, systems, and/or components thereof involves direct delivery of the compositions, systems, and/or components thereof to cell types in their native tissues. In vivo polynucleotide modification via compositions, 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 compositions, 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 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 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 system in vivo. Generally, to reduce the potential for off-target effects, it is optimal but not necessarily required, that the amount of the 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 system or component thereof. In embodiments, where the immunogenicity of the system or component thereof is of concern, the immunogenicity system or component thereof can be reduced. By way of example only, the immunogenicity of the 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 in the host species (human or other species). 
     Xenotransplantation 
     The present invention also contemplates use of the CRISPR-Cas system described herein, e.g. Cas effector protein systems, 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 a (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. 
     Embodiments of the invention also relate to methods and compositions related to knocking out genes, amplifying genes and repairing particular mutations associated with DNA repeat instability and neurological disorders (Robert D. Wells, Tetsuo Ashizawa, Genetic Instabilities and Neurological Diseases, Second Edition, Academic Press, Oct. 13, 2011—Medical). Specific aspects of tandem repeat sequences have been found to be responsible for more than twenty human diseases (New insights into repeat instability: role of RNA·DNA hybrids. McIvor E I, Polak U, Napierala M. RNA Biol. 2010 September-October; 7(5):551-8). The present effector protein systems may be harnessed to correct these defects of genomic instability. 
     Several further aspects of the invention relate to correcting defects associated with a wide range of genetic diseases which are further described on the website of the National Institutes of Health under the topic subsection Genetic Disorders (website at health.nih.gov/topic/GeneticDisorders). The genetic brain diseases may include but are not limited to Adrenoleukodystrophy, Agenesis of the Corpus Callosum, Aicardi Syndrome, Alpers&#39; Disease, Alzheimer&#39;s Disease, Barth Syndrome, Batten Disease, CADASIL, Cerebellar Degeneration, Fabry&#39;s Disease, Gerstmann-Straussler-Scheinker Disease, Huntington&#39;s Disease and other Triplet Repeat Disorders, Leigh&#39;s Disease, Lesch-Nyhan Syndrome, Menkes Disease, Mitochondrial Myopathies and NINDS Colpocephaly. These diseases are further described on the website of the National Institutes of Health under the subsection Genetic Brain Disorders. 
     Applications in Plants and Fungi 
     The 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 Aug. 17, 2013; 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. Dec. 29, 2011; 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 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: 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, Rafflesiales, 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, Malta, 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, Nannochloropsis,  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 recombination 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, S1DMR6-1), 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, the 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, Tf Dof1, 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-Al, TaMLO-Bl and TaMLO-Dl 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. Mar. 4, 2019; 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-phosphate dehydrogenase (G3PDH), Enoyl-acyl carrier protein reductase (Enoyl-ACP-reductase), glycerol-3-phosphate acyltransferase, lysophosphatidic acyl transferase or diacylglycerol acyltransferase, phospholipid: diacylglycerol acyltransferase, phosphatidate 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 International Patent Publication No. WO 2015/086795. 
     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, fatAl, 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, S1 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 C1 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. cerervisiae,  Kluyveromyces marxianus, Issatchenkia orientalis, Candida  spp. (e.g.,  Candida albicans ),  Yarrowia  spp. (e.g.,  Yarrowia  hpolytica),  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. Aug. 1, 2017; 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. Dec. 19, 2013; 155(7):1479-91), targeted gene disruption positive-selection in vitro and in vivo (as described in Malina A et al., Genes Dev. Dec. 1, 2013; 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. May 12, 2015; 112(19):6164-9; Ramanan V et. al., Sci Rep. Jun. 2, 2015; 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. Sep. 8, 2015; 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. Oct. 29, 2014; 42(19):e147), development of kits for multiplex genome editing (as described in Xing H L et al., BMC Plant Biol. Nov. 29, 2014; 14:327), starch production (as described in Hebelstrup K H et al., Front Plant Sci. Apr. 23, 2015; 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. Oct. 11, 2013; 9(1):39; Harrison M M, et al., Genes Dev. Sep. 1, 2014; 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. Nov. 26, 2018; 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. Apr. 16, 2020. 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  lichenformis) 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. Oct. 17, 2017; 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. May 20, 2014; 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. Oct. 8, 2013; 110(41):16526-31;  Mali  P, et al., Science. Feb. 15, 2013; 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. Feb. 1, 2015; 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. 
     Kits 
     In one aspect, the invention provides kits containing any one or more of the elements disclosed in the above methods and compositions. In one aspect, the invention provides a kit comprising one or more of the components described herein. In some embodiments, the kit comprises the CRISPR system components (Cas protein or polynucleotide encoding Cas protein and guide RNA or polynucleotide encoding guide RNA, as well as any other components or polynucleotides encoding said components) and instructions for using the kit. In some embodiments, the kit comprises a vector system and instructions for using the kit. In some embodiments, the kit comprises a delivery system and instructions for using the kit. In some embodiments, the kit comprises a vector system and instructions for using the kit. Elements may be provided individually or in combinations, and may be provided in any suitable container, such as a vial, a bottle, or a tube. The kits may include the gRNA and the unbound protector strand as described herein. The kits may include the gRNA with the protector strand bound to at least partially to the guide sequence (i.e. pgRNA). Thus the kits may include the pgRNA in the form of a partially double stranded nucleotide sequence as described here. In some embodiments, the kit includes instructions in one or more languages, for example in more than one language. The instructions may be specific to the applications and methods described herein. 
     In some embodiments, a kit comprises one or more reagents for use in a process utilizing one or more of the elements described herein. Reagents may be provided in any suitable container. For example, a kit may provide one or more reaction or storage buffers. Reagents may be provided in a form that is usable in a particular assay, or in a form that requires addition of one or more other components before use (e.g., in concentrate or lyophilized form). A buffer can be any buffer, including but not limited to a sodium carbonate buffer, a sodium bicarbonate buffer, a borate buffer, a Tris buffer, a MOPS buffer, a HEPES buffer, and combinations thereof. In some embodiments, the buffer is alkaline. In some embodiments, the buffer has a pH from about 7 to about 10. In some embodiments, the kit comprises one or more oligonucleotides corresponding to a guide sequence for insertion into a vector so as to operably link the guide sequence and a regulatory element. In some embodiments, the kit comprises a homologous recombination template polynucleotide. In some embodiments, the kit comprises one or more of the vectors and/or one or more of the polynucleotides described herein. The kit may advantageously allow to provide all elements of the systems of the invention. 
     Other Example Embodiments 
     The present application also provides aspects and embodiments as set forth in the following numbered Statements: 
     Statement 1. An engineered Cas protein comprising a region providing access to a location of target polynucleotide binding. 
     Statement 2. The engineered Cas protein of Statement 1 comprising no more than 600, no more than 700, or no more than 800 amino acids. 
     Statement 3. The engineered Cas protein of any one of the proceeding Statements, wherein the protein lacks or substantially lacks a Rec 1 and/or Rec2 domain or the structural equivalent thereof. 
     Statement 4. The engineered Cas protein of any one of the proceeding Statements, wherein the protein lacks or substantially lacks a Rec lobe or structural equivalent thereof. 
     Statement 5. The engineered Cas protein of any one of the proceeding Statements, wherein the protein comprises at least one nuclease domain. 
     Statement 6. The engineered Cas protein of any one of the proceeding Statements, wherein the Cas protein comprises an HNH and a RuvC nuclease domain. 
     Statement 7. The engineered Cas protein of Statement 6, wherein the RuvC domain comprises RuvCI, RuvCII, and/or RuvCIII, preferably all. 
     Statement 8. The engineered Cas protein of any one of the proceeding Statements, wherein the Cas protein targets DNA. 
     Statement 9. The engineered Cas protein of Statement 8, wherein the Cas protein targets dsDNA. 
     Statement 10. The engineered Cas protein of any one of the proceeding Statements 1, wherein the Cas protein comprises a region that has 10-45% identity to IscB. 
     Statement 11. The engineered Cas protein of Statement 10, wherein Cas protein comprises a region that has 20-25% identity to IscB. 
     Statement 12. The engineered Cas protein of any one of the proceeding Statements, the Cas protein has at least 10%, at least 20%, at least 30%, at least 40% or at least 45% identity to SpCas9 or is at least 10%, preferably at least 20%, shorter than SpCas9. 
     Statement 13. The engineered Cas protein of any one of the proceeding Statements, wherein the Cas protein is a Class 2, Type II CRISPR-Cas protein. 
     Statement 14. The engineered Cas protein of any one of the proceeding Statements, wherein the Cas protein is a Cas9. 
     Statement 15. The engineered Cas protein of any one of the proceeding Statements, wherein one or both nuclease domains are catalytically inactive or modified to be catalytically inactive, or wherein the protein is a nickase. 
     Statement 16. The engineered Cas protein of any one of the proceeding Statements, wherein both nuclease domains are catalytically inactive. 
     Statement 17. The engineered Cas protein of any one of the proceeding Statements, wherein the Cas protein comprise a region that has at least 80% identity to IscB. 
     Statement 18. The engineered Cas protein of Statement 17, wherein the region is at N-terminus of the Cas protein. 
     Statement 19. An engineered CRISPR-Cas system comprising the Cas protein of any one of the proceeding Statements and a guide molecule capable of forming a complex with the Cas protein and directing site-specific binding of the complex to a target sequence of a target polypeptide. 
     Statement 20. The system or Cas protein of any one of the preceding Statements, wherein the Cas protein and/or guide molecule further comprise a functional domain. 
     Statement 21. The system or Cas protein of Statement 20, wherein the functional domain comprises base editing activity, nucleotide deaminase activity, methylase activity, demethylase activity, translation activation activity, translation repression activity, transcription activation activity, transcription repression activity, transcription release factor activity, chromatin modifying or remodeling 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, nucleic acid binding activity, detectable activity, or any combination thereof. 
     Statement 22. The system or Cas protein of Statement 21, wherein the functional domain is a nucleotide deaminase linked or fused to the Cas protein. 
     Statement 23. The system of Cas protein of Statement 22, wherein said deaminase is an adenosine deaminase or a cytidine deaminase. 
     Statement 24. The system or Cas protein of any one of the preceding Statements, further comprising one or more nucleic acid modifying proteins or domains. 
     Statement 25. The system or Cas protein of Statement 24, wherein the one or more DNA modifying proteins comprises DNA polymerase, recombinase, ribonucleotide reductase, methyltransferase, diadenosine tetraphosphate hydrolase, DNA helicase, or RNA helicase. 
     Statement 26. The system of any one of the preceding Statements, wherein the target sequence comprises a PAM sequence. 
     Statement 27. The system of Statement 26, wherein the PAM sequence is NGG. 
     Statement 28. A polynucleotide molecule that encodes one or more components of the CRISPR-Cas system or Cas protein of any one of the proceeding Statements. 
     Statement 29. The polynucleotide of Statements 28, wherein one or more regions of the polynucleotide is codon optimized for expression in a eukaryotic cell, such as a mammalian or plant cell. 
     Statement 30. A vector comprising the polynucleotide of any one of Statements 28-29. 
     Statement 31. A vector system comprising two or more vectors of Statement 30. 
     Statement 32. A cell comprising a polynucleotide, the vector, or vector system of any one of Statements 28 to 31. 
     Statement 33. The cell of Statement 32, wherein the cell is a eukaryotic cell, a prokaryotic cell, or a plant cell. 
     Statement 34. A plant or non-human animal comprising one or more polynucleotide, the vector, vector system, or cells of any one of Statements 28 to 33. 
     Statement 35. A method of targeting a polynucleotide, comprising contacting a sample that comprises the polynucleotide with the system or Cas protein, the polynucleotide, the vector, or the vector system of any one of Statements 1 to 34. 
     Statement 36. The method of Statement 35, further comprising detecting binding of the complex to the polynucleotide. 
     Statement 37. The method of Statement 35 or 36, wherein contacting results in modification of a gene product or modification of the amount or expression of a gene product. 
     Statement 38. The method of any one of Statements 35-37, wherein the target sequence of the polynucleotide is a disease-associated target sequence. 
     Statement 39. A method of modifying an adenine or cytidine in a target polynucleotide sequence, comprising contacting said target polynucleotide with the system or Cas protein of any one of Statements 1-38. 
     Statement 40. An antiviral composition comprising the system or Cas protein of any one of Statements 1-39. 
     Statement 41. A method for treating, preventing, suppressing and/or alleviating viral pathogenesis, infection, propagation, and/or replication in a subject in need thereof, comprising administering to a subject in need thereof the composition of Statement 40. 
     EXAMPLES 
     Example 1 
       FIG. 9  shows an exemplary method for identifying and characterizing novel CRISPR-Cas systems and other RNA-guided nucleases.  FIG. 10  shows exemplary CRISPR-Cas systems and other RNA-guided nucleases. 
     Example 2 
       FIG. 11  shows the locus of an exemplary Cas, which is associated with various enzymes (e.g., nucleic acid modifying enzyme). PAM of an exemplary Cas was tested ( FIG. 12 ). Processed crRNA and tracrRNA were revealed by dRNA-seq ( FIG. 13 ). The Cas protein was purified using MBP tags ( FIG. 14 ). 
     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.