Patent Publication Number: US-2021189388-A1

Title: DNA-Guided Gene Editing and Regulation

Description:
RELATED APPLICATION DATA 
     This application claims priority to U.S. Provisional Application No. 62/292,962 filed on Feb. 9, 2016 and to U.S. Provisional Application No. 62/396,418 filed on Sep. 19, 2016 which are hereby incorporated herein by reference in their entirety for all purposes. 
    
    
     STATEMENT OF GOVERNMENT INTERESTS 
     This invention was made with government support under Grant No. P50 HG005550 from the National Institutes of Health. The government has certain rights in the invention. 
    
    
     SEQUENCE LISTING 
     The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Feb. 7, 2017, is named 010498_00905-WO_SL.txt and is 26,801 bytes in size. 
     BACKGROUND 
     Argonaute (Ago) proteins, involved in RNA interference pathways, use small single-stranded RNA (ssRNA) to facilitate RNA interference of complementary ssRNA targets. See G. Meister, Argonaute proteins: functional insights and emerging roles.  Nature reviews. Genetics  14, 447-459 (2013). Ago from  T thermophiles  (TtAgo) was reported to prevent infection and propagation of foreign DNA by loading 5′-phosphorylated small interfering DNA (siDNA) guides approximately 20 base pairs (bp) in length to cut complementary single stranded DNA. See D. C. Swarts, M. M. Jore, E. R. Westra, Y. Zhu, J. H. Janssen, A. P. Snijders, Y. Wang, D. J. Patel, J. Berenguer, S. J. Brouns, J. van der Oost, DNA-guided DNA interference by a prokaryotic Argonaute.  Nature  507, 258-261 (2014); G. Sheng, H. Zhao, J. Wang, Y. Rao, W. Tian, D. C. Swarts, J. van der Oost, D. J. Patel, Y. Wang, Structure-based cleavage mechanism of  Thermus thermophilus  Argonaute DNA guide strand-mediated DNA target cleavage.  Proc Nail Acad Sci U S A  111, 652-657 (2014); K. Nakanishi, D. E. Weinberg, D. P. Bartel, D. J. Patel, Structure of yeast Argonaute with guide RNA.  Nature  486, 368-374 (2012); Y. Wang, S. Juranek, H. Li, G. Sheng, T. Tuschl, D. J. Patel, Structure of an argonaute silencing complex with a seed-containing guide DNA and target RNA duplex.  Nature  456, 921-926 (2008). 
     SUMMARY 
     Aspects of the present disclosure are directed to a method of altering genomic nucleic acids in a eukaryotic cell using an Argonaute (Ago) protein and a guide DNA which form a complex with a target nucleic acid sequence. According to one aspect, an Ago system, i.e. one or more guide DNA and an Ago protein and/or alternatively a donor nucleic acid sequence, are provided to a eukaryotic cell to a target nucleic acid sequence. According to this aspect, the Ago system is used to cut or nick the target nucleic acid sequence or otherwise deliver a transcriptional regulator, such as a transcriptional activator or transcriptional repressor, to the target nucleic acid sequence, or otherwise insert the donor nucleic acid into the target nucleic acid sequence. 
     According to certain aspects, a method of modulating expression of a target nucleic acid sequence in a eukaryotic cell is provided including introducing into the cell a first foreign nucleic acid encoding one or more guide DNAs complementary to one or more target nucleic acids, introducing into the cell a second foreign nucleic acid encoding an Ago protein, such as an Ago enzyme, an Ago nuclease or a nuclease null Ago protein, introducing into the cell a third foreign nucleic acid encoding a transcriptional regulator protein or domain, wherein the Ago protein and the transcriptional regulator protein or domain are expressed, wherein the one or more guide DNAs, the Ago protein and the transcriptional regulator protein or domain co-localize to the target nucleic acid sequence and wherein the transcriptional regulator protein or domain regulates expression of the target nucleic acid sequence. 
     According to certain aspects, a method of integrating foreign DNA into a nucleic acid sequence of a eukaryotic cell is provided including providing to the cell a guide DNA sequence complementary to a target nucleic acid sequence, providing to the cell a donor sequence, providing to the cell an Ago enzyme that interacts with the guide DNA sequence and cleaves the target nucleic acid sequence in a site specific manner, wherein the guide DNA sequence binds to the complementary target nucleic acid sequence and the Ago enzyme cleaves the target nucleic acid sequence in a site specific manner; and wherein the donor sequence is integrated into the target nucleic acid sequence. 
     According to one aspect, the foreign nucleic acid encoding the Ago protein further encodes the transcriptional regulator protein or domain to create an expression product or fusion of the Ago protein attached to the transcriptional regulator protein or domain. According to this aspect, a third foreign nucleic acid encoding a transcriptional regulator or domain is not utilized. According to one aspect, the foreign nucleic acid further encodes a target of an RNA-binding domain and the foreign nucleic acid encoding the transcriptional regulator protein or domain further encodes an RNA-binding domain fused to the transcriptional regulator protein or domain. In this aspect, the guide DNA and the expressed transcriptional regulator protein or domain connect via the target of the RNA-binding domain and the RNA-binding domain. Other methods of linking a guide DNA and a transcriptional regulator protein or domain are known to those of skill in the art. 
     According to one aspect, the transcriptional regulator protein or domain is a transcriptional activator. According to one aspect, the transcriptional regulator protein or domain upregulates expression of the target nucleic acid sequence. According to one aspect, the transcriptional regulator protein or domain upregulates expression of the target nucleic acid sequence to treat a disease or detrimental condition. According to one aspect, the target nucleic acid sequence is associated with a disease or detrimental condition. According to one aspect, the transcriptional regulator protein or domain is a transcriptional repressor. According to one aspect, the transcriptional regulator protein or domain downregulates expression of the target nucleic acid sequence. According to one aspect, the transcriptional regulator protein or domain downregulates expression of the target nucleic acid sequence to treat a disease or detrimental condition. According to one aspect, the target nucleic acid sequence is associated with a disease or detrimental condition. According to one aspect, the transcriptional regulator or domain is a chromatin modifier, remodeller, or histone modifier including enzymes involved in histone acetylation, methylation, demethylation, phosphorylation, ubiquitination, sumoylation, ADP-ribosylation, deimination, and proline isomerization. According to one aspect, the transcriptional regulator protein or domain upregulates expression of the target nucleic acid sequence. According to one aspect, the transcriptional regulator protein or domain upregulates expression of the target nucleic acid sequence to treat a disease or detrimental condition. 
     According to one aspect of altering a target nucleic acid, two or more guide DNAs may be used with an Ago nuclease wherein the Ago nuclease co-localizes with the two or more guide DNAs to the target nucleic acid sequence and cuts the target nucleic acid sequence resulting in two or more adjacent cuts. According to one aspect, the two or more adjacent cuts are on the same strand of the double stranded target nucleic acid sequence. According to one aspect, the two or more adjacent cuts are on the same strand of the double stranded target nucleic acid sequence and result in homologous recombination. According to one aspect, the two or more adjacent cuts are on different strands of the double stranded target nucleic acid sequence. According to one aspect, the two or more adjacent cuts are on different strands of the double stranded target nucleic acid sequence and create double stranded breaks. According to one aspect, the two or more adjacent cuts are on different strands of the double stranded target nucleic acid sequence and create double stranded breaks resulting in nonhomologous end joining. According to one aspect, the two or more adjacent cuts are on different strands of the double stranded target nucleic acid sequence and are offset with respect to one another. According to one aspect, the two or more adjacent cuts are on different strands of the double stranded target nucleic acid sequence and are offset with respect to one another and create double stranded breaks. According to one aspect, the two or more adjacent cuts are on different strands of the double stranded target nucleic acid sequence and are offset with respect to one another and create double stranded breaks resulting in nonhomologous end joining. 
     According to one aspect, the two or more adjacent cuts are on different strands of the double stranded target nucleic acid and create double stranded breaks resulting in fragmentation of the target nucleic acid sequence thereby preventing expression of the target nucleic acid sequence. 
     According to one aspect, the method further includes introducing into the cell a third foreign nucleic acid encoding a donor nucleic acid sequence wherein the two or more cuts results in incorporation of the donor nucleic acid sequence into the target nucleic acid sequence through homologous recombination or nonhomologous end joining. 
     According to one aspect, a method of altering a eukaryotic cell is provided including the steps of providing to the eukaryotic cell a guide DNA sequence complementary to a target nucleic acid sequence, providing to the eukaryotic cell an Ago enzyme that interacts with the guide DNA sequence and cleaves the target nucleic acid sequence in a site specific manner, wherein the guide DNA sequence binds to the complementary target nucleic acid sequence and the Ago enzyme cleaves the target nucleic acid sequence in a site specific manner 
     According to one aspect, methods described herein include providing a helicase to the cell where the helicase unwinds a helicase targeting target nucleic acid sequence. According to one aspect, methods described herein include providing a helicase to the cell where the helicase unwinds a helicase targeting target nucleic acid sequence where the helicase targeting nucleic acid sequence and the complementary target nucleic acid sequence are about 75 to 150 base pairs apart. According to one aspect, methods described herein include providing a nuclease null Cas9 protein and a guide RNA, wherein the guide RNA is complementary to a guide RNA targeting target nucleic acid sequence, wherein the nuclease null Cas9 and the guide RNA colocalize at the guide RNA targeting target nucleic acid sequence and the guide RNA targeting target nucleic acid sequence is unwound. According to one aspect, methods described herein include providing a nuclease null Cas9 protein and a guide RNA, wherein the guide RNA is complementary to a guide RNA targeting target nucleic acid sequence, wherein the nuclease null Cas9 and the guide RNA colocalize at the guide RNA targeting target nucleic acid sequence and the guide RNA targeting target nucleic acid sequence is unwound, and wherein the guide RNA targeting target nucleic acid sequence and the complementary target nucleic acid sequence are about 75 to 150 base pairs apart. According to one aspect, methods described herein include providing a donor sequence and wherein the donor sequence is inserted into the target nucleic acid sequence. According to one aspect, methods described herein include providing a plurality of guide DNAs into the cell that are complementary to different target nucleic acid sequences and the Ago enzyme cleaves the different target nucleic acid sequences in a site specific manner According to methods described herein, the eukaryotic cell is a yeast cell, a plant cell, a mammalian cell or a human cell. 
     Further features and advantages of certain embodiments of the present invention will become more fully apparent in the following description of embodiments and drawings thereof, and from the claims. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publications with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. The foregoing and other features and advantages of the present embodiments will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings in which: 
         FIG. 1  depicts results of a gel shifting assay (SEQ ID NOS 96, 97, 98, and 25, respectively, in order of appearance). 
         FIG. 2A  to  FIG. 2D  are directed to engineered TtAgo NN -VP64 activating human endogenous genes.  FIG. 2A  is a schematic depicting construction of TtAgo-VP64 having a CMV promoter driving an Ago double mutant D478A, D546A to remove nuclease activity fused with VP64 transcriptional domain with nuclear localization signal (NLS).  FIG. 2B  is a schematic of siDNA (guide DNA) targeted to TFAM promoter for activation of genomic locus. TFAM-7 and TFAM-8 were designed to target the promoter region of TFAM in the genome, 148 bp and 200 bp upstream of the transcriptional start site, respectively.  FIG. 2C  depicts data demonstrating that a TtAgo-VP64 system activates TFAM similarly to Cas9ST-VP64 system as measured by RT-QPCR of TFAM.  FIG. 2D  depicts data demonstrating that activation of endogenous genes decreases with the GC % of the 5′-phosphorylated 20 bp siDNA. A reverse correlation of GC % of siDNA targeting sequence with TtAgo-VP64/siDNA activation activities was observed. 
         FIG. 3A  depicts a schematic of guide RNA designed to localize nuclease null Cas9 at various distances from TtAgo-VP64/siDNA_TFAM-4.  FIG. 3B  depicts data of Fold TFAM Activation. 
         FIG. 4  depicts immunoblots and data of DNA-guided activation TFAM (25 kDa). 
         FIG. 5  is directed to data demonstrating that DNA-guided TtAgo NN -VP64 activates a panel of genes. 3-6 siDNA targeting sequences were designed for the promoter region of 9 genes. The melting temperature (Tm), which is expressed as GC %, of each siDNA affects the efficiency of endogenous activation. All levels of activation were measured by RT-QPCR and normalized to experiments transfected by TtAgo NN -VP64 alone. The 18s housekeeping gene was used as a control. Plotted values represent the average. Error bars represent the standard deviation from the mean (N=3). 
         FIG. 6  is directed to data demonstrating that DNA-guided TtAgo NN  represses a panel of genes. 3 siDNA targeting sequences were designed for exon 1 or 2 of 9 genes. GC % of each siDNA is listed. All levels of suppression were normalized to experiments transfected by TtAgo NN  alone. The 18s housekeeping gene was used as a control. Plotted values represent the average. Error bars represent the standard deviation from the mean (N=3). 
         FIG. 7  depicts data 5 depicts data for TtAgo NN -VP64/siDNA sequence specificity as measured by TFAM activation activities by TtAgo NN -VP64/siDNA coupled with different siDNAs (SEQ ID NOS 99, 23, 100, and 101, respectively, in order of appearance). 
     
    
    
     DETAILED DESCRIPTION 
     Embodiments of the present disclosure are directed to methods of targeting one or more target nucleic acid sequences for editing or transcriptional regulation using an Ago protein and a guide DNA. 
     According to one aspect, an Ago system, i.e. an Ago enzyme and a guide DNA sequence, is provided to a eukaryotic cell. The guide DNA sequence and the Ago enzyme co-localize to the target nucleic acid sequence or otherwise co-localize with the target nucleic acid sequence, and the Ago cuts or cleaves the target nucleic acid sequence in a site specific manner. The Ago enzyme and the guide DNA sequence are referred to as a co-localization complex as that term is understood by one of skill in the art to the extent that the guide RNA and the Ago enzyme complex with a target nucleic acid sequence. 
     According to one aspect, an Ago system, i.e. an Ago enzyme and a guide DNA sequence, is provided to a eukaryotic cell. A donor nucleic acid sequence is also provided to the cell. The guide DNA sequence and the Ago enzyme co-localize to the target nucleic acid sequence or otherwise co-localize with the target nucleic acid sequence, and the Ago cuts or cleaves the target nucleic acid sequence in a site specific manner The donor nucleic acid is inserted into the target nucleic acid sequence, such as by homologous recombination or non-homologous end joining. The Ago enzyme and the guide DNA sequence are referred to as a co-localization complex as that term is understood by one of skill in the art to the extent that the guide DNA sequence and the Ago enzyme complex with a target nucleic acid sequence. 
     According to one aspect, an Ago system, i.e. an Ago enzyme and a guide DNA sequence, is provided to a eukaryotic cell. A donor nucleic acid sequence is also provided to the cell, such as by being attached, connected or otherwise bound to either the Ago enzyme or the guide DNA sequence. The guide DNA sequence and the Ago enzyme co-localize to the target nucleic acid sequence or otherwise co-localize with the target nucleic acid sequence, and the Ago cuts or cleaves the target nucleic acid sequence in a site specific manner. The donor nucleic acid sequence is inserted into the target nucleic acid sequence, such as by homologous recombination or non-homologous end joining. The Ago enzyme and the guide DNA sequence are referred to as a co-localization complex as that term is understood by one of skill in the art to the extent that the guide DNA sequence and the Ago enzyme complex with a target nucleic acid sequence. 
     According to one aspect, an Ago system, i.e. a nuclease null Ago protein, i.e., an Ago protein that lacks enzymatic activity, and a guide DNA sequence, is provided to a eukaryotic cell. A transcriptional regulator is also provided to the cell. The guide DNA sequence and the nuclease null Ago protein co-localize to the target nucleic acid sequence or otherwise co-localize with the target nucleic acid sequence, and the transcriptional regulator regulates expression of the target nucleic acid sequence. The nuclease null Ago protein and the guide DNA sequence are referred to as a co-localization complex as that term is understood by one of skill in the art to the extent that the guide DNA sequence and the nuclease null Ago protein complex with a target nucleic acid sequence. 
     According to one aspect, an Ago system, i.e. an Ago nuclease and a guide DNA sequence, is provided to a eukaryotic cell. The guide DNA sequence and the Ago nuclease co-localize to the target nucleic acid sequence or otherwise co-localize with the target nucleic acid sequence, and the Ago nuclease cuts the target nucleic acid sequence in a site specific manner. According to one aspect, an Ago system, i.e. an Ago nuclease and a two or more guide DNA sequences, is provided to a eukaryotic cell. The guide DNA sequences and the Ago nuclease co-localize to target nucleic acid sequences at locations determined by the two guide DNA sequences or otherwise co-localize with the target nucleic acid sequences at locations determined by the two guide DNA sequences, and the Ago nuclease cuts the target nucleic acid at two adjacent sites. According to one aspect, a donor nucleic acid sequence may be provided which is inserted into the target nucleic acid sequence, such as by homologous recombination or non-homologous end joining. The Ago nuclease and the guide DNA sequence are referred to as a co-localization complex as that term is understood by one of skill in the art to the extent that the guide RNA and the Ago nuclease complex with a target nucleic acid. 
     According to one aspect, any of the methods using an Ago system described herein may also be used with a helicase. According to this aspect, the helicase is provided to the cell and may be used to unwind a target nucleic acid to facilitate the Ago system with cutting, nicking or delivering a transcriptional regulator to the target nucleic acid sequence. A suitable helicase is a nuclease null Cas9 protein. Such a nuclease null Cas9 protein can be used with a guide RNA to form a co-localization complex at or with a target nucleic acid sequence so as to unwind the target nucleic acid sequence into two separate single strand portions of the double stranded target nucleic acid. The unwound target nucleic acid sequence then facilitates co-localization of an Ago system at or near the target nucleic acid sequence. 
     According to one aspect, a eukaryotic cell is provided including a first nucleic acid sequence that is complementary to a target nucleic acid sequence, a second nucleic acid sequence encoding an Ago enzyme that interacts with the first nucleic acid sequence and cleaves the target nucleic acid sequence in a site specific manner, wherein the first nucleic acid sequence is a guide DNA, and wherein the cell expresses the Ago enzyme, and wherein the guide DNA binds to the complementary target nucleic acid and the Ago enzyme cleaves the target nucleic acid sequence in a site specific manner. In one embodiment, the eukaryotic cell is a yeast cell, a plant cell or a mammalian cell. In another embodiment, the eukaryotic cell is a human cell. In one embodiment, the eukaryotic cell further includes a plurality of nucleic acids encoding a plurality of guide DNA sequences complementary to different target nucleic acid sequences. In another embodiment, the eukaryotic cell of further comprises a helicase where the helicase unwinds a helicase targeting target nucleic acid sequence. In yet another embodiment, the eukaryotic cell further comprises a helicase where the helicase unwinds a helicase targeting target nucleic acid sequence where the helicase targeting nucleic acid sequence and the complementary target nucleic acid sequence are about 75 to 150 base pairs apart. In another embodiment, the eukaryotic cell further comprises a nuclease null Cas9 protein and a guide RNA, wherein the guide RNA is complementary to a guide RNA targeting target nucleic acid sequence, wherein the nuclease null Cas9 and the guide RNA colocalize at the guide RNA targeting target nucleic acid sequence and unwind the guide RNA targeting target nucleic acid sequence. In a further embodiment, the eukaryotic cell further comprises a nuclease null Cas9 protein and a guide RNA, wherein the guide RNA is complementary to a guide RNA targeting target nucleic acid sequence, wherein the nuclease null Cas9 and the guide RNA colocalize at the guide RNA targeting target nucleic acid sequence and unwind the guide RNA targeting target nucleic acid sequence, and wherein the guide RNA targeting target nucleic acid sequence and the complementary target nucleic acid sequence are about 75 to 150 base pairs apart. In another embodiment, the eukaryotic cell further comprises a donor sequence and wherein the donor sequence is inserted into the target nucleic acid sequence. 
     According to another aspect, a eukaryotic cell is provided including a first nucleic acid sequence that is complementary to a target nucleic acid sequence, a second nucleic acid sequence encoding a nuclease null Ago protein, wherein the nuclease null Ago protein includes a transcriptional regulator domain connected thereto for modulating target nucleic acid expression, wherein the guide DNA and the nuclease null Ago protein including the transcriptional regulator domain co-localize at the target nucleic acid sequence and wherein the transcriptional regulator domain modulates expression of the target nucleic acid sequence. In one embodiment, the eukaryotic cell further comprises a helicase where the helicase unwinds a helicase targeting target nucleic acid sequence. In another embodiment, the eukaryotic cell further comprises a helicase where the helicase unwinds a helicase targeting target nucleic acid sequence where the helicase targeting nucleic acid sequence and the complementary target nucleic acid sequence are about 75 to 150 base pairs apart. In one embodiment, the eukaryotic cell further comprises a nuclease null Cas9 protein and a guide RNA, wherein the guide RNA is complementary to a guide RNA targeting target nucleic acid sequence, wherein the nuclease null Cas9 and the guide RNA colocalize at the guide RNA targeting target nucleic acid sequence and unwind the guide RNA targeting target nucleic acid sequence. In another embodiment, the eukaryotic cell further comprises a nuclease null Cas9 protein and a guide RNA, wherein the guide RNA is complementary to a guide RNA targeting target nucleic acid sequence, wherein the nuclease null Cas9 and the guide RNA colocalize at the guide RNA targeting target nucleic acid sequence and unwind the guide RNA targeting target nucleic acid sequence, and wherein the guide RNA targeting target nucleic acid sequence and the complementary target nucleic acid sequence are about 75 to 150 base pairs apart. In one embodiment, the transcriptional regulator is a transcriptional activator or a transcriptional repressor. In another embodiment, the eukaryotic cell further comprises a plurality of guide DNAs that are complementary to different target nucleic acid sequences and the nuclease null Ago protein modulates expression of the different target nucleic acid sequences in a site specific manner In one embodiment, the eukaryotic cell is a yeast cell, a plant cell or a mammalian cell. In another embodiment, the eukaryotic cell is a human cell. 
     According to one aspect, one or more vectors are used to introduce one or more nucleic acids encoding an Ago system, i.e. an Ago protein and a guide DNA sequence, and optionally a donor nucleic acid sequence, into a eukaryotic cell for editing or transcriptional regulation. A transcriptional regulator may be provided to the cell separately or may be attached, connected or otherwise bound to either the Ago protein or the guide DNA for delivery to the target nucleic acid sequence to be regulated. The nucleic acids are expressed and the Ago system cuts or nicks the target nucleic acid sequence or otherwise delivers a transcriptional regulator to the target nucleic acid sequence. Together, a guide DNA and an Ago protein are referred to as a co-localization complex as that term is understood by one of skill in the art to the extent that the guide DNA sequence and the Ago protein complex with a target nucleic acid sequence. According to certain aspects, a vector may include one or more nucleic acids encoding an Ago protein, a guide DNA, a donor nucleic acid sequence and/or a transcriptional regulator. The vectors may include any additional genetic regulatory elements as known to those of skill in the art, such as regulators, promoters, nuclear localization signals (NLS), start codons, stop codons, a transgene etc. According to certain aspects, one or more nucleic acids encoding an Ago protein, a guide DNA sequence, a donor nucleic acid sequence and/or a transcriptional regulator may be present within the same vector or present within different vectors. 
     Vectors according to the present disclosure include those known in the art as being useful in delivering genetic material into a cell and would include regulators, promoters, nuclear localization signals (NLS), start codons, stop codons, a transgene etc., and any other genetic elements useful for integration and expression, as are known to those of skill in the art. 
     AGO Description 
     Argonaute (Ago) proteins, involved in RNA interference pathways, use small single-stranded RNA (ssRNA) to facilitate RNA interference of complementary ssRNA targets. See G. Meister, Argonaute proteins: functional insights and emerging roles.  Nature reviews. Genetics  14, 447-459 (2013). Ago from  T. thermophiles  (TtAgo) was reported to prevent infection and propagation of foreign DNA by loading 5′-phosphorylated small interfering DNA (siDNA) guides approximately 20 base pairs (bp) in length to cut complementary single stranded DNA. See D. C. Swarts, M. M. Jore, E. R. Westra, Y. Zhu, J. H. Janssen, A. P. Snijders, Y. Wang, D. J. Patel, J. Berenguer, S. J. Brouns, J. van der Oost, DNA-guided DNA interference by a prokaryotic Argonaute.  Nature  507, 258-261 (2014); G. Sheng, H. Zhao, J. Wang, Y. Rao, W. Tian, D. C. Swarts, J. van der Oost, D. J. Patel, Y. Wang, Structure-based cleavage mechanism of  Thermus thermophilus  Argonaute DNA guide strand-mediated DNA target cleavage.  Proc Nail Acad Sci U S A  111, 652-657 (2014); K. Nakanishi, D. E. Weinberg, D. P. Bartel, D. J. Patel, Structure of yeast Argonaute with guide RNA.  Nature  486, 368-374 (2012); Y. Wang, S. Juranek, H. Li, G. Sheng, T. Tuschl, D. J. Patel, Structure of an argonaute silencing complex with a seed-containing guide DNA and target RNA duplex.  Nature  456, 921-926 (2008). 
     An exemplary Ago protein is Ago from Thermus thermophiles (TtAgo) or a modification or homolog thereof. Sequences of Ago protein species are known to those of skill in the art. According to one aspect, the Ago enzyme, or the nuclease null Ago includes homologs and orthologs thereof and which retain the ability of the protein to bind to the DNA and be guided by the guide DNA sequence. According to one aspect, the Ago protein includes the sequence as known for naturally occurring Thermus thermophiles Ago and protein sequences having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% homology thereto and being a DNA binding protein. According to one aspect, the Ago protein includes protein sequences having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% homology to the naturally occurring Thermus thermophiles Ago and being a DNA binding protein. An exemplary Ago protein is Ago from haloalkaliphilic archaebacterium  N. gregoryi  SP2 (NgAgo) or a modification or homolog thereof. A non-exhaustive list of protein sequences that have been identified as relevant Ago proteins by searching the public databases such as (NCBI, GENBANK and UniProt) with the TtAgo sequence is provided below. 
     
       
         
           
               
               
               
             
               
                   
               
               
                 UniProt Sequence 
                   
                   
               
               
                 Entry Name 
                 UniProt Submitted Name 
                 Organism 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 Q53W94_THET8 
                 Uncharacterized protein 
                 Thermus thermophilus (strain 
                 0.00E+00 
               
               
                   
                   
                 HB8/ATCC 27634/DSM 579) 
               
               
                 Q8U3D2_PYRFU 
                 Uncharacterized protein 
                 Pyrococcus furiosus (strain 
                 0.00E+00 
               
               
                   
                   
                 ATCC 43587/DSM 3638/ 
               
               
                   
                   
                 JCM 8422/Vc1) 
               
               
                 K9XZC6_STAC7 
                 Stem cell self-renewal protein 
                 Stanieria cyanosphaera 
                 7.60E−60 
               
               
                   
                 Piwi 
                 (strain ATCC 29371/PCC 
               
               
                   
                   
                 7437) 
               
               
                 B1XJG0_SYNP2 
                 Uncharacterized protein 
                   Synechococcus  sp. (strain 
                 1.30E−58 
               
               
                   
                   
                 ATCC 27264/PCC 7002/PR-6) 
               
               
                 A0A0C1VHB4_9CYAN 
                 Uncharacterized protein 
                 Lyngbya confervoides 
                 1.60E−58 
               
               
                   
                   
                 BDU141951 
               
               
                 E0UCX3_CYAP2 
                 Stem cell self-renewal protein 
                   Cyanothece  sp. (strain PCC 
                 1.60E−55 
               
               
                   
                 Piwi domain protein 
                 7822) 
               
               
                 Q2JI93_SYNJB 
                 Piwi domain protein 
                   Synechococcus  sp. (strain JA- 
                 2.30E−45 
               
               
                   
                   
                 2-3B′a(2-13)) 
               
               
                 Q8DKB1_THEEB 
                 TII0948 protein 
                 Thermosynechococcus 
                 3.50E−44 
               
               
                   
                   
                 elongatus (strain BP-1) 
               
               
                 Q31N05_SYNE7 
                 Uncharacterized protein 
                 Synechococcus elongatus 
                 1.30E−42 
               
               
                   
                   
                 (strain PCC 7942) 
               
               
                 D3S0S6_FERPA 
                 Stem cell self-renewal protein 
                 Ferroglobus placidus (strain 
                 1.60E−40 
               
               
                   
                 Piwi domain protein 
                 DSM 10642/AEDII12DO) 
               
               
                 Y1321_METJA 
                 Uncharacterized protein 
                 Methanocaldococcus 
                 1.50E−35 
               
               
                   
                 MJ1321 
                 jannaschii (strain ATCC 43067/ 
               
               
                   
                   
                 DSM 2661/JAL-1/JCM 
               
               
                   
                   
                 10045/NBRC 100440) 
               
               
                 I3TE64_THEC1 
                 Uncharacterized protein 
                 Thermogladius cellulolyticus 
                 2.70E−30 
               
               
                   
                   
                 (strain 1633) 
               
               
                 Q5P0R2_AROAE 
                 Uncharacterized protein 
                 Aromatoleum aromaticum 
                 9.10E−22 
               
               
                   
                   
                 (strain EbN1) 
               
               
                 L0JS08_NATP1 
                 Uncharacterized protein 
                 Natrinema pellirubrum (strain 
                 2.60E−21 
               
               
                   
                   
                 DSM 15624/JCM 10476/ 
               
               
                   
                   
                 NCIMB 786) 
               
               
                 R5NJJ8_9FIRM 
                 Uncharacterized protein 
                   Eubacterium  sp. CAG: 603 
                 3.40E−21 
               
               
                 R7RTW6_9CLOT 
                 Uncharacterized protein 
                 Thermobrachium celere DSM 
                 1.50E−19 
               
               
                   
                   
                 8682 
               
               
                 R5XQK4_9CLOT 
                 Piwi domain protein 
                 Clostridium bartlettii 
                 1.50E−19 
               
               
                   
                   
                 CAG: 1329 
               
               
                 F8D8G0_HALXS 
                 Stem cell self-renewal protein 
                 Halopiger xanaduensis (strain 
                 1.20E−18 
               
               
                   
                 Piwi 
                 DSM 18323/JCM 14033/SH-6) 
               
               
                 Q53W94_THET9 
                 Uncharacterized protein 
                 Thermus thermophilus (strain 
                 0.00E+00 
               
               
                   
                   
                 HB8/ATCC 27634/DSM 579) 
               
               
                 Q8U3D2_PYRFU 
                 Uncharacterized protein 
                 Pyrococcus furiosus (strain 
                 0.00E+00 
               
               
                   
                   
                 ATCC 43587/DSM 3638/ 
               
               
                   
                   
                 JCM 8422/Vc1) 
               
               
                 K9XZC6_STAC8 
                 Stem cell self-renewal protein 
                 Stanieria cyanosphaera 
                 0.00E+00 
               
               
                   
                 Piwi 
                 (strain ATCC 29371/PCC 
               
               
                   
                   
                 7437) 
               
               
                 B1XJG0_SYNP3 
                 Uncharacterized protein 
                   Synechococcus  sp. (strain 
                 0.00E+00 
               
               
                   
                   
                 ATCC 27264/PCC 7002/PR-6) 
               
               
                 A0A0C1VHB4_9CYAN 
                 Uncharacterized protein 
                 Lyngbya confervoides 
                 0.00E+00 
               
               
                   
                   
                 BDU141952 
               
               
                 E0UCX3_CYAP3 
                 Stem cell self-renewal protein 
                   Cyanothece  sp. (strain PCC 
                 0.00E+00 
               
               
                   
                 Piwi domain protein 
                 7822) 
               
               
                 Q2JI93_SYNJB 
                 Piwi domain protein 
                   Synechococcus  sp. (strain JA- 
                 0.00E+00 
               
               
                   
                   
                 2-3B′a(2-13)) 
               
               
                 Q8DKB1_THEEB 
                 TII0948 protein 
                 Thermosynechococcus 
                 0.00E+00 
               
               
                   
                   
                 elongatus (strain BP-1) 
               
               
                 Q31N05_SYNE8 
                 Uncharacterized protein 
                 Synechococcus elongatus 
                 0.00E+00 
               
               
                   
                   
                 (strain PCC 7942) 
               
               
                 Q53W94_THET9 
                 Uncharacterized protein 
                 Thermus thermophilus (strain 
                 0.00E+00 
               
               
                   
                   
                 HB8/ATCC 27634/DSM 579) 
               
               
                 Q8U3D2_PYRFU 
                 Uncharacterized protein 
                 Pyrococcus furiosus (strain 
                 0.00E+00 
               
               
                   
                   
                 ATCC 43587/DSM 3638/ 
               
               
                   
                   
                 JCM 8422/Vc1) 
               
               
                 K9XZC6_STAC8 
                 Stem cell self-renewal protein 
                 Stanieria cyanosphaera 
                 0.00E+00 
               
               
                   
                 Piwi 
                 (strain ATCC 29371/PCC 
               
               
                   
                   
                 7437) 
               
               
                 B1XJG0_SYNP3 
                 Uncharacterized protein 
                   Synechococcus  sp. (strain 
                 0.00E+00 
               
               
                   
                   
                 ATCC 27264/PCC 7002/PR-6) 
               
               
                 A0A0C1VHB4_9CYAN 
                 Uncharacterized protein 
                 Lyngbya confervoides 
                 0.00E+00 
               
               
                   
                   
                 BDU141952 
               
               
                 E0UCX3_CYAP3 
                 Stem cell self-renewal protein 
                   Cyanothece  sp. (strain PCC 
                 0.00E+00 
               
               
                   
                 Piwi domain protein 
                 7822) 
               
               
                 Q2JI93_SYNJB 
                 Piwi domain protein 
                   Synechococcus  sp. (strain JA- 
                 0.00E+00 
               
               
                   
                   
                 2-3B′a(2-13)) 
               
               
                 Q8DKB1_THEEB 
                 TII0948 protein 
                 Thermosynechococcus 
                 0.00E+00 
               
               
                   
                   
                 elongatus (strain BP-1) 
               
               
                 Q31N05_SYNE8 
                 Uncharacterized protein 
                 Synechococcus elongatus 
                 0.00E+00 
               
               
                   
                   
                 (strain PCC 7942) 
               
               
                 D3S0S6_FERPA 
                 Stem cell self-renewal protein 
                 Ferroglobus placidus (strain 
                 0.00E+00 
               
               
                   
                 Piwi domain protein 
                 DSM 10642/AEDII12DO) 
               
               
                 Y1321_METJA 
                 Uncharacterized protein 
                 Methanocaldococcus 
                 0.00E+00 
               
               
                   
                 MJ1322 
                 jannaschii (strain ATCC 43067/ 
               
               
                   
                   
                 DSM 2661/JAL-1/JCM 
               
               
                   
                   
                 10045/NBRC 100440) 
               
               
                 I3TE64_THEC2 
                 Uncharacterized protein 
                 Thermogladius cellulolyticus 
                 0.00E+00 
               
               
                   
                   
                 (strain 1633) 
               
               
                 Q5P0R2_AROAE 
                 Uncharacterized protein 
                 Aromatoleum aromaticum 
                 0.00E+00 
               
               
                   
                   
                 (strain EbN1) 
               
               
                 L0JS08_NATP2 
                 Uncharacterized protein 
                 Natrinema pellirubrum (strain 
                 0.00E+00 
               
               
                   
                   
                 DSM 15624/JCM 10476/ 
               
               
                   
                   
                 NCIMB 786) 
               
               
                 R5NJJ8_9FIRM 
                 Uncharacterized protein 
                   Eubacterium  sp. CAG: 604 
                 0.00E+00 
               
               
                 Q53W94_THET9 
                 Uncharacterized protein 
                 Thermus thermophilus (strain 
                 0.00E+00 
               
               
                   
                   
                 HB8/ATCC 27634/DSM 579) 
               
               
                 Q8U3D2_PYRFU 
                 Uncharacterized protein 
                 Pyrococcus furiosus (strain 
                 0.00E+00 
               
               
                   
                   
                 ATCC 43587/DSM 3638/ 
               
               
                   
                   
                 JCM 8422/Vc1) 
               
               
                 K9XZC6_STAC8 
                 Stem cell self-renewal protein 
                 Stanieria cyanosphaera 
                 0.00E+00 
               
               
                   
                 Piwi 
                 (strain ATCC 29371/PCC 
               
               
                   
                   
                 7437) 
               
               
                 B1XJG0_SYNP3 
                 Uncharacterized protein 
                   Synechococcus  sp. (strain 
                 0.00E+00 
               
               
                   
                   
                 ATCC 27264/PCC 7002/PR-6) 
               
               
                 A0A0C1VHB4_9CYAN 
                 Uncharacterized protein 
                 Lyngbya confervoides 
                 0.00E+00 
               
               
                   
                   
                 BDU141952 
               
               
                 E0UCX3_CYAP3 
                 Stem cell self-renewal protein 
                   Cyanothece  sp. (strain PCC 
                 0.00E+00 
               
               
                   
                 Piwi domain protein 
                 7822) 
               
               
                 Q2JI93_SYNJB 
                 Piwi domain protein 
                   Synechococcus  sp. (strain JA- 
                 0.00E+00 
               
               
                   
                   
                 2-3B′a(2-13)) 
               
               
                 Q8DKB1_THEEB 
                 TII0948 protein 
                 Thermosynechococcus 
                 0.00E+00 
               
               
                   
                   
                 elongatus (strain BP-1) 
               
               
                 Q31N05_SYNE8 
                 Uncharacterized protein 
                 Synechococcus elongatus 
                 0.00E+00 
               
               
                   
                   
                 (strain PCC 7942) 
               
               
                 D3S0S6_FERPA 
                 Stem cell self-renewal protein 
                 Ferroglobus placidus (strain 
                 0.00E+00 
               
               
                   
                 Piwi domain protein 
                 DSM 10642/AEDII12DO) 
               
               
                 Q53W94_THET9 
                 Uncharacterized protein 
                 Thermus thermophilus (strain 
                 0.00E+00 
               
               
                   
                   
                 HB8/ATCC 27634/DSM 579) 
               
               
                 Q8U3D2_PYRFU 
                 Uncharacterized protein 
                 Pyrococcus furiosus (strain 
                 0.00E+00 
               
               
                   
                   
                 ATCC 43587/DSM 3638/ 
               
               
                   
                   
                 JCM 8422/Vc1) 
               
               
                 K9XZC6_STAC8 
                 Stem cell self-renewal protein 
                 Stanieria cyanosphaera 
                 0.00E+00 
               
               
                   
                 Piwi 
                 (strain ATCC 29371/PCC 
               
               
                   
                   
                 7437) 
               
               
                 B1XJG0_SYNP3 
                 Uncharacterized protein 
                   Synechococcus  sp. (strain 
                 0.00E+00 
               
               
                   
                   
                 ATCC 27264/PCC 7002/PR-6) 
               
               
                 A0A0C1VHB4_9CYAN 
                 Uncharacterized protein 
                 Lyngbya confervoides 
                 0.00E+00 
               
               
                   
                   
                 BDU141952 
               
               
                 E0UCX3_CYAP3 
                 Stem cell self-renewal protein 
                   Cyanothece  sp. (strain PCC 
                 0.00E+00 
               
               
                   
                 Piwi domain protein 
                 7822) 
               
               
                 Q2JI93_SYNJB 
                 Piwi domain protein 
                   Synechococcus  sp. (strain JA- 
                 0.00E+00 
               
               
                   
                   
                 2-3B′a(2-13)) 
               
               
                 Q8DKB1_THEEB 
                 TII0948 protein 
                 Thermosynechococcus 
                 0.00E+00 
               
               
                   
                   
                 elongatus (strain BP-1) 
               
               
                 Q31N05_SYNE8 
                 Uncharacterized protein 
                 Synechococcus elongatus 
                 0.00E+00 
               
               
                   
                   
                 (strain PCC 7942) 
               
               
                 D3S0S6_FERPA 
                 Stem cell self-renewal protein 
                 Ferroglobus placidus (strain 
                 0.00E+00 
               
               
                   
                 Piwi domain protein 
                 DSM 10642/AEDII12DO) 
               
               
                 Y1321_METJA 
                 Uncharacterized protein 
                 Methanocaldococcus 
                 0.00E+00 
               
               
                   
                 MJ1322 
                 jannaschii (strain ATCC 43067/ 
               
               
                   
                   
                 DSM 2661/JAL-1/JCM 
               
               
                   
                   
                 10045/NBRC 100440) 
               
               
                 I3TE64_THEC2 
                 Uncharacterized protein 
                 Thermogladius cellulolyticus 
                 0.00E+00 
               
               
                   
                   
                 (strain 1633) 
               
               
                 Q5P0R2_AROAE 
                 Uncharacterized protein 
                 Aromatoleum aromaticum 
                 0.00E+00 
               
               
                   
                   
                 (strain EbN1) 
               
               
                 L0JS08_NATP2 
                 Uncharacterized protein 
                 Natrinema pellirubrum (strain 
                 0.00E+00 
               
               
                   
                   
                 DSM 15624/JCM 10476/ 
               
               
                   
                   
                 NCIMB 786) 
               
               
                 R5NJJ8_9FIRM 
                 Uncharacterized protein 
                   Eubacterium  sp. CAG: 604 
                 0.00E+00 
               
               
                 R7RTW6_9CLOT 
                 Uncharacterized protein 
                 Thermobrachium celere DSM 
                 0.00E+00 
               
               
                   
                   
                 8683 
               
               
                 Q53W94_THET9 
                 Uncharacterized protein 
                 Thermus thermophilus (strain 
                 0.00E+00 
               
               
                   
                   
                 HB8/ATCC 27634/DSM 579) 
               
               
                 Q8U3D2_PYRFU 
                 Uncharacterized protein 
                 Pyrococcus furiosus (strain 
                 0.00E+00 
               
               
                   
                   
                 ATCC 43587/DSM 3638/ 
               
               
                   
                   
                 JCM 8422/Vc1) 
               
               
                 K9XZC6_STAC8 
                 Stem cell self-renewal protein 
                 Stanieria cyanosphaera 
                 0.00E+00 
               
               
                   
                 Piwi 
                 (strain ATCC 29371/PCC 
               
               
                   
                   
                 7437) 
               
               
                 B1XJG0_SYNP3 
                 Uncharacterized protein 
                   Synechococcus  sp. (strain 
                 0.00E+00 
               
               
                   
                   
                 ATCC 27264/PCC 7002/PR-6) 
               
               
                 A0A0C1VHB4_9CYAN 
                 Uncharacterized protein 
                 Lyngbya confervoides 
                 0.00E+00 
               
               
                   
                   
                 BDU141952 
               
               
                 Q53W94_THET9 
                 Uncharacterized protein 
                 Thermus thermophilus (strain 
                 0.00E+00 
               
               
                   
                   
                 HB8/ATCC 27634/DSM 579) 
               
               
                 Q8U3D2_PYRFU 
                 Uncharacterized protein 
                 Pyrococcus furiosus (strain 
                 0.00E+00 
               
               
                   
                   
                 ATCC 43587/DSM 3638/ 
               
               
                   
                   
                 JCM 8422/Vc1) 
               
               
                 K9XZC6_STAC8 
                 Stem cell self-renewal protein 
                 Stanieria cyanosphaera 
                 0.00E+00 
               
               
                   
                 Piwi 
                 (strain ATCC 29371/PCC 
               
               
                   
                   
                 7437) 
               
               
                 B1XJG0_SYNP3 
                 Uncharacterized protein 
                   Synechococcus  sp. (strain 
                 0.00E+00 
               
               
                   
                   
                 ATCC 27264/PCC 7002/PR-6) 
               
               
                 A0A0C1VHB4_9CYAN 
                 Uncharacterized protein 
                 Lyngbya confervoides 
                 0.00E+00 
               
               
                   
                   
                 BDU141952 
               
               
                   
               
            
           
         
       
     
     It is well known in the art that eukaryotic Argonaute proteins are involved in small RNA-mediated silencing mechanisms such as RNA interference (RNAi), microRNA repression and piRNA-mediated transposon silencing. Argonautes bind small RNAs that guide them to RNA targets in order to regulate gene expression and repress invasive genomic elements. Carthew R W, Sontheimer E J., Origins and Mechanisms of miRNAs and siRNAs,  Cell . Vol. 136, Pp 642-655, (2009). Prokaryotic Argonaute proteins are found in many bacterial and archaeal species and are well documented in the art. Exemplary prokaryotic Argonaute proteins described in the following references including supplementary information are hereby incorporated by reference in their entireties. For example, bioinformatics analysis and the phylogenetic distribution of Argonaute proteins in bacteria and archaea indicate that about 20% of sequenced strains contain at least one Ago gene. Makarova K S, et al., Prokaryotic homologs of Argonaute proteins are predicted to function as key components of a novel system of defense against mobile genetic elements.  Biology direct.  4:29, (2009). Molloy S, Bacterial argonaute sets sail,  Nature Reviews Microbiology , Vol. 11, Pp 743, (2013). Bacterial  Aquifex aeolicus  and archaeal  Pyrococcus furiosus  Ago proteins have also been reported and well known, e.g., via structural studies. Song J J, et al., Crystal structure of Argonaute and its implications for RISC slicer activity, Science, Vol. 305, Pp 1434-1437, (2004). Yuan Y R, et al., Crystal structure of  A. aeolicus  argonaute, a site-specific DNA-guided endoribonuclease, provides insights into RISC-mediated mRNA cleavage,  Mol Cell., Vol.  19, Pp 405-419, (2005). It has been known in the art that several prokaryotic Agos, unlike their eukaryotic counterparts, bind single-stranded DNA guides in vitro for cleavage of RNA targets. The presence of an Ago gene correlated with other known genome protection systems suggests roles of prokaryotic Agos for protecting cells from invasive nucleic acids. 
     Argonautes have also been known in the gram-negative bacteria  Rhodobacter sphaeroides  (RsAgo) and  Thermus thermophilus  (TtAgo). Olovnikov I, et al., Bacterial argonaute samples the transcriptome to identify foreign DNA,  Molecular cell ., Vol. 51, Pp: 594-605, (2013). Swarts DC, et al. DNA-guided DNA interference by a prokaryotic Argonaute,  Nature , Vol. 507, Pp: 258-261, (2014). These Agos from both bacterial species directly target DNA molecules and protect the genome against foreign and possibly invasive genomic elements, such as plasmids. However, there are important differences between Agos from both bacterial species in that RsAgo uses small RNAs as guide molecules, whereas TtAgo uses small DNAs as guide molecules. The Ago 5′ binding pocket discriminates its guide by the nature of the first nucleotide. For example, in  Rhodobacter sphaeroides , there is a strong nucleotide bias for uridine at the 5′ position for RsAgo-associated small RNA guide molecules. Whereas in Thermus thermophiles, there is a strong nucleotide bias for deoxycytidine at the 5′ position for TtAgo-associated small DNA guide molecules. In the case of TtAgo, its endonuclease activity cleaves DNA targets in the middle of the region targeted by the guide DNA. Unlike the CRISPR-Cas system, which is another prokaryotic genomic host defense system, where effector proteins associate with host-encoded RNA guides to determine DNA cleavage loci, the Agos in both bacteria  Rhodobacter sphaeroides  and  Thermus thermophilus  recognize properties inherent to invading DNAs and silence them without using small RNA or DNA guides encoded in separate genomic loci. Hur, J K., et al., Prokaryotic Argonautes defend genomes against invasive DNA,  Trends Biochem Sci ., June, Vol. 39(6): 257-259, (2014), Wiedenheft B, et al., RNA-guided genetic silencing systems in bacteria and archaea,  Nature , Vol. 482, Pp 331-338, (2012). 
     According to certain aspects, an Ago enzyme is altered or otherwise modified to inactivate the nuclease activity. Such alteration or modification includes altering one or more amino acids to inactivate the nuclease activity or the nuclease domain. For example, modifications at D478A and D546A result in a nuclease null Ago protein. Such modification includes removing the polypeptide sequence or polypeptide sequences exhibiting nuclease activity, i.e. the nuclease domain, such that the polypeptide sequence or polypeptide sequences exhibiting nuclease activity, i.e. nuclease domain, are absent from the Ago protein. Other modifications to inactivate nuclease activity will be readily apparent to one of skill in the art based on the present disclosure. The nuclease-null Ago protein retains the ability to bind to DNA even though the nuclease activity has been inactivated. Accordingly, the nuclease null Ago protein includes the polypeptide sequence or sequences required for DNA binding but may lack the one or more or all of the nuclease sequences exhibiting nuclease activity or may be altered to render the nuclease sequences inactive. 
     According to certain aspects, the Ago protein may be delivered directly to a cell as a native species by methods known to those of skill in the art, including injection or lipofection, or as transcribed from its cognate DNA, with the cognate DNA introduced into cells through electroporation, transient and stable transfection (including lipofection) and viral transduction. 
     According to one aspect, the Ago protein is exogenous to the cell. According to one aspect, the Ago protein is foreign to the cell. According to one aspect, the Ago protein is non-naturally occurring within the cell. 
     According to one aspect, the nucleic acid sequence encoding the TtAgo protein or its variants is codon optimized for optimal host cell expression. In one embodiment, the nucleic acid sequence encoding the TtAgo protein or its variants is codon optimized for human cell expression. 
     CAS9 Helicase Description 
     Cas9 proteins and Type II CRISPR systems are well documented in the art. See Makarova et al.,  Nature Reviews, Microbiology , Vol. 9, June 2011, pp. 467-477 including all supplementary information hereby incorporated by reference in its entirety. 
     In general, bacterial and archaeal CRISPR-Cas systems rely on short guide RNAs in complex with Cas proteins to direct degradation of complementary sequences present within invading foreign nucleic acid. See Deltcheva, E. et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.  Nature  471, 602-607 (2011); Gasiunas, G., Barrangou, R., Horvath, P. &amp; Siksnys, V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria.  Proceedings of the National Academy of Sciences of the United States of America  109, E2579-2586 (2012); Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.  Science  337, 816-821 (2012); Sapranauskas, R. et al. The  Streptococcus thermophilus  CRISPR/Cas system provides immunity in  Escherichia coli. Nucleic acids research  39, 9275-9282 (2011); and Bhaya, D., Davison, M. &amp; Barrangou, R. CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation.  Annual review of genetics  45, 273-297 (2011). A recent in vitro reconstitution of the  S. pyogenes  type II CRISPR system demonstrated that crRNA (“CRISPR RNA”) fused to a normally trans-encoded tracrRNA (“trans-activating CRISPR RNA”) is sufficient to direct Cas9 protein to sequence-specifically cleave target DNA sequences matching the crRNA. Expressing a gRNA homologous to a target site results in Cas9 recruitment and degradation of the target DNA. See H. Deveau et al., Phage response to CRISPR-encoded resistance in  Streptococcus thermophilus. Journal of Bacteriology  190, 1390 (February 2008). 
     Three classes of CRISPR systems are generally known and are referred to as Type I, Type II or Type III). According to one aspect, a particular useful enzyme according to the present disclosure to cleave dsDNA is the single effector enzyme, Cas9, common to Type II. See K. S. Makarova et al., Evolution and classification of the CRISPR-Cas systems.  Nature reviews. Microbiology  9, 467 (June 2011) hereby incorporated by reference in its entirety. Within bacteria, the Type II effector system consists of a long pre-crRNA transcribed from the spacer-containing CRISPR locus, the multifunctional Cas9 protein, and a tracrRNA important for gRNA processing. The tracrRNAs hybridize to the repeat regions separating the spacers of the pre-crRNA, initiating dsRNA cleavage by endogenous RNase III, which is followed by a second cleavage event within each spacer by Cas9, producing mature crRNAs that remain associated with the tracrRNA and Cas9. TracrRNA-crRNA fusions are contemplated for use in the present methods. 
     Cas9 unwinds the DNA duplex and searches for sequences matching the crRNA to cleave. Target recognition occurs upon detection of complementarity between a “protospacer” sequence in the target DNA and the remaining spacer sequence in the crRNA. Importantly, Cas9 cuts the DNA only if a correct protospacer-adjacent motif (PAM) is also present at the 3′ end. According to certain aspects, different protospacer-adjacent motif can be utilized. For example, the  S. pyogenes  system requires an NGG sequence, where N can be any nucleotide.  S. thermophilus  Type II systems require NGGNG (see P. Horvath, R. Barrangou, CRISPR/Cas, the immune system of bacteria and archaea.  Science  327, 167 (Jan. 8, 2010) hereby incorporated by reference in its entirety and NNAGAAW (see H. Deveau et al., Phage response to CRISPR-encoded resistance in  Streptococcus thermophilus. Journal of bacteriology  190, 1390 (Feb, 2008) hereby incorporated by reference in its entirety), respectively, while different  S. mutans  systems tolerate NGG or NAAR (see J. R. van der Ploeg, Analysis of CRISPR in  Streptococcus  mutans suggests frequent occurrence of acquired immunity against infection by M102-like bacteriophages.  Microbiology  155, 1966 (June 2009) hereby incorporated by reference in its entirety. Bioinformatic analyses have generated extensive databases of CRISPR loci in a variety of bacteria that may serve to identify additional useful PAMs and expand the set of CRISPR-targetable sequences (see M. Rho, Y. W. Wu, H. Tang, T. G. Doak, Y. Ye, Diverse CRISPRs evolving in human microbiomes.  PLoS genetics  8, e1002441 (2012) and D. T. Pride et al., Analysis of streptococcal CRISPRs from human saliva reveals substantial sequence diversity within and between subjects over time.  Genome research  21, 126 (Jan, 2011) each of which are hereby incorporated by reference in their entireties. 
     In  S. pyogenes , Cas9 generates a blunt-ended double-stranded break 3 bp upstream of the protospacer-adjacent motif (PAM) via a process mediated by two catalytic domains in the protein: an HNH domain that cleaves the complementary strand of the DNA and a RuvC-like domain that cleaves the non-complementary strand. See Jinek et al.,  Science  337, 816-821 (2012) hereby incorporated by reference in its entirety. Cas9 proteins are known to exist in many Type II CRISPR systems including the following as identified in the supplementary information to Makarova et al., Nature Reviews, Microbiology, Vol. 9, June 2011, pp. 467-477:  Methanococcus maripaludis  C7;  Corynebacterium diphtheriae; Corynebacterium efficiens  YS-314;  Corynebacterium glutamicum  ATCC 13032 Kitasato;  Corynebacterium glutamicum  ATCC 13032 Bielefeld;  Corynebacterium glutamicum  R;  Corynebacterium kroppenstedtii  DSM 44385;  Mycobacterium abscessus  ATCC 19977;  Nocardia farcinica  IFM10152;  Rhodococcus erythropolis  PR4;  Rhodococcus jostii  RHA1;  Rhodococcus opacus  B4 uid36573;  Acidothermus cellulolyticus  11B;  Arthrobacter chlorophenolicus  A6;  Kribbella flavida  DSM 17836 uid43465;  Thermomonospora curvata  DSM 43183;  Bifidobacterium dentium  Bd1;  Bifidobacterium longum  DJO10A;  Slackia heliotrinireducens  DSM 20476;  Persephonella marina  EX H1 ; Bacteroides fragilis  NCTC 9434;  Capnocytophaga ochracea  DSM 7271;  Flavobacterium psychrophilum  JIP02 86;  Akkermansia muciniphila  ATCC BAA 835;  Roseiflexus castenholzii  DSM 13941;  Roseiflexus  RS1;  Synechocystis  PCC6803;  Elusimicrobium minutum  Pei191; uncultured Termite group 1 bacterium phylotype Rs D17;  Fibrobacter succinogenes  S85;  Bacillus cereus  ATCC 10987;  Listeria innocua; Lactobacillus casei; Lactobacillus rhamnosus  GG;  Lactobacillus salivarius  UCC118;  Streptococcus agalactiae  A909;  Streptococcus agalactiae  NEM316;  Streptococcus agalactiae  2603;  Streptococcus dysgalactiae equisimilis  GGS 124;  Streptococcus equi zooepidemicus  MGCS10565;  Streptococcus gallolyticus  UCN34 uid46061;  Streptococcus gordonii  Challis subst CH1;  Streptococcus mutans  NN2025 uid46353;  Streptococcus mutans; Streptococcus pyogenes  M1 GAS;  Streptococcus pyogenes  MGAS5005;  Streptococcus pyogenes  MGAS2096;  Streptococcus pyogenes  MGAS9429;  Streptococcus pyogenes  MGAS10270;  Streptococcus pyogenes  MGAS6180;  Streptococcus pyogenes  MGAS315;  Streptococcus pyogenes  SSI-1;  Streptococcus pyogenes  MGAS10750;  Streptococcus pyogenes  NZ131;  Streptococcus thermophiles  CNRZ1066;  Streptococcus thermophiles  LMD-9;  Streptococcus thermophiles  LMG 18311;  Clostridium botulinum  A3 Loch Maree;  Clostridium botulinum  B Eklund 17B;  Clostridium botulinum  Ba4 657;  Clostridium botulinum  F Langeland;  Clostridium cellulolyticum  H10;  Finegoldia magna  ATCC 29328;  Eubacterium rectale  ATCC 33656;  Mycoplasma gallisepticum; Mycoplasma mobile  163K;  Mycoplasma penetrans; Mycoplasma synoviae  53;  Streptobacillus moniliformis  DSM 12112;  Bradyrhizobium  BTAi 1;  Nitrobacter hamburgensis  X14;  Rhodopseudomonas palustris  BisB18;  Rhodopseudomonas palustris  BisB5;  Parvibaculum lavamentivorans  DS-1;  Dinoroseobacter shibae  DFL 12;  Gluconacetobacter diazotrophicus  Pal 5 FAPERJ;  Gluconacetobacter diazotrophicus  Pal 5 JGI;  Azospirillum B 510 uid46085;  Rhodospirillum rubrum  ATCC 11170;  Diaphorobacter  TPSY uid29975;  Verminephrobacter eiseniae  EF01-2;  Neisseria meningitides  053442;  Neisseria meningitides  alphal4;  Neisseria meningitides  Z2491;  Desulfovibrio salexigens  DSM 2638;  Campylobacter jejuni doylei  269 97;  Campylobacter jejuni  81116;  Campylobacter jejuni; Campylobacter lari  RM2100;  Helicobacter hepaticus; Wolinella succinogenes; Tolumonas auensis  DSM 9187;  Pseudoalteromonas atlantica  T6c;  Shewanella pealeana  ATCC 700345;  Legionella pneumophila  Paris;  Actinobacillus succinogenes  130Z;  Pasteurella multocida; Francisella tularensis novicida  U112;  Francisella tularensis holarctica; Francisella tularensis  FSC 198;  Francisella tularensis tularensis; Francisella tularensis  WY96-3418; and  Treponema denticola  ATCC 35405. The Cas9 protein may be referred by one of skill in the art in the literature as Csn1. An exemplary  S. pyogenes  Cas9 protein sequence is provided in Deltcheva et al.,  Nature  471, 602-607 (2011) hereby incorporated by reference in its entirety. 
     Modification to the Cas9 protein is a representative embodiment of the present disclosure. CRISPR systems useful in the present disclosure are described in R. Barrangou, P. Horvath, CRISPR: new horizons in phage resistance and strain identification.  Annual review of food science and technology  3, 143 (2012) and B. Wiedenheft, S. H. Sternberg, J. A. Doudna, RNA-guided genetic silencing systems in bacteria and archaea.  Nature  482, 331 (Feb. 16, 2012) each of which are hereby incorporated by reference in their entireties. 
     According to certain aspects, Cas9 is altered or otherwise modified to inactivate the nuclease activity. Such alteration or modification includes altering one or more amino acids to inactivate the nuclease activity or the nuclease domain. Such modification includes removing the polypeptide sequence or polypeptide sequences exhibiting nuclease activity, i.e. the nuclease domain, such that the polypeptide sequence or polypeptide sequences exhibiting nuclease activity, i.e. nuclease domain, are absent from the DNA binding protein. Other modifications to inactivate nuclease activity will be readily apparent to one of skill in the art based on the present disclosure. Accordingly, a nuclease-null DNA binding protein, such as a nuclease null Cas9 protein, includes polypeptide sequences modified to inactivate nuclease activity or removal of a polypeptide sequence or sequences to inactivate nuclease activity. The nuclease-null Cas9 retains the ability to bind to DNA even though the nuclease activity has been inactivated. Accordingly, the nuclease null Cas9 includes the polypeptide sequence or sequences required for DNA binding but may lack the one or more or all of the nuclease sequences exhibiting nuclease activity. 
     According to an additional aspect, nuclease-null Cas9 proteins are provided where one or more amino acids in Cas9 are altered or otherwise removed to provide nuclease-null Cas9 proteins. According to one aspect, the amino acids include D10 and H840. See Jinek et al.,  Science  337, 816-821 (2012). According to an additional aspect, the amino acids include D839 and N863. According to one aspect, one or more or all of D10, H840, D839 and H863 are substituted with an amino acid which reduces, substantially eliminates or eliminates nuclease activity. According to one aspect, one or more or all of D10, H840, D839 and H863 are substituted with alanine. According to one aspect, a Cas9 protein having one or more or all of D10, H840, D839 and H863 substituted with an amino acid which reduces, substantially eliminates or eliminates nuclease activity, such as alanine, is referred to as a nuclease-null Cas9 (“Cas9Nuc”) and exhibits reduced or eliminated nuclease activity, or nuclease activity is absent or substantially absent within levels of detection. According to this aspect, nuclease activity for a Cas9Nuc may be undetectable using known assays, i.e. below the level of detection of known assays. 
     An exemplary CRISPR system includes the  S. pyogenes  Cas9 nuclease (Sp. Cas9), an extremely high-affinity (see Sternberg, S. H., Redding, S., Jinek, M., Greene, E. C. &amp; Doudna, J. A. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507, 62-67 (2014) hereby incorporated by reference in its entirety), programmable DNA-binding protein isolated from a type II CRISPR-associated system (see Garneau, J. E. et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA.  Nature  468, 67-71 (2010) and Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.  Science  337, 816-821 (2012) each of which are hereby incorporated by reference in its entirety). According to certain aspects, a nuclease null or nuclease deficient Cas 9 can be used in the methods described herein. Such nuclease null or nuclease deficient Cas9 proteins are described in Gilbert, L. A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes.  Cell  154, 442-451 (2013); Mali, P. et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering.  Nature biotechnology  31, 833-838 (2013); Maeder, M. L. et al. CRISPR RNA-guided activation of endogenous human genes.  Nature methods  10, 977-979 (2013); and Perez-Pinera, P. et al. RNA-guided gene activation by CRISPR-Cas9-based transcription factors.  Nature methods  10, 973-976 (2013) each of which are hereby incorporated by reference in its entirety. The DNA locus targeted by Cas9 (and by its nuclease-deficient mutant, “dCas9” precedes a three nucleotide (nt) 5′-NGG-3′“PAM” sequence, and matches a 15-22-nt guide or spacer sequence within a Cas9-bound RNA cofactor, referred to herein and in the art as a guide RNA. Altering this guide RNA is sufficient to target Cas9 or a nuclease deficient Cas9 to a target nucleic acid. In a multitude of CRISPR-based biotechnology applications (see Mali, P., Esvelt, K. M. &amp; Church, G. M. Cas9 as a versatile tool for engineering biology.  Nature methods  10, 957-963 (2013); Hsu, P. D., Lander, E. S. &amp; Zhang, F. Development and Applications of CRISPR-Cas9 for Genome Engineering.  Cell  157, 1262-1278 (2014); Chen, B. et al. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system.  Cell  155, 1479-1491 (2013); Shalem, O. et al. Genome-scale CRISPR-Cas9 knockout screening in human cells.  Science  343, 84-87 (2014); Wang, T., Wei, J. J., Sabatini, D. M. &amp; Lander, E. S. Genetic screens in human cells using the CRISPR-Cas9 system.  Science  343, 80-84 (2014); Nissim, L., Perli, S. D., Fridkin, A., Perez-Pinera, P. &amp; Lu, T. K. Multiplexed and Programmable Regulation of Gene Networks with an Integrated RNA and CRISPR/Cas Toolkit in Human Cells.  Molecular cell  54, 698-710 (2014); Ryan, O. W. et al. Selection of chromosomal DNA libraries using a multiplex CRISPR system.  eLife  3 (2014); Gilbert, L. A. et al. Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation.  Cell  (2014); and Citorik, R. J., Mimee, M. &amp; Lu, T. K. Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases.  Nature biotechnology  (2014) each of which are hereby incorporated by reference in its entirety), the guide is often presented in a so-called sgRNA (single guide RNA), wherein the two natural Cas9 RNA cofactors (gRNA and tracrRNA) are fused via an engineered loop or linker. 
     According to certain aspects, the helicase, such as a nuclease null Cas9, may be delivered directly to a cell as a native species by methods known to those of skill in the art, including injection or lipofection, or as transcribed from its cognate DNA, with the cognate DNA introduced into cells through electroporation, transient and stable transfection (including lipofection) and viral transduction. 
     According to one aspect, the Cas9 protein is exogenous to the cell. According to one aspect, the Cas9 protein is foreign to the cell. According to one aspect, the Cas9 protein is non-naturally occurring within the cell. 
     Guide DNA Description 
     Embodiments of the present disclosure are directed to the use of an Ago system and, in particular, a guide DNA which includes a spacer sequence. The term spacer sequence is understood by those of skill in the art and may include any polynucleotide having sufficient complementarity with a target nucleic acid sequence to hybridize with the target nucleic acid sequence and direct sequence-specific binding of an Ago protein to the target nucleic acid sequence. According to certain aspects, the spacer sequence is between about 1 and about 5 nucleotides in length. Aspects of the present disclosure are directed to methods of making or providing such guide DNA sequences as described herein by expressing constructs encoding such guide DNA using promoters and terminators and optionally other genetic elements as described herein. 
     According to certain aspects, the guide DNA may be delivered directly to a cell as a native species by methods known to those of skill in the art, including injection or lipofection, or as transcribed from its cognate DNA, with the cognate DNA introduced into cells through electroporation, transient and stable transfection (including lipofection) and viral transduction. 
     According to one aspect, an exemplary guide DNA is a 5′-phosphorylated DNA sequence. According to one aspect, an exemplary guide DNA is a 20 bp DNA sequence. According to one aspect, an exemplary guide DNA is a 5′-phosphorylated 20 bp DNA sequence. According to one aspect, the spacer sequence need not hybridize 100% to the target nucleic acid sequence. According to one aspect, the spacer sequence may include between 1 to 5 mismatches with the target nucleic acid sequence. 
     According to one aspect, the guide DNA is exogenous to the cell. According to one aspect, the guide DNA is foreign to the cell. According to one aspect, the guide DNA is non-naturally occurring within the cell. 
     Donor Description 
     The term “donor nucleic acid” includes a nucleic acid sequence which is to be inserted into a target nucleic acid sequence which has been cut or nicked according to methods described herein. The donor nucleic acid sequence may be expressed by the cell. 
     According to one aspect, the donor nucleic acid is exogenous to the cell. According to one aspect, the donor nucleic acid is foreign to the cell. According to one aspect, the donor nucleic acid is non-naturally occurring within the cell. 
     Transcription Regulator Description 
     According to one aspect, an engineered Ago protein and guide DNA system is provided which enables DNA-guided nucleic acid regulation, such as genomic DNA regulation, in eukaryotic cells by tethering transcriptional regulatory proteins or domains to either a nuclease-null Ago protein or to a guide DNA. According to one aspect of the present disclosure, one or more transcriptional regulatory proteins or domains (such terms are used interchangeably) are joined or otherwise connected to a nuclease-deficient or nuclease null Ago protein or one or more guide DNA (gDNA). The transcriptional regulatory domains correspond to targeted loci. Accordingly, aspects of the present disclosure include methods and materials for localizing transcriptional regulatory domains to targeted loci by fusing, connecting or joining such domains to either a nuclease null Ago or to the gDNA. 
     According to one aspect, a nuclease null Ago-transcriptional regulator fusion capable of transcriptional regulation, either transcriptional activation or transcriptional repression, is provided. According to one aspect, a VP64 activation domain is joined, fused, connected or otherwise tethered to the C terminus of a nuclease null Ago protein. According to one method, the transcriptional regulatory domain is provided to the site of a target nucleic acid sequence by the nuclease null Ago protein and a guide DNA. According to one method, a nuclease null Ago protein attached, connected, bound or fused to a transcriptional regulatory domain is provided within a cell along with one or more guide DNA sequences. The nuclease null Ago protein with the transcriptional regulatory domain attached, connected, bound or fused thereto co-localize with a guide DNA at or near a target nucleic acid sequence. The transcriptional regulatory domain regulates expression of the target nucleic acid sequence. According to a specific aspect, a nuclease null Ago-VP64 construct activates transcription of a gene, such as a reporter construct, when combined with a gDNA(s) targeting a sequence(s) near the promoter, thereby displaying DNA-guided transcriptional activation. According to a specific aspect, a nuclease null Ago-repressor construct represses transcription of a gene, such as a reporter construct, when combined with a gDNA(s) targeting a sequence(s) near the promoter, thereby displaying DNA-guided transcriptional repression. According to a specific aspect, a nuclease null Ago represses transcription of a gene, such as a reporter construct, when combined with a gDNA(s) targeting a sequence(s) near the promoter, thereby displaying DNA-guided transcriptional repression by blocking transcription due to binding. 
     According to one aspect, a gDNA-transcriptional regulator fusion capable of transcriptional regulation, either transcriptional activation or transcriptional repression, is provided. According to one aspect, a VP64 activation domain is joined, fused, connected or otherwise tethered to a gDNA sequence. According to one method, the transcriptional regulatory domain is provided to the site of a target nucleic acid sequence by the guide DNA sequence. According to one method, a nuclease null Ago protein is provided within a cell along with a guide DNA sequence attached, connected, bound or fused to a transcriptional regulatory domain. The nuclease null Ago protein co-localizes with a guide DNA at or near a target nucleic acid sequence. The transcriptional regulatory domain regulates expression of the target nucleic acid sequence. According to a specific aspect, a gDNA-VP64 construct activates transcription of a gene, such as a reporter construct, when co-localized with a nuclease null Ago targeting a sequence(s) near the promoter, thereby displaying DNA-guided transcriptional activation. According to a specific aspect, a gDNA-repressor construct represses transcription of a gene, such as a reporter construct, when co-localized with a nuclease null Ago targeting a sequence(s) near the promoter, thereby displaying DNA-guided transcriptional repression. 
     Transcriptional regulator proteins or domains which are transcriptional activators include VP16 and VP64 and others readily identifiable by those skilled in the art based on the present disclosure. 
     According to one aspect, the transcriptional regulator or domain is a chromatin modifier, remodeller, or histone modifier including enzymes involved in histone acetylation, methylation, demethylation, phosphorylation, ubiquitination, sumoylation, ADP-ribosylation, deimination, and proline isomerization. Exemplary regulators include DNA methyltransferases, histone methyltransferases and demethylases, histone acetyltransferase and deacetylases, etc. are well known in the art. Non-limiting disclosures as found in the following references are hereby incorporated by reference in their entireties. Taiping Chen &amp; Sharon Y. R. Dent, Chromatin modifiers and remodellers: regulators of cellular differentiation,  Nature Reviews Genetics , Vol. 15, Pp: 93-106 (2014). Andrew J Bannister and Tony Kouzarides, Regulation of chromatin by histone modifications, Cell Research, Vol. 21, Pp 381-395, (2011). 
     According to one aspect, the transcriptional regulator protein or domain upregulates expression of the target nucleic acid sequence. 
     According to one aspect, the transcriptional regulator protein or domain upregulates expression of the target nucleic acid sequence to treat a disease or detrimental condition. 
     According to one aspect, the transcriptional regulator is exogenous to the cell. According to one aspect, the transcriptional regulator is foreign to the cell. According to one aspect, the transcriptional regulator is non-naturally occurring within the cell. 
     Target Nucleic Acid Sequence 
     A target nucleic acid sequence includes any nucleic acid sequence, such as a genomic nucleic acid sequence or a gene to which a co-localization complex as described herein can be useful to either cut, nick or regulate. Target nucleic acids include nucleic acid sequences capable of being expressed into proteins. According to one aspect, the target nucleic acid is genomic DNA, mitochondrial DNA, plastid DNA, viral DNA, exogenous DNA or cellular RNA. 
     For purposes of the present disclosure, DNA, such as genomic DNA, can include the target nucleic acid sequence and a co-localization complex can bind to or otherwise co-localize with the target nucleic acid sequence or adjacent or near the target nucleic acid sequence and in a manner in which the co-localization complex may have a desired effect on the target nucleic acid sequence. One of skill based on the present disclosure will readily be able to identify or design guide DNAs and Ago proteins which co-localize to a target nucleic acid sequence. According to one aspect, the target nucleic acid is an A-T rich nucleic acid sequence with an initiating 5′-phosphorylated nucleotide C. 
     Foreign Nucleic Acids Description 
     Foreign nucleic acids (i.e. those which are not part of a cell&#39;s natural nucleic acid composition) may be introduced into a cell using any method known to those skilled in the art for such introduction. Such methods include transfection, transduction, viral transduction, microinjection, lipofection, nucleofection, nanoparticle bombardment, transformation, conjugation and the like. One of skill in the art will readily understand and adapt such methods using readily identifiable literature sources. According to one aspect, a foreignnucleic acid is exogenous to the cell. According to one aspect, a foreign nucleic acid is non-naturally occurring within the cell. 
     Cells 
     Cells according to the present disclosure include any cell into which foreign nucleic acids can be introduced and expressed as described herein. It is to be understood that the basic concepts of the present disclosure described herein are not limited by cell type. Cells according to the present disclosure include eukaryotic cells, prokaryotic cells, animal cells, plant cells, fungal cells, archael cells, eubacterial cells and the like. Cells include eukaryotic cells such as yeast cells, plant cells, and animal cells. Particular cells include mammalian cells. Further, cells include any in which it would be beneficial or desirable to cut, nick or regulate a target nucleic acid. Such cells may include those which are deficient in expression of a particular protein leading to a disease or detrimental condition. Such diseases or detrimental conditions are readily known to those of skill in the art. According to the present disclosure, the nucleic acid responsible for expressing the particular protein may be targeted by the methods described herein and a transcriptional activator resulting in upregulation of the target nucleic acid and corresponding expression of the particular protein. In this manner, the methods described herein provide therapeutic treatment. Such cells may include those which over express a particular protein leading to a disease or detrimental condition. Such diseases or detrimental conditions are readily known to those of skill in the art. According to the present disclosure, the nucleic acid responsible for expressing the particular protein may be targeted by the methods described herein and a transcriptional depressor resulting in downregulation of the target nucleic acid and corresponding expression of the particular protein. In this manner, the methods described herein provide therapeutic treatment. 
     According to one aspect, the cell is a eukaryotic cell or a prokaryotic cell. According to one aspect, the cell is a yeast cell, bacterial cell, fungal cell, a plant cell or an animal cell. According to one aspect, the cell is a mammalian cell. According to one aspect, the cell is a human cell. According to one aspect, the cell is a stem cell whether adult or embryonic. According to one aspect, the cell is a pluripotent stem cell. According to one aspect, the cell is an induced pluripotent stem cell. According to one aspect, the cell is a human induced pluripotent stem cell. According to one aspect, the cell is in vitro, in vivo or ex vivo. 
     Vectors 
     Vectors are contemplated for use with the methods and constructs described herein. The term “vector” includes a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors used to deliver the nucleic acids to cells as described herein include vectors known to those of skill in the art and used for such purposes. Certain exemplary vectors may be plasmids, lentiviruses or adeno-associated viruses known to those of skill in the art. Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g. circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, wherein virally-derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g. retroviruses, lentiviruses, 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. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.” Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. Recombinant expression vectors can comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably linked” 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). 
     Methods of non-viral delivery of nucleic acids or native DNA binding protein, native guide RNA or other native species include lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Felgner, WO 91/17424; WO 91/16024. Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration). The term native includes the protein, enzyme or guide RNA species itself and not the nucleic acid encoding the species. 
     Regulatory Elements and Terminators and Tags 
     Regulatory elements are contemplated for use with the methods and constructs described herein. The term “regulatory element” is intended to 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. In some embodiments, a vector may comprise one or more pol III promoter (e.g. 1, 2, 3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g. 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g. 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof. Examples of pol III promoters include, but are not limited to, U6 and H1 promoters. Examples of pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1α promoter and Pol II promoters described herein. Also encompassed by the term “regulatory element” are enhancer elements, such as WPRE; CMV enhancers; the R-U5′ segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression desired, etc. A vector can be introduced into host cells to thereby produce transcripts, proteins, or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., clustered regularly interspersed short palindromic repeats (CRISPR) transcripts, proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.). 
     Aspects of the methods described herein may make use of terminator sequences. A terminator sequence includes a section of nucleic acid sequence that marks the end of a gene or operon in genomic DNA during transcription. This sequence mediates transcriptional termination by providing signals in the newly synthesized mRNA that trigger processes which release the mRNA from the transcriptional complex. These processes include the direct interaction of the mRNA secondary structure with the complex and/or the indirect activities of recruited termination factors. Release of the transcriptional complex frees RNA polymerase and related transcriptional machinery to begin transcription of new mRNAs. Terminator sequences include those known in the art and identified and described herein. 
     Aspects of the methods described herein may make use of epitope tags and reporter gene sequences. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase, betaglucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP). 
     Delivery Description 
     Embodiments of the present disclosure are directed to a method of delivering an Ago protein to cells within a subject comprising administering to the subject, such as systemically administering to the subject, such as by intravenous administration or injection, intraperitoneal administration or injection, intramuscular administration or injection, intracranial administration or injection, intraocular administration or injection, subcutaneous administration or injection, an Ago protein or a nucleic acid encoding the Ago protein. 
     Embodiments of the present disclosure are directed to a method of delivering a guide DNA to cells within a subject comprising administering to the subject, such as systemically administering to the subject, such as by intravenous administration or injection, intraperitoneal administration or injection, intramuscular administration or injection, intracranial administration or injection, intraocular administration or injection, subcutaneous administration or injection, a guide DNA or a nucleic acid encoding the guide DNA. 
     Embodiments of the present disclosure are directed to a method of delivering an Ago protein and a guide DNA to cells within a subject comprising administering to the subject, such as systemically administering to the subject, such as by intravenous administration or injection, intraperitoneal administration or injection, intramuscular administration or injection, intracranial administration or injection, intraocular administration or injection, subcutaneous administration or injection, an Ago protein or a nucleic acid encoding the Ago protein and a guide DNA or a nucleic acid encoding the guide DNA. 
     Diseases and Conditions 
     Diseases and detrimental conditions may be characterized by abnormal loss of expression or underexpression of a particular protein or abnormal gain or overexpression of a particular protein. Such diseases or detrimental conditions can be treated by upregulation or down regulation of the particular protein. Accordingly, methods of treating a disease or detrimental condition are provided where the co-localization complex as described herein associates or otherwise binds to a target nucleic acid and the transcriptional activator of the co-localization complex upregulates expression of the target nucleic acid or the transcriptional repressor of the co-localization complex downregulates expression of the target nucleic acid. One of skill in the art will readily identify such diseases and detrimental conditions based on the present disclosure. 
     The following examples are set forth as being representative of the present disclosure. These examples are not to be construed as limiting the scope of the present disclosure as these and other equivalent embodiments will be apparent in view of the present disclosure, figures and accompanying claims. 
     EXAMPLE I 
     Materials and Methods 
     Cell Lines 
     The 293 FT HEK cells were grown and maintained in Dulbecco&#39;s modified Eagle&#39;s medium (DMEM, Invitrogen) high glucose supplemented with 10% fetal bovine serum (Invitrogen), penicillin/streptomycin (pen/strep, Invitrogen) and nonessential amino acids (Invitrogen). 
     Primer Design 
     
         
         List of primers used herein are provided below. 
       
    
     
       
         
           
               
               
               
               
             
               
                   
                   
               
               
                   
                 NCBI Gene 
                   
                   
               
               
                   
                 Symbol 
                 Sequence 
                 SEQ ID NO: 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                   
                 TFAM 
                 TGGGAAGGTCTGGAGCA 
                 1 
               
               
                   
                   
                 GCCAAGACAGATGAAAACCAC 
                 2 
               
               
                   
                   
               
               
                   
                 NANOG 
                 ATGTCTTCTGCTGAGATGCC 
                 3 
               
               
                   
                   
                 GAAGTGGGTTGTTTGCCTTTG 
                 4 
               
               
                   
                   
               
               
                   
                 IL1B 
                 CAGCCAATCTTCATTGCTCAAG 
                 5 
               
               
                   
                   
                 GAACAAGTCATCCTCATTGCC 
                 6 
               
               
                   
                   
               
               
                   
                 TERT 
                 CATGCCAAGCTCTCGCT 
                 7 
               
               
                   
                   
                 GATCTCCTCACGCAGACG 
                 8 
               
               
                   
                   
               
               
                   
                 SOX2 
                 GCCTCACTTACAAGACAGCTC 
                 9 
               
               
                   
                   
                 GCCTCCAAGACCTAGCCTA 
                 10 
               
               
                   
                   
               
               
                   
                 KLF4 
                 TTGACTTTGGGGTTCAGGTG 
                 11 
               
               
                   
                   
                 GCGAACGTGGAGAAAGATGG 
                 12 
               
               
                   
                   
               
               
                   
                 cMYC 
                 TTCGGGTAGTGGAAAACCAG 
                 13 
               
               
                   
                   
                 CAGTAGAAATACGGCTGCAC 
                 14 
               
               
                   
                   
               
               
                   
                 RNA18s5 
                 GAGACTCTGGCATGCTAACTAG 
                 15 
               
               
                   
                   
                 GGACATCTAAGGGCATCACAG 
                 16 
               
               
                   
                   
               
               
                   
                 HBG1 
                 TGAATGTGGAAGATGCTGGA 
                 17 
               
               
                   
                   
                 TTGCCATGTGCCTTGACTT 
                 18 
               
               
                   
                   
               
               
                   
                 Oct4 
                 AATCCAGTCCCAGGACATCA 
                 19 
               
               
                   
                   
                 CGTTTGGCTGAATACCTTCC 
                 20 
               
               
                   
                   
               
               
                   
                 ASCL 
                 GGAGCTTCTCGACTTCACCA 
                 21 
               
               
                   
                   
                 AACGCCACTGACAAGAAAGC 
                 22 
               
               
                   
                   
               
            
           
         
       
         
         siDNA (guide DNA) 
         List of siDNAs used herein are provided below. 
       
    
     
       
         
           
               
             
               
                   
               
             
            
               
                 TtAgo NN -VP64 system 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 SEQ 
               
               
                 Name of 
                 Gene to  
                   
                 ID 
               
               
                 siDNA 
                 activate 
                 siDNA Sequence 
                 NO: 
               
               
                   
               
               
                 TFAM-1 
                 TFAM 
                 /5Phos/caaattattttagaaatcaa 
                 23 
               
               
                   
               
               
                 TFAM-4 
                 TFAM 
                 /5Phos/cagcggagcgtctcagttca 
                 24 
               
               
                   
               
               
                 TFAM-5 
                 TFAM 
                 /5Phos/cagccctggcttgaactgag 
                 25 
               
               
                   
               
               
                 TFAM-7 
                 TFAM 
                 /5Phos/cagaaatagtaacgggagag 
                 26 
               
               
                   
               
               
                 TFAM-8 
                 TFAM 
                 /5Phos/cacggaattaagctctgcgg 
                 27 
               
               
                   
               
               
                 Ago-GC- 
                 TFAM 
                 /5Phos/catgttttataaagtaatta 
                 28 
               
               
                 15-1 
                   
                   
                   
               
               
                   
               
               
                 Ago-GC- 
                 TFAM 
                 /5Phos/tattaaactttttgttgttt 
                 29 
               
               
                 15-2 
                   
                   
                   
               
               
                   
               
               
                 Ago3-top 
                 TFAM 
                 /5Phos/caaaaagtttaatatccaaa 
                 30 
               
               
                   
               
               
                 Ago-GC- 
                 TFAM 
                 /5Phos/catggtatataattacttta 
                 31 
               
               
                 20-1 
                   
                   
                   
               
               
                   
               
               
                 Ago-3top2 
                 TFAM 
                 /5Phos/catttgaaagaacaaaaaca 
                 32 
               
               
                   
               
               
                 Ago2-bot 
                 TFAM 
                 /5Phos/catttgtataaatcttgctg 
                 33 
               
               
                   
               
               
                 Target 3  
                 TFAM 
                 /5Phos/caacttaaatgtaacttctg 
                 34 
               
               
                 TtAgo 
                   
                   
                   
               
               
                   
               
               
                 Ago-GC-13 
                 TFAM 
                 /5Phos/catgttttataaagtaattatat 
                 35 
               
               
                   
               
               
                 Ago-GC-19 
                 TFAM 
                 /5Phos/caaaaagtttaatatccaaaa 
                 36 
               
               
                   
               
               
                 Ago-GC-14 
                 TFAM 
                 /5Phos/caaattattttagaaatcaactt 
                 37 
               
               
                   
                   
                 aaat 
                   
               
               
                   
               
               
                 ASLC1_1 
                 ASLC1 
                 /5Phos/caattcctagagccatttgtc 
                 38 
               
               
                   
               
               
                 ASLC1_2 
                 ASLC2 
                 /5Phos/cattttttctgcccaaaccctt 
                 39 
               
               
                   
               
               
                 ASLC1_3 
                 ASLC3 
                 /5Phos/caagttcttagtagaatccaa 
                 40 
               
               
                   
               
               
                 ASLC1_4 
                 ASLC4 
                 /5Phos/cactttttttcactgttctgg 
                 41 
               
               
                   
               
               
                 Nanog_l 
                 NANOG 
                 /5Phos/cagctacttttgcattacaat 
                 42 
               
               
                   
               
               
                 Nanog_2 
                 NANOG 
                 /5Phos/caggttctgttgctcggtttt 
                 43 
               
               
                   
               
               
                 Nanog_3 
                 NANOG 
                 /5Phos/cattcctgttgaaccatattc 
                 44 
               
               
                   
               
               
                 Nanog_4 
                 NANOG 
                 /5Phos/ctaatttttgtatttttagta 
                 45 
               
               
                   
               
               
                 HBG1_1 
                 HBG1 
                 /5Phos/caatagtcttagagtatcca 
                 46 
               
               
                   
               
               
                 HBG1_2 
                 HB G2 
                 /5Phos/caaaggctataaaaaaaatta 
                 47 
               
               
                   
               
               
                 HBG1_3 
                 HBG3 
                 /5Phos/cagtttttctctaatttattc 
                 48 
               
               
                   
               
               
                 HBG1_4 
                 HB G4 
                 /5Phos/caagaaggtaaaaaacggct 
                 49 
               
               
                   
               
               
                 IL1B_1 
                 IL1B 
                 /5Phos/cataaaaacagcgagggagaa 
                 50 
               
               
                   
               
               
                 IL1B_2 
                 IL1B 
                 /5Phos/caggtattcaacagagaaatt 
                 51 
               
               
                   
               
               
                 IL1B_3 
                 IL1B 
                 /5Phos/caatactcttttcccctttcc 
                 52 
               
               
                   
               
               
                 IL1B_4 
                 IL1B 
                 /5Phos/caggaaaacaatgcatatttg 
                 53 
               
               
                   
               
               
                 TERT_1 
                 TERT 
                 /5Phos/catagaaaacacaattttaaaa 
                 54 
               
               
                   
               
               
                 TERT_2 
                 TERT 
                 /5Phos/cagtttctgaaagtaggaaa 
                 55 
               
               
                   
               
               
                 TERT_3 
                 TERT 
                 /5Phos/catttcccaccctttctcga 
                 56 
               
               
                   
               
               
                 TERT_4 
                 TERT 
                 /5Phos/catttctctttgcaggttct 
                 57 
               
               
                   
               
               
                 GDF11-1 
                 GDF11 
                 /5phos/ atttttgtctccactctaac 
                 58 
               
               
                   
               
               
                 GDF11-2 
                 GDF11 
                 /5phos/ ttcctccttcttggttctgt 
                 59 
               
               
                   
               
               
                 GDF11-3 
                 GDF11 
                 /5phos/ tcttcttttcctcctttgtc 
                 60 
               
               
                   
               
            
           
           
               
            
               
                 Cas9 NN -VP64 system 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 SEQ 
               
               
                   
                   
                   
                 ID 
               
            
           
           
               
               
               
               
               
            
               
                 Name of gRNA 
                 Gene 
                 gRNA targeting sequence 
                 PAM 
                 NO: 
               
               
                   
               
               
                 gRNA2 
                 TFAM 
                 ctcggagttcagaaatagta 
                 acggga 
                 61 
               
               
                   
               
               
                 gRNA3 
                 TFAM 
                 ttaagctctgcggtaaggcc 
                 ttggaa 
                 62 
               
               
                   
               
               
                 SP1 
                 TFAM 
                 acctaaaaatgcagattcca 
                 agg 
                 63 
               
               
                   
               
               
                 SP2 
                 TFAM 
                 cgcctctcccgttactattt 
                 ggg 
                 64 
               
               
                   
               
               
                 SP3 
                 TFAM 
                 acacacggaattaagctctg 
                 cgg 
                 65 
               
               
                   
               
               
                 SP4 
                 TFAM 
                 tcgctaaaatgaagattaat 
                 ggg 
                 66 
               
               
                   
               
               
                 SPS 
                 TFAM 
                 ttagaacttgttaaattctg 
                 ggg 
                 67 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
            
               
                   
               
               
                 TtAgo NN  repressor system 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 SEQ 
                   
               
               
                   
                 Name of 
                 ID 
                   
               
               
                   
                 siDNA 
                 NO: 
                 siDNA Sequence 
               
               
                   
                   
               
               
                   
                 TFAM-1 
                 68 
                 /5Phos/ctacagaactaattagaaga 
               
               
                   
                   
               
               
                   
                 TFAM-2 
                 69 
                 /5Phos/cttcctgattcaaagaaaaa 
               
               
                   
                   
               
               
                   
                 TFAM-3 
                 70 
                 /5Phos/cttctttatatacctgccac 
               
               
                   
                   
               
               
                   
                 Sox2-1 
                 71 
                 /5Phos/caacatgatggagacggagc 
               
               
                   
                   
               
               
                   
                 Sox2-2 
                 72 
                 /5Phos/catgatgcaggaccagctgg 
               
               
                   
                   
               
               
                   
                 Sox2-3 
                 73 
                 /5Phos/cttcacatgtcccagcacta 
               
               
                   
                   
               
               
                   
                 Oct4.1 
                 74 
                 /5Phos/ctctttctgtcctttcacga 
               
               
                   
                   
               
               
                   
                 Oct4.2 
                 75 
                 /5Phos/ctacagactattccttgggg 
               
               
                   
                   
               
               
                   
                 Oct4.3 
                 76 
                 /5Phos/cttacaagtcttctgccttt 
               
               
                   
                   
               
               
                   
                 KLf-2.1 
                 77 
                 /5Phos/cttctccactttcgccagcc 
               
               
                   
                   
               
               
                   
                 KLf-2.2 
                 78 
                 /5Phos/ctttctgctggcgccgcccg 
               
               
                   
                   
               
               
                   
                 KLf-2.3 
                 79 
                 /5Phos/cttcggtctcttcgacgacg 
               
               
                   
                   
               
               
                   
                 cmyc.1 
                 80 
                 /5Phos/ctggatttttttcgggtagt 
               
               
                   
                   
               
               
                   
                 cmyc.2 
                 81 
                 /5Phos/ctcaacgttagcttcaccaa 
               
               
                   
                   
               
               
                   
                 cmyc.3 
                 82 
                 /5Phos/cagccgtatttctactgcga 
               
               
                   
                   
               
               
                   
                 NANOG.1 
                 83 
                 /5Phos/cttgctttgaagcatccgac 
               
               
                   
                   
               
               
                   
                 NANOG.2 
                 84 
                 /5Phos/cttcacctatgcctgtgatt 
               
               
                   
                   
               
               
                   
                 NANOG.3 
                 85 
                 /5Phos/caaatgtcttctgctgagat 
               
               
                   
                   
               
               
                   
                 HBG1.1 
                 86 
                 /5Phos/catttcacagaggaggacaa 
               
               
                   
                   
               
               
                   
                 HBG1.2 
                 87 
                 /5Phos/ctggaggagaaaccctggga 
               
               
                   
                   
               
               
                   
                 HBG1.3 
                 88 
                 /5Phos/ctttgacagctttggcaacc 
               
               
                   
                   
               
               
                   
                 IL1B.1 
                 89 
                 /5Phos/ctcgccagtgaaatgatggc 
               
               
                   
                   
               
               
                   
                 IL1B.2 
                 90 
                 /5Phos/caatgaggatgacttgttct 
               
               
                   
                   
               
               
                   
                 IL1B.3 
                 91 
                 /5Phos/ctttgaagctgatggcccta 
               
               
                   
                   
               
               
                   
                 TERT.1 
                 92 
                 /5Phos/ctccccgctgccgagccgtg 
               
               
                   
                   
               
               
                   
                 TERT.2 
                 93 
                 /5Phos/ctgcgcagccactaccgcga 
               
               
                   
                   
               
               
                   
                 TERT.3 
                 94 
                 /5Phos/ctttccgcgcgctggtggcc 
               
               
                   
                   
               
            
           
         
       
     
     Plasmids 
     The ttAGo-VP64 and ttAgo fragments (Genewiz) were synthesized and cloned into pCdna3.1 topo ct-gfp (Invitrogen) plasmids according to the manufacture&#39;s′ instruction. The sequence information of the vector is provided below.
     ttAgo-VP64 sequence (NLS-VP64 is highlighted in bold and italics)   

     
       
         
           
               
               
            
               
                 (SEQ ID NO: 95) 
                   
               
               
                 ATGAACCACCTGGGTAAAACTGAAGTATTCCTGAACCGTTTCGCACTGagaccacttaat 
                   
               
               
                   
               
               
                 CCGGAAGAACTGCGTCCGTGGCGTCTGGAAGTTGTTCTGGACCCGCCGCCGGGTC 
               
               
                   
               
               
                 GTGAAGAAGTTTACCCGCTGCTGGCTCAGGTTGCTCGTCGTGCTGGTGGTGTTAC 
               
               
                   
               
               
                 CGTTCGTATGGGTGACGGTCTGGCTTCTTGGTCTCCGCCGGAAgtattagtacttGAAGG 
               
               
                   
               
               
                 TACCCTGGCTCGTATGGGTCAGACCTACgcatatcgtttatatccaAAAGGTCGTCGTCCGCT 
               
               
                   
               
               
                 GgatccgaaggatcctGGTGAACGTTCTGTTCTGTCTgcattagctagaCGTCTGCTGCAGGAAC 
               
               
                   
               
               
                 GTCTGCGTCGTCTGGAAGGTGTTTGGGTTGAAGGTCTGGCTGTTTACCGTCGTGA 
               
               
                   
               
               
                 ACACGCTCGTGGTCCGGGTTGGCGTGTTCTGGGTGGTGCTGTTCTGGACCTGTGG 
               
               
                   
               
               
                 GTTTCTGACTCTGGTGCTTTCCTGCTGGAAGTTGACCCGGCTTACCGTATCCTGTG 
               
               
                   
               
               
                 CGAAATGTCTCTGGAAGCTTGGCTGGCTCAGGGTCACCCGCTGCCGAAACGTGTT 
               
               
                   
               
               
                 CGTAACGCTTACGACCGTCGTACCTGGGAActgctaagattaGGTGAAGAAGACCCGAA 
               
               
                   
               
               
                 AGAACTGCCGCTGCCGGGTGGTCTGTCTCTGCTGGACTACCACGCTTCTAAAGGT 
               
               
                   
               
               
                 CGTCTGCAGGGTCGTGAAggcgggagagtaGCTTGGGTTGCTGACCCGAAAGACCCGC 
               
               
                   
               
               
                 GTAAACCGATCCCGCACCTGACCGGTCTGCTGGTTCCGGTTCTGACCCTGGAAGA 
               
               
                   
               
               
                 CCTGCACGAGGAAGAAGGTTCTCTGGCTCTGTCTCTGCCGTGGGAAGAACGTCGT 
               
               
                   
               
               
                 CGTCGTACCCGTGAAATCGCTTCTTGGATCGGTCGTCGTCTGGGTCTGGGTACCC 
               
               
                   
               
               
                 CGGAAGCTGTTCGTGCTCAGGCTTACCGTCTGTCTATCCCGAAACTGATGGGTCG 
               
               
                   
               
               
                 TCGTGCTGTTTCTAAACCGGCTGACGCTCTGCGTGTTGGTTTCTACCGTGCTCAGG 
               
               
                   
               
               
                 AAACCgcgttagcccttttacgaCTGGACGGTGCTCAGGGTTGGCCGGAATTCCTGCGTCGG 
               
               
                   
               
               
                 GCTCTGCTGCGTGCTTTCGGTGCTTCTGGTGCTTCTCTGCGTCTGCACACCCTGCA 
               
               
                   
               
               
                 CGCTCACCCGTCTCAGGGTCTGGCTTTCCGTGAAGCTCTGCGTAAAGCTAAAGAA 
               
               
                   
               
               
                 GAAGGTGTTCAGGCTGTTCTGGTTCTGACCCCGCCGATGGCTTGGGAAGACCGTA 
               
               
                   
               
               
                 ACCGTCTGAAAGCTcttttactgagagagggcctgccgagcCAGATCCTGAACGTTCCGCTGCGT 
               
               
                   
               
               
                 GAAGAAGAACGTCACCGTTGGGAAAACGCTCTGCTGGGTCTGCTGGCTAAAGCT 
               
               
                   
               
               
                 GGTCTGCAGGTTGTTGCTCTGTCTGGTGCTTACCCGGCTGAACTGGCTGTTGGTTT 
               
               
                   
               
               
                 CGcCGCTGGTGGTCGTGAATCTTTCCGTTTCGGTGGTGCTGCTTGCGCTGTTGGTG 
               
               
                   
               
               
                 GTGACGGTGGTCACCTGCTGTGGACCCTGCCGGAAGCTCAGGCTGGTGAACGTA 
               
               
                   
               
               
                 TCCCGCAGGAAGTTGTTTGGGACCTGCTGGAAGAAACCCTGTGGGCTTTCCGTCG 
               
               
                   
               
               
                 TAAAGCTGGTCGTCTGCCGTCTCGTGTTCTGCTGCTGCGTGcCGGTCGTGTTCCGC 
               
               
                   
               
               
                 AGGACGAATTCGCTCTGGCTttagaagcgttagcgCGTGAAGGTATCGCTTACGACCTGGT 
               
               
                   
               
               
                 TTCTGTTCGTAAATCTGGTGGTGGTCGTGTTTACCCGGTTCAGGGTCGTCTGGCTG 
               
               
                   
               
               
                 ACGGTCTGTACGTTCCGCTGGAAGACAAAACCTTCCTGCTGCTGACCGTTCACCG 
               
               
                   
               
               
                 TGACTTCCGTGGTACCCCGCGTCCGCTGAAACTGGTTCACGAAGCTGGTGACACC 
               
               
                   
               
               
                 CCGCTGGAAGCTCTGGCTCACCAGATCTTCCACCTGACCCGTCTGTACCCGGCTT 
               
               
                   
               
               
                 CTGGTTTCGCTTTCCCGCGTCTGCCGGCTCCGCTGCACCTGGCTGACCGTCTGGTT 
               
               
                   
               
               
                 AAAGAAGTTGGTCGTCTGGGTATCCGTCACCTGAAAGAAGTTGACCGTGAAAAA 
               
               
                   
               
               
                 CTGTTCTTCGTTCCCAAGAAGAAGAGAAAGGTGGAGGCCAGCGGTTCCGGACGGG 
               
               
                   
               
               
                 CTGACGCATTGGACGATTTTGATCTGGATATGCTGGGAAGTGACGCCCTCGATGAT 
               
               
                   
               
               
                 TTTGACCTTGACATGCTTGGTTCGGATGCCCTTGATGACTTTGACCTCGACATGCT 
               
               
                   
               
               
                 CGGCAGTGACGCCCTTGATGATTTCGACCTGGACATGCTGATTAAC 
               
            
           
         
       
     
     Transfection and QPCR Analysis 
     293 T reporter cells were seeded at densities of 5 *10 4 cells per well in 96-well plate and transfected them with 300 ng of each ttAgo-VP64 plasmid and 300 ng siDNA using Lipofectamine 2000 following the manufacturer&#39;s protocols. The cells were harvested 36 hours after transfection and RT-QPCR was performed using cDNA to CT II kit (Invitrogen). All the RT-QPCR data is normalized with 18sRNA control and the sequence of the primers are provided herein. 
     Western Blot 
     Cell Harvest 
     7 million 293 T reporter cells were seeded in each T175 plate and were transfected with 12 μg of TtAgo-VP64 plasmid and 12 μg siDNA using 60 μL of Lipofectamine 2000 following the manufacturer&#39;s protocols. The cells were harvested 1 week after transfection by removing culture medium from the adherent cells, rinsing the cells in PBS, and adding M-PER (Pierce) reagent according to protocol. The lysate was collected and transferred into a microcentifuge tube. The samples were spun at 14,000×g for 10 minutes at 4° C. to pellet the cell debris. The supernatant was then transferred to another tube for analysis and frozen at −20° C. overnight. 
     Western Blot 
     Supernatant was thawed on ice. The Pierce™ BCA Protein Assay Kit was used to quantify protein content (1:12.5 dilution) in each sample according to the manufacturer&#39;s protocol. The samples were then normalized to lowest concentration. Next, Laemmli Buffer was added at 1:6 dilution and boiled at 95° C. for 5 minutes. Gels were then loaded with 30 μL of sample at 60 V to stack for 20 minutes and then 100 V for 1 hour. PVDF was pre-treated with 20% methanol and then transfer buffer (25 mM Tris, 192 mM glycine, 10% methanol). The wet transfer sandwich was then prepared according to manufacturer&#39;s protocol and transferred for 1 hour at 100 V. Membranes were then trimmed and incubated in 5% milk TBST (Tris-buffered saline, 0.1% Tween-20). The membrane was then incubated in either TFAM antibody (Aviva Systems Biology #0AAF02353 or Tubulin (TUBB3) antibody (Aviva Systems #OAAD00318) in 5% milk overnight at 4° C. The membranes were then washed 3 times in 1×TBST for 5 minutes each. Next, the membranes were incubated in anti-Rabbit in 5% milk for 1 hour and washed 3 times for 5 minutes in 1×TBST. The membranes were then exposed to the Bio-Rad Clarity™ ECL substrate according to manufacturer&#39;s instructions and imaged using x-ray film. Densitometry of each band was quantified using ImageJ toolkit. 
     Gel Shifting Assay 
     Protein Purification 
     Plasmid pWUR-702 and pWUR-703 containing strept-tagged TtAgo proteins from Addgene are transformed into Rosetta™ 2(DE3) strain (Agilent) and grown in 5 mL of 2YT media with 100 μg/mL of Spectinomycin overnight. 250 mL media are inoculated with the overnight cultures and protein expression is induced with 1 mM of IPTG at OD600 around 0.6-0.8. The cells are incubated at shaker at 20° C. for 16 h and harvest by centrifuge. Cell pellet is then resuspended in Buffer A (20 mM Tris-HCl pH 7.6, 1 M NaCl and 2 mM MgCl 2 ) and lysed with french pressure cell. The protein is purified over 3 mL Strep-Tactin® Superflow® resin with protocol suggested by vendor. Protein are eluted into Buffer A plus 2.5 mM d-Desthiobiotin and stored in −80° C. upon adding one volume of 80% glycerol. 
     Gel Shift Assay 
     5′-Biotinylated or unlabeled competing substrate ssDNA, dsDNA and 5′-phosphorylated guide DNA are purchased from Integrated DNA Technology without further purification. Binding reactions contain the following components: 1 μM of 5′-biontin-substrate, 1 μM of guide DNA, 0.5 mg/mL of Salmon sperm DNA (Invitrogen), 5 μM of TtAgo protein, 100 μM of competing substrate (optional), 20 mM HEPES-KOH pH 7.6, 100 mM KCl, 5 mM MgCl 2 , 1 mM DTT. The reactions are incubated at 37° C. for 1 h and resolved on 6% DNA retardation gel (Invitrogen) with running buffer containing 1×TBE and 5 mM MgCl 2 . The DNA are transferred to nitrocellulose membrane by iBlot (Invitrogen) dry transfer, cross-linked under UV and stained by the LightShift Chemiluminescent EMSA Kit (Thermo Scientific) according to vendor&#39;s manual instruction. 
     EXAMPLE II 
     Engineered Ago and Guide DNA 
     A DNA-guided Ago system is provided for genome engineering. A TtAgo protein and siDNA components are engineered to both activate and repress gene expression in human cells. As indicated in  FIG. 1 , it was confirmed that TtAgo/siDNA has sequence specific single stranded DNA (ssDNA) binding activity in vitro at 37° C. Purified ttAgo protein was incubated with either a dsDNA (double stranded DNA) or ssDNA (single stranded DNA) substrate wt/wo siDNA at 37° C. and the binding of ttAgo with the substrate was examined by gel shifting assay. Binding of ttAgo with ssDNA with the presence of siDNA (lane 9 from the left) was detected. The sequence of substrate and siDNA are identified in  FIG. 1 . 
     TtAgo/siDNA systems were engineered for gene suppression and activation in human cells. The gene suppression system features a CMV promoter driving a human optimized codon Ago double site mutant TtAgo NN (D478A, D546A), which removes its nuclease activity, fused to a C-terminal SV40 nuclear localization signal (NLS). The activation system features an activation domain, VP64, on the C-terminus ( FIG. 2A ). Guide siDNA were selected in AT rich sequences within the promoter (for activation) or exon 1/2 (for suppression) region with a 5′dC initiating nucleotide. See D. C. Swarts, M. M. Jore, E. R. Westra, Y. Zhu, J. H. Janssen, A. P. Snijders, Y. Wang, D. J. Patel, J. Berenguer, S. J. Brouns, J. van der Oost, DNA-guided DNA interference by a prokaryotic Argonaute. Nature 507, 258-261 (2014) hereby incorporated by reference in its entirety. 
     To test if TtAgo NN-VP 64/siDNA activates human endogenous genes, two siDNAs (siDNA TFAM-7 and siDNA TFAM-8) were designed to target the promoter regions of transcription factor A mitochondria (TFAM). See  FIG. 2B  and sequences provided below. 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                   
                   
                   
                 SEQ 
               
               
                   
                   
                   
                 ID 
               
               
                   
                   
                   
                 NO: 
               
               
                   
               
             
            
               
                 Name 
                 Gene 
                 siDNA sequence for TtAgo system 
                   
               
               
                   
               
               
                 TFAM-7 
                 TFAM 
                 /5Phos/cagaaatagtaacgggagag 
                 26 
               
               
                   
               
               
                 TFAM-8 
                 TFAM 
                 /5Phos/cacggaattaagctctgcgg 
                 27 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 gRNA targeting sequence for 
                   
                   
               
               
                 Name 
                 Gene 
                 CRISPR/Cas-ST 
                 PAM 
                   
               
               
                   
               
               
                 gRNA 2 
                 TFAM 
                 ctcggagttcagaaatagta 
                 acggga 
                 61 
               
               
                   
               
               
                 gRNA 3 
                 TFAM 
                 ttaagctctgcggtaaggcc 
                 ttggaa 
                 62 
               
               
                   
               
            
           
         
       
     
     As a comparison, we designed two  Streptococcus thermophiles  Cas9-VP64 (Cas9ST-VP64)/gRNA constructs were designed according to K. M. Esvelt, P. Mali, J. L. Braff, M. Moosburner, S. J. Yaung, G. M. Church, Orthogonal Cas9 proteins for RNA-guided gene regulation and editing.  Nat Methods  10, 1116-1121 (2013) hereby incorporated by reference in its entirety (gRNA 2 and gRNA 3) targeting the same regions. See  FIG. 2C . Delivery of TtAgo NN -VP64/siDNA into HEK 293 cells demonstrated robust TFAM activation of 20-fold and 60-fold increase in gene expression respectively, comparable to the level achieved by Cas9-VP64/gRNA targeting the same regions (see  FIG. 2C ). TtAgo NN -VP64 provided higher activation than the Cas9ST-VP64 system. 
     According to one aspect, lower melting temperature (Tm) AT-rich regions of target nucleic acids are targeted by the Ago systems described herein using TtAgo NN , which does not have a helicase domain, and therefore increases the likelihood for activation. 14 siDNAs targeting the TFAM promoter as a function of Tm were designed as indicated herein. A reverse correlation of Tm of the targeting sites and TtAgo NN -VP64/siDNA activation activities were observed as indicated in  FIG. 2D ). Because TtAgo lacks a helicase domain, co-transfection of the  S. pyogenes  Cas9 null mutant (Cas9 sp-NN ) (see P. Mali, L. Yang, K. M. Esvelt, J. Aach, M. Guell, J. E. DiCarlo, J. E. Norville, G. M. Church, RNA-guided human genome engineering via Cas9.  Science  339, 823-826 (2013) hereby incorporated by reference in its entirety) improves activation efficiency of the TtAgo NN -VP64 system by unwinding the dsDNA substrate. To this end, a siDNA with low activation performance (TFAM-4, Activation ˜1±0.2 compared to TtAgo NN -VP64 alone) was selected and co-transfected with Cas9SP NN /gRNA systems as described in  Science  339, 823-826 (2013) targeting between 46 and 187 bp away from the siDNA target site. Binding of Cas9SP NN /gRNA nearby improves activation of TtAgo NN -VP64/siDNA, and the optimal efficiency was seen when the two systems are ˜128 bp apart. See  FIG. 3A and 3B . This optimal distance most likely reduces the effects of steric hindrance between the two systems and allows for TtAgo NN -VP64 to access its ssDNA target which otherwise is in the dsDNA form. TtAgo NN -VP64 activates genes in the human genome, preferably at A-T rich regions, when the ssDNA substrate (target nucleic acid sequence) is more accessible. 
     As shown in  FIG. 3A and 3B , the DNA helicase domain of the null mutant Cas9 NN-SP /gRNA systems improves activation of TtAgo NN -VP64/siDNA from 1- to 4-fold when the Cas9 system binds 77 bp-161 bp upstream of TtAgo binding on the TFAM promoter.  FIG. 3A  is a schematic of a design of 5 gRNAs to localize Cas9 NN-SP  at varying distances from TtAgo-VP64/siDNA_TFAM-4. Each gRNA is named ‘SPX’, where SP denotes the species of Cas9 protein, and X is 1, 2, 3, 4, or 5 to identify the gRNA used. The legend highlights that the gRNA and Cas9 NN-SP  combine as a unit to localize the system to a specific area in the genome. Similarly, the TtAgo-VP64 and siDNA (TFAM-4) combine as a unit to localize the system to a specific area in the genome. The specific gRNA targeting sequences are described herein.  FIG. 3B  is data directed to TtAgo NN -VP64/siDNA_TFAM-4 activation activities when combining with Cas9 NN-SP /gRNA as measured by RT-PCR to amplify a 150 bp region of exon 1 of TFAM. Cas9 NN-SP /gRNAs with distance 46 bp-187 bp to siDNA_TFAM-4 binding site increases the TtAgo NN -VP64/siDNA_TFAM-4 activities 4 fold. 
     The methods described herein increased mRNA levels using TtAgo NN -VP64/siDNA. Methods described herein using TtAgo NN -VP64/siDNA also increased protein levels.  FIG. 4  depicts immunoblots showing DNA-guided activation of TFAM (25 kDa). Tubulin (50 kDA) served as a control. Intensity of each blot band, I, was calculated using ImageJ software. Densitometry of TFAM protein was quantified using the equation above. Activators for TFAM (TFAM-1 and TFAM-3 siDNA, respectively) were selected to quantify the amount of TFAM protein in each population after 3 days of transfection. As shown in  FIG. 4 , a 2.3-fold and 1.9-fold increase of TFAM protein, respectively, was confirmed using the TtAgo NN -VP64/TFAM-1 siDNA. 
     To test the effectiveness of TtAgo NN -VP64 activation system across the genome, a panel of endogenous genes (GDF11, ASLC, NANOG, HBG1, IL1B, TERT, SOX2, KLF4, cMYC, and OCT4) was selected and between 3-6 siDNAs were designed targeting AT-rich areas of the respective promoter region (See  FIG. 5 ). Among the ten genes tested, nine genes were successfully activated by a single TtAgo NN -VP64/siDNA complex and the activation level showed predicable fashion based on AT content (See  FIG. 5 ). Notably, genes such as TERT and NANOG, that were previously not able to be activated by a single gRNA in the CRISPR/Cas9 system (see A. W. Cheng, H. Wang, H. Yang, L. Shi, Y. Katz, T. W. Theunissen, S. Rangarajan, C. S. Shivalila, D. B. Dadon, R. Jaenisch, Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system.  Cell research  23, 1163-1171 (2013); P. Perez-Pinera, D. D. Kocak, C. M. Vockley, A. F. Adler, A. M. Kabadi, L. R. Polstein, P. I. Thakore, K. A. Glass, D. G. Ousterout, K. W. Leong, F. Guilak, G. E. Crawford, T. E. Reddy, C. A. Gersbach, RNA-guided gene activation by CRISPR-Cas9-based transcription factors.  Nat Methods  10, 973-976 (2013)) have been robustly activated by a single siDNA in the TtAgo NN -VP64/siDNA system. Moreover, TtAgo NN -VP64/siDNA activated ASLC, HBG1, SOX2, and OCT4 better than Cas9/gRNA systems. Of note, HBG1-3 siDNA (20% GC, 143 fold activation) significantly outperformed HBG1-2 and HBG1-4 (each 15% GC, 43 and 43 fold activation). 
     The methods described herein utilize an Ago system to suppress gene expression. Gene suppression activity was demonstrated by interfering with or otherwise blocking the translational machinery. Three siDNA targeting exon 1 and/or 2 of each gene in the panel described previously (TFAM, NANOG, HBG1, IL1B, TERT, SOX2, KLF4, cMYC, and OCT4) were designed and HEK293 cells were transfected with TtAgo NN  with siDNA respectively. Notably, 6 out of the 9 constructs tested demonstrated robust gene repression by 10-90% (see  FIG. 6 ). TtAgo NN /siDNAs targeting the exon 1 of HBG1 and OCT4 activated gene expression, while TtAgo NN /siDNAs targeting exon 2 of HBG1 and OCT4 sufficiently repressed gene expression, likely resulting from interaction of TtAgo NN  with transcription initiation complex. 
       FIG. 7  is directed to guide DNA sequence specificity. TtAgo NN -VP64/siDNA sequence specificity as measured by TFAM activation activities by TtAgo NN -VP64/siDNA coupled with different siDNAs was determined. As indicated in  FIG. 7 , a 5′ phosphate on the guide DNA provided TtAgo NN -VP64 activities. Mismatches may be allowed under certain circumstances. A mismatch in the middle of the siDNA recognition or spacer sequence showed reduced TtAgo NN -VP64/siDNA activity.