Patent Publication Number: US-2022235382-A1

Title: Genome Engineering

Description:
RELATED APPLICATION DATA 
     This application is a continuation application which claims priority to U.S. application Ser. No. 14/319,498, filed on Jun. 30, 2014, which claims priority to U.S. Provisional Patent Application No. 61/858,866, filed on Jul. 26, 2013 and is hereby incorporated herein by reference in its entirety for all purposes. 
    
    
     STATEMENT OF GOVERNMENT INTERESTS 
     This invention was made with government support under P50 HG003170 from the National Human Genome Research Center for Excellence in Genomics Science. The government has certain rights in the invention. 
    
    
     BACKGROUND 
     Genome editing via sequence-specific nucleases is known. See references 1, 2, and 3 hereby incorporated by reference in their entireties. A nuclease-mediated double-stranded DNA (dsDNA) break in the genome can be repaired by two main mechanisms: Non-Homologous End Joining (NHEJ), which frequently results in the introduction of non-specific insertions and deletions (indels), or homology directed repair (HDR), which incorporates a homologous strand as a repair template. See reference 4 hereby incorporated by reference in its entirety. When a sequence-specific nuclease is delivered along with a homologous donor DNA construct containing the desired mutations, gene targeting efficiencies are increased by 1000-fold compared to just the donor construct alone. See reference 5 hereby incorporated by reference in its entirety. Use of single stranded oligodeoxyribonucleotides (“ssODNs”) as DNA donors has been reported. See references 21 and 22 hereby incorporated by reference in their entireties. 
     Despite large advances in gene editing tools, many challenges and questions remain regarding the use of custom-engineered nucleases in human induced pluripotent stem cell (“hiPSC”) engineering. First, despite their design simplicity, Transcription Activator-Like Effectors Nucleases (TALENs) target particular DNA sequences with tandem copies of Repeat Variable Diresidue (RVD) domains. See reference 6 hereby incorporated by reference in its entirety. While the modular nature of RVDs simplifies TALEN design, their repetitive sequences complicate methods for synthesizing their DNA constructs (see references 2, 9, and 15-19 hereby incorporated by reference in their entireties) and also impair their use with lentiviral gene delivery vehicles. See reference 13 hereby incorporated by reference in its entirety. 
     In current practice, NHEJ and HDR are frequently evaluated using separate assays. Mismatch-sensitive endonuclease assays (see reference 14 hereby incorporated by reference in its entirety) are often used for assessing NHEJ, but the quantitative accuracy of this method is variable and the sensitivity is limited to NHEJ frequencies greater than ˜3%. See reference 15 hereby incorporated by reference in its entirety. HDR is frequently assessed by cloning and sequencing, a completely different and often cumbersome procedure. Sensitivity is still an issue because, while high editing frequencies on the order of 50% are frequently reported for some cell types, such as U2OS and K562 (see references 12 and 14 hereby incorporated by reference in their entireties), frequencies are generally lower in hiPSCs. See reference 10 hereby incorporated by reference in its entirety. Recently, high editing frequencies have been reported in hiPSC and hESC using TALENs (see reference 9 hereby incorporated by reference in its entirety), and even higher frequencies with the CRISPR Cas9-gRNA system (see references 16-19 hereby incorporated by reference in their entireties. However, editing rates at different sites appear to vary widely (see reference 17 hereby incorporated by reference in its entirety), and editing is sometimes not detectable at all at some sites (see reference 20 hereby incorporated by reference in its entirety). 
     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). 
     SUMMARY 
     Aspects of the present disclosure are directed to the use of modified Transcription Activator-Like Effector Nucleases (TALENs) for genetically modifying a cell, such as a somatic cell or a stem cell. TALENs are known to include repeat sequences. Aspects of the present disclosure are directed to a method of altering target DNA in a cell including introducing into a cell a TALEN lacking repeat sequences 100 bp or longer wherein the TALEN cleaves the target DNA and the cell undergoes nonhomologous end joining to produce altered DNA in the cell. According to certain aspects, repeat sequences of desired length have been removed from a TALEN. According to certain aspects, the TALEN is devoid of repeat sequences of certain desired length. According to certain aspects, a TALEN is provided with repeat sequences of desired length removed. According to certain aspects, a TALEN is modified to remove repeat sequences of desired length. According to certain aspects, a TALEN is engineered to remove repeat sequences of desired length. 
     Aspects of the present disclosure include methods of altering target DNA in a cell including combining within a cell a TALEN lacking repeat sequences 100 bp or longer and a donor nucleic acid sequence wherein the TALEN cleaves the target DNA and the donor nucleic acid sequence is inserted into the DNA in the cell. Aspects of the present disclosure are directed to a virus including a nucleic acid sequence encoding a TALEN lacking repeat sequences 100 bp or longer. Aspects of the present disclosure are directed to a cell including a nucleic acid sequence encoding a TALEN lacking repeat sequences 100 bp or longer. According to certain aspects described herein, the TALEN lacks repeat sequences 100 bp or longer, 90 bp or longer, 80 bp or longer, 70 bp or longer, 60 bp or longer, 50 bp or longer, 40 bp or longer, 30 bp or longer, 20 bp or longer, 19 bp or longer, 18 bp or longer, 17 bp or longer, 16 bp or longer, 15 bp or longer, 14 bp or longer, 13 bp or longer, 12 bp or longer, 11 bp or longer, or 10 bp or longer. 
     Aspects of the present disclosure are directed to making a TALE including combining an endonuclease, a DNA polymerase, a DNA ligase, an exonuclease, a plurality of nucleic acid dimer blocks encoding repeat variable diresidue domains and a TALE-N/TF backbone vector including an endonuclease cutting site, activating the endonuclease to cut the TALE-N/TF backbone vector at the endonuclease cutting site to produce a first end and a second end, activating the exonuclease to create a 3′ and a 5′ overhang on the TALE-N/TF backbone vector and the plurality of nucleic acid dimer blocks and to anneal the TALE-N/TF backbone vector and the plurality of nucleic acid dimer blocks in a desired order, activating the DNA polymerase and the DNA ligase to connect the TALE-N/TF backbone vector and the plurality of nucleic acid dimer blocks. One of skill in the art will readily based on the present disclosure be able to identify suitable endonucleases, DNA polymerases, DNA ligases, exonucleases, nucleic acid dimer blocks encoding repeat variable diresidue domains and TALE-N/TF backbone vectors. 
     Aspects of the present disclosure are directed to a method of altering target DNA in a stem cell expressing an enzyme that forms a co-localization complex with RNA complementary to the target DNA and that cleaves the target DNA in a site specific manner including (a) introducing into the stem cell a first foreign nucleic acid encoding an RNA complementary to the target DNA and which guides the enzyme to the target DNA, wherein the RNA and the enzyme are members of a co-localization complex for the target DNA, introducing into the stem cell a second foreign nucleic acid encoding a donor nucleic acid sequence, wherein the RNA and the donor nucleic acid sequences are expressed, wherein the RNA and the enzyme co-localize to the target DNA, the enzyme cleaves the target DNA and the donor nucleic acid is inserted into the target DNA to produce altered DNA in the stem cell. 
     Aspects of the present disclosure are directed to a stem cell including a first foreign nucleic acid encoding for an enzyme that forms a co-localization complex with RNA complementary to target DNA and that cleaves the target DNA in a site specific manner. 
     Aspects of the present disclosure are directed to a cell including a first foreign nucleic acid encoding for an enzyme that forms a co-localization complex with RNA complementary to target DNA and that cleaves the target DNA in a site specific manner and including an inducible promoter for promoting expression of the enzyme. In this manner, expression can be regulated, for example, it can be started and it can be stopped. 
     Aspects of the present disclosure are directed to a cell including a first foreign nucleic acid encoding for an enzyme that forms a co-localization complex with RNA complementary to target DNA and that cleaves the target DNA in a site specific manner, wherein the first foreign nucleic acid is removable from genomic DNA of the cell using a removal enzyme, such as a transposase. 
     Aspects of the present disclosure are directed to a method of altering target DNA in a cell expressing an enzyme that forms a co-localization complex with RNA complementary to the target DNA and that cleaves the target DNA in a site specific manner including (a) introducing into the cell a first foreign nucleic acid encoding a donor nucleic acid sequence, introducing into the cell from media surrounding the cell an RNA complementary to the target DNA and which guides the enzyme to the target DNA, wherein the RNA and the enzyme are members of a co-localization complex for the target DNA, wherein the donor nucleic acid sequence is expressed, wherein the RNA and the enzyme co-localize to the target DNA, the enzyme cleaves the target DNA and the donor nucleic acid is inserted into the target DNA to produce altered DNA in the cell. 
     Aspects of the present disclosure are directed to the use of an RNA guided DNA binding protein for genetically modifying a stem cell. In one aspect, the stem cell has been genetically modified to include a nucleic acid encoding for the RNA guided DNA binding protein and the stem cell expresses the RNA guided DNA binding protein. According to a certain aspect, donor nucleic acids for introducing specific mutations are optimized for genome editing using either the modified TALENs or the RNA guided DNA binding protein. 
     Aspects of the present disclosure are directed to the modification of DNA, such as multiplex modification of DNA, in a stem cell using one or more guide RNAs (ribonucleic acids) to direct an enzyme having nuclease activity expressed by the stem cell, such as a DNA binding protein having nuclease activity, to a target location on the DNA (deoxyribonucleic acid) wherein the enzyme cuts the DNA and an exogenous donor nucleic acid is inserted into the DNA, such as by homologous recombination. Aspects of the present disclosure include cycling or repeating steps of DNA modification on a stem cell to create a stem cell having multiple modifications of DNA within the cell. Modifications may include insertion of exogenous donor nucleic acids. 
     Multiple exogenous nucleic acid insertions can be accomplished by a single step of introducing into a stem cell, which expresses the enzyme, nucleic acids encoding a plurality of RNAs and a plurality of exogenous donor nucleic acids, such as by co-transformation, wherein the RNAs are expressed and wherein each RNA in the plurality guides the enzyme to a particular site of the DNA, the enzyme cuts the DNA and one of the plurality of exogenous nucleic acids is inserted into the DNA at the cut site. According to this aspect, many alterations or modification of the DNA in the cell are created in a single cycle. 
     Multiple exogenous nucleic acid insertions can be accomplished in a cell by repeated steps or cycles of introducing into a stem cell, which expresses the enzyme, one or more nucleic acids encoding one or more RNAs or a plurality of RNAs and one or more exogenous nucleic acids or a plurality of exogenous nucleic acids wherein the RNA is expressed and guides the enzyme to a particular site of the DNA, the enzyme cuts the DNA and the exogenous nucleic acid is inserted into the DNA at the cut site, so as to result in a cell having multiple alterations or insertions of exogenous DNA into the DNA within the stem cell. According to one aspect, the stem cell expressing the enzyme has been genetically altered to express the enzyme such as by introducing into the cell a nucleic acid encoding the enzyme and which can be expressed by the stem cell. In this manner, aspects of the present disclosure include cycling the steps of introducing RNA into a stem cell which expresses the enzyme, introducing exogenous donor nucleic acid into the stem cell, expressing the RNA, forming a co-localization complex of the RNA, the enzyme and the DNA, enzymatic cutting of the DNA by the enzyme, and insertion of the donor nucleic acid into the DNA. Cycling or repeating of the above steps results in multiplexed genetic modification of a stem cell at multiple loci, i.e., a stem cell having multiple genetic modifications. 
     According to certain aspects, DNA binding proteins or enzymes within the scope of the present disclosure include a protein that forms a complex with the guide RNA and with the guide RNA guiding the complex to a double stranded DNA sequence wherein the complex binds to the DNA sequence. According to one aspect, the enzyme can be an RNA guided DNA binding protein, such as an RNA guided DNA binding protein of a Type II CRISPR System that binds to the DNA and is guided by RNA. According to one aspect, the RNA guided DNA binding protein is a Cas9 protein. 
     This aspect of the present disclosure may be referred to as co-localization of the RNA and DNA binding protein to or with the double stranded DNA. In this manner, a DNA binding protein-guide RNA complex may be used to cut multiple sites of the double stranded DNA so as to create a stem cell with multiple genetic modifications, such as multiple insertions of exogenous donor DNA. 
     According to certain aspects, a method of making multiple alterations to target DNA in a stem cell expressing an enzyme that forms a co-localization complex with RNA complementary to the target DNA and that cleaves the target DNA in a site specific manner is provided including (a) introducing into the stem cell a first foreign nucleic acid encoding one or more RNAs complementary to the target DNA and which guide the enzyme to the target DNA, wherein the one or more RNAs and the enzyme are members of a co-localization complex for the target DNA, introducing into the stem cell a second foreign nucleic acid encoding one or more donor nucleic acid sequences, wherein the one or more RNAs and the one or more donor nucleic acid sequences are expressed, wherein the one or more RNAs and the enzyme co-localize to the target DNA, the enzyme cleaves the target DNA and the donor nucleic acid is inserted into the target DNA to produce altered DNA in the stem cell, and repeating step (a) multiple times to produce multiple alterations to the DNA in the stem cell. 
     According to one aspect, the RNA is between about 10 to about 500 nucleotides. According to one aspect, the RNA is between about 20 to about 100 nucleotides. 
     According to one aspect, the one or more RNAs is a guide RNA. According to one aspect, the one or more RNAs is a tracrRNA-crRNA fusion. 
     According to one aspect, the DNA is genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA. 
     According to one aspect, a cell may be genetically modified to reversibly include a nucleic acid encoding a DNA binding enzyme using a vector which can be easily removed using an enzyme. Useful vectors methods are known to those of skill in the art and include lentivirus, adeno associated virus, nuclease and integrase mediated tarteget insertion methods and transposon mediated insertion methods. According to one aspect, the nucleic acid encoding a DNA binding enzyme that has been added, such as by using a cassette or vector can be removed in its entirety along with the cassette and vector and without leaving a portion of such nucleic acid, cassette or vector in the genomic DNA, for example. Such removal is referred to in the art as “scarless” removal, as the genome is the same as it was before addition of the nucleic acid, cassette or vector. One exemplary embodiment for insertion and scarless removal is a PiggyBac vector commercially available from System Biosciences. 
     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 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( a )-( e )  are directed to functional tests of re-TALENs in human somatic and stem cells. 
         FIG. 1( a )  Schematic representation of experimental design for testing genome targeting efficiency. A genomically integrated GFP coding sequence is disrupted by the insertion of a stop codon and a 68 bp genomic fragment derived from the AAVS1 locus (bottom). Restoration of the GFP sequence by nuclease-mediated homologous recombination with tGFP donor (top) results in GFP+ cells that can be quantitated by FACS. Re-TALENs and TALENs target identical sequences within AAVS1 fragments.
 
 FIG. 1( b )  Bar graph depicting GFP+ cell percentage introduced by tGFP donor alone, TALENs with tGFP donor, and re-TALENs with tGFP donor at the target locus, as measured by FACS. (N=3, error bar=SD) Representative FACS plots are shown below.
 
 FIG. 1( c )  Schematic overview depicting the targeting strategy for the native AAVS1 locus. The donor plasmid, containing splicing acceptor (SA)-2A (self-cleaving peptides), puromycin resistant gene (PURO) and GFP were described (see reference 10 hereby incorporated by reference in its entirety. The locations of PCR primers used to detect successful editing events are depicted as blue arrows.
 
 FIG. 1( d )  Successfully targeted clones of PGP1 hiPSCs were selected with puromycin (0.5ug/mL) for 2 weeks. Microscopy images of three representative GFP+ clones are shown. Cells were also stained for the pluripotency markers TRA-1-60. Scale bar: 200 μm.
 
 FIG. 1( e )  PCR assays performed on these the monoclonal GFP+ hiPSC clones demonstrated successful insertions of the donor cassettes at the AAVS1 site (lane 1,2,3), whereas plain hiPSCs show no evidence of successful insertion (lane C).
 
 FIG. 1( f )  depicts SEQ ID NO:1
 
         FIG. 2( a )-( c )  relate to a comparison of reTALENs and Cas9-gRNAs genome targeting efficiency on CCR5 in iPSCs. 
         FIG. 2( a )  Schematic representation of genome engineering experimental design. At the re-TALEN pair or Cas9-gRNA targeting site, a 90 mer ssODN carrying a 2 bp mismatch against the genomic DNA was delivered along with the reTALEN or Cas9-gRNA constructs into PGP1 hiPSCs. The cutting sites of the nucleases are depicted as red arrows in the figure.
 
 FIG. 2( b )  Deep sequencing analysis of HDR and NHEJ efficiencies for re-TALEN pairs (CCR5 #3) and ssODN, or the Cas9-gRNA and ssODN. Alterations in the genome of hiPSCs were analyzed from high-throughput sequence data by GEAS. Top: HDR was quantified from the fraction of reads that contained a 2 bp point mutation built into the center of the ssODN (blue), and NHEJ activity was quantified from the fraction of deletions (grey)/Insertions (red) at each specific position in the genome. For the reTALEN and ssODN graphs, green dashed lines are plotted to mark the outer boundary of the re-TALEN pair&#39;s binding sites, which are at positions −26 bp and +26 bp relative to the center of the two re-TALEN binding sites. For Cas9-gRNA and ssODN graphs, the green dashed lines mark the outer boundary of the gRNA targeting site, which are at positions −20 and −1 bp relative to the PAM sequence. Bottom: Deletion/Insertion size distribution in hiPSCs analyzed from the entire NHEJ population with treatments indicated above.
 
 FIG. 2( c )  The genome editing efficiency of re-TALENs and Cas9-gRNAs targeting CCR5 in PGP1 hiPSCs.
 
Top: schematic representation of the targeted genome editing sites in CCR5. The 15 targeting sites are illustrated by blue arrows below. For each site, cells were co-transfected with a pair of re-TALENs and their corresponding ssODN donor carrying 2 bp mismatches against the genomic DNA. Genome editing efficiencies were assayed 6 days after transfection. Similarly, 15 Cas9-gRNAs were transfected with their corresponding ssODNs individually into PGP1-hiPSCs to target the same 15 sites and analyzed the efficiency 6 days after transfection. Bottom: the genome editing efficiency of re-TALENs and Cas9-gRNAs targeting CCR5 in PGP1 hiPSCs. Panel 1 and 2 indicate NHEJ and HDR efficiencies mediated by reTALENs. Panel 3 and 4 indicate NHEJ and HDR efficiencies mediated by Cas9-gRNAs. NHEJ rates were calculated by the frequency of genomic alleles carrying deletions or insertions at the targeting region; HDR rates were calculated by the frequency of genomic alleles carrying 2 bp mismatches. Panel 5, the DNaseI HS profile of a hiPSC cell line from ENCODE database (Duke DNase HS, iPS NIHi7 DS). Of note, the scales of different panels are different.
 
         FIG. 3( a )-( d )  are directed to a study of functional parameters governing ssODN-mediated HDR with re-TALENs ir Cas9-gRNAs in PGP1 hiPSCs. 
         FIG. 3( a )  PGP1 hiPSCs were co-transfected with re-TALENs pair (#3) and ssODNs of different lengths (50, 70, 90, 110, 130, 150, 170 nts). All ssODNs possessed an identical 2 bp mismatch against the genomic DNA in the middle of their sequence. A 90 mer ssODN achieved optimal HDR in the targeted genome. The assessment of HDR, NHEJ-incurred deletion and insertion efficiency is as described herein.
 
 FIG. 3( b )  90 mer ssODNs corresponding to re-TALEN pair #3 each containing a 2 bp mismatch (A) in the center and an additional 2 bp mismatch (B) at different positions offset from A (where offsets varied from −30 bp→30 bp) were used to test the effects of deviations from homology along the ssODN. Genome editing efficiency of each ssODN was assessed in PGP1 hiPSCs. The bottom bar graph shows the incorporation frequency of A only, B only, and A+B in the targeted genome. HDR rates decrease as the distance of homology deviations from the center increase.
 
 FIG. 3( c )  ssODNs targeted to sites with varying distances (−620 bp˜480 bp) away from the target site of re-TALEN pair #3 were tested to assess the maximum distance within which ssODNs can be placed to introduce mutations. All ssODNs carried a 2 bp mismatch in the middle of their sequences. Minimal HDR efficiency (&lt;=0.06%) was observed when the ssODN mismatch was positioned 40 bp away from the middle of re-TALEN pair&#39;s binding site.
 
 FIG. 3( d )  PGP1 hiPSCs were co-transfected with Cas9-gRNA (AAVS1) and ssODNs of different orientation (O c : complement to gRNA; O n : non-complement to gRNA) and different lengths (30, 50, 70, 90, 110 nt). All ssODNs possessed an identical 2 bp mismatch against the genomic DNA in the middle of their sequence. A 70mer O c  achieved optimal HDR in the targeted genome.
 
         FIG. 4( a )-( e )  are directed to using re-TALENs and ssODNs to obtain monoclonal genome edited hiPSC without selection. 
         FIG. 4( a )  Timeline of the experiment.
 
 FIG. 4( b )  Genome engineering efficiency of re-TALENs pair and ssODN (#3) assessed by the NGS platform described in  FIG. 2   b.  
 
 FIG. 4( c )  Sanger sequencing results of monoclonal hiPSC colonies after genome editing. The 2 bp heterogeneous genotype (CT/CT→TA/CT) was successfully introduced into the genome of PGP1-iPS-3-11, PGP1-iPS-3-13 colonies.
 
 FIG. 4( d )  Immunofluorescence staining of targeted PGP1-iPS-3-11. Cells were stained for the pluripotency markers Tra-1-60 and SSEA4.
 
 FIG. 4( e )  Hematoxylin and eosin staining of teratoma sections generated from monoclonal PGP1-iPS-3-11 cells.
 
         FIG. 5( a )-( b ) . Design of reTALE.  FIG. 5( a )  Sequence alignment of the original TALE RVD monomer with monomers in re-TALE-16.5 (re-TALE-M1→re-TALE-M17) (SEQ ID NOs:2-19 and 222). Nucleotide alterations from the original sequence are highlighted in gray.  FIG. 5( b )  Test of repetitiveness of re-TALE by PCR. Top panel illustrates the structure of re-TALE/TALE and positions of the primers in the PCR reaction. Bottom panel illustrates PCR bands with condition indicated below. The PCR laddering presents with the original TALE template (right lane). 
         FIG. 6( a )-( c ) . Design and practice of TALE Single-incubation Assembly (TASA) assembly. 
         FIG. 6( a )  Schematic representation of the library of re-TALE dimer blocks for TASA assembly. There is a library of 10 re-TALE dimer blocks encoding two RVDs. Within each block, all 16 dimers share the same DNA sequence except the RVD encoding sequences; Dimers in different blocks have distinct sequences but are designed such that they share 32 bp overlaps with the adjacent blocks. DNA (SEQ ID NO:223) and amino acid sequence (SEQ ID NO:224) of one dimer (Block6_AC) are listed on the right.
 
 FIG. 6( b )  Schematic representation of TASA assembly. The left panel illustrates the TASA assembly method: a one-pot incubation reaction is conducted with an enzyme mixture/re-TALE blocks/re-TALE-N/TF backbone vectors. The reaction product can be used directly for bacterial transformation. The right panel illustrates the mechanism of TASA. The destination vector is linearized by an endonuclease at 37° C. to cut off ccdB counter-selection cassette; the exonuclease, which processes the end of blocks and linearized vectors, exposes ssDNA overhangs at the end of fragments to allow blocks and vector backbones to anneal in a designated order. When the temperature rises up to 50° C., polymerases and ligases work together to seal the gap, producing the final constructs ready for transformation.
 
 FIG. 6( c )  TASA assembly efficiency for re-TALEs possessing different monomer lengths. The blocks used for assembly are illustrated on the left and the assembly efficiency is presented on the right.
 
         FIG. 7( a )-( d )  relate to the functionality and sequence integrity of Lenti-reTALEs. 
         FIG. 8( a )-( b )  relate to the sensitivity and reproducibility of LEAS. 
         FIG. 8( a )  Information-based analysis of HDR detection limit. Given the dataset of re-TALENs (#10)/ssODN, the reads containing the expected editing (HDR) were identified and these HDR reads were systematically removed to generate different artificial datasets with a “diluted” editing signal. Datasets with 100, 99.8, 99.9, 98.9, 97.8, 89.2, 78.4, 64.9, 21.6, 10.8, 2.2, 1.1, 0.2. 0.1, 0.02, and 0% removal of HDR reads were generated to generate artificial datasets with HR efficiency ranging from 0˜0.67%. For each individual dataset, mutual information (MI) of the background signal (in purple) and the signal obtained in the targeting site (in green) was estimated. MI at the targeting site is remarkably higher than the background when the HDR efficiency is above 0.0014%. A limit of HDR detection between 0.0014% and 0.0071% was estimated. MI calculation is described herein.
 
 FIG. 8( b )  The test of reproducibility of genome editing assessment system. The pairs of plots (Top and Bottom) show the HDR and NHEJ assessment results of two replicates with re-TALENs pair and cell type indicated above. For each experiment, nucleofection, targeted genome amplification, deep-sequencing and data analysis were conducted independently. The genome editing assessment variation of replicates was calculated as √2 (|HDR1−HDR2|)/((HDR+HDR2)/2)=ΔHDR/HDR and √2 (|NHEJ1−NHEJ2|)/((NHEJ1+NHEJ2)/2)=ΔNHEJ/NHEJ and the variation results are listed below the plots. The average variation of the system was (19%+11%+4%+9%+10%+35%)/6=15%. Factors that may contribute to the variations include the status of cells under nucleofection, nucleofection efficiency, and sequencing coverage and quality.
 
         FIG. 9( a )-( c )  relate to statistical analysis of NHEJ and HDR efficiencies by reTALENs and Cas9-gRNAs on CCR5. 
         FIG. 9( a )  The correlation of HR and NHEJ efficiencies mediated by reTALENs at identical sites in iPSCs (r=0.91, P&lt;1×10 −5 .
 
 FIG. 9( b )  The correlation of HR and NHEJ efficiencies mediated by Cas9-gRNA at identical sites in iPSCs (r=0.74, P=0.002).
 
 FIG. 9 ( c .) The correlation of NHEJ efficiencies mediated by Cas9-gRNA and the Tm temperature of gRNA targeting site in iPSCs (r=0.52, P=0.04)
 
         FIG. 10 . The correlation analysis of genome editing efficiency and epigenetic state. 
       Pearson correlation was used to study possible associations between DNase I sensitivity and genome engineering efficiencies (HR, NHEJ). The observed correlation was compared to a randomized set (N=100000). Observed correlations higher than the 95th percentile, or lower than the 5th percentile of the simulated distribution were considered as potential associations. No remarkable correlation between DNase1 sensitivity and NHEJ/HR efficiencies was observed. 
         FIG. 11( a )-( c )  relate to the impact of homology pairing in the ssODN-mediated genome editing. 
         FIG. 11( a )  In the experiment described in  FIG. 3 b   , overall HDR as measured by the rate at which the middle 2b mismatch (A) was incorporated decreased as the secondary mismatches B increased their distance from the A (relative position of B to A varies from −30→30 bp). The higher rates of incorporation when B is only 10 bp away from A (−10 bp and +10 b) may reflect a lesser need for pairing of the ssODN against genomic DNA proximal to the dsDNA break.
 
 FIG. 11( b )  Distribution of gene conversion lengths along the ssODN. At each distance of B from A, a fraction of HDR events incorporates only A while another fraction incorporates both A and B. These two events may be interpretable in terms of gene conversion tracts (Elliott et al., 1998), whereby A+B events represent long conversion tracts that extend beyond B and A-only events represent shorter ones that do not reach to B. Under this interpretation, a distribution of gene conversion lengths in both directions along the oligo can be estimated (the middle of ssODN is defined as 0, conversion tracks towards the 5′ end of ssODN as − direction, and 3′ end as + direction). Gene conversion tracts progressively decrease in incidence as their lengths increase, a result very similar to gene conversion tract distributions seen with dsDNA donors, but on a highly compressed distance scale of tens of bp for the ssDNA oligo vs. hundreds of bases for dsDNA donors.
 
 FIG. 11( c )  Assays for gene conversion tracts using a single ssODN that contains a series of mutations and measuring contiguous series of incorporations. A ssODN donor with three pairs of 2 bp mismatches (orange) spaced at intervals of 10 nt on either side of the central 2 bp mismatch (Top) was used. Few genomic sequencing reads were detected (see reference 62 hereby incorporated by reference in its entirety) carrying &gt;=1 mismatches defined by ssODN among &gt;300,000 reads sequencing this region. All these reads were plotted (bottom) and the sequence of the reads was color coded. Orange: defined mismatches; green: wild type sequence. Genome editing with this ssODN gave rise to a pattern in which middle mutation alone was incorporated 85% (53/62) of the time, with multiple B mismatches incorporated at other times. Although numbers of B incorporation events were too low to estimate a distribution of tract lengths &gt;10 bp, it is clear that the short tract region from −10-10 bp predominates.
 
         FIG. 12 . Cas9-gRNA nuclease and nickases genome editing efficiencies. 
       PGP1 iPSCs were co-transfected with combination of nuclease (C 2 ) (Cas9-gRNA) or nickase (C c ) (Cas9D10A-gRNA) and ssODNs of different orientation (Oc and On). All ssODNs possessed an identical 2 bp mismatch against the genomic DNA in the middle of their sequence. The assessment of HDR is described herein. 
         FIG. 13 . The design and optimization of re-TALE sequence. 
       The re-TALE sequence was evolved in several design cycles to eliminate repeats. In each cycle, synonymous sequences from each repeat are evaluated. Those with the largest hamming distance to the evolving DNA are selected. The final sequence with cai=0.59 ΔG=−9.8 kcal/mol. An R package was provided to carry out this general framework for synthetic protein design. 
         FIG. 14  is a gel image showing PCR validation of the genomic insertion of Cas 9 in PGP1 cells. Line 3, 6, 9, 12 are PCR product of plain PGP1 cell lines. 
         FIG. 15  is a graph of the mRNA expression level of Cas9 mRNA under the induction. 
         FIG. 16  is a graph showing genome targeting efficiency by different RNA designs. 
         FIG. 17  is a graph showing genome targeting efficiency of 44% homologous recombination achieved by a guide RNA-donor DNA fusion. 
         FIG. 18  is a diagram showing the genotype of isogenic PGP1 cell lines generated by system described herein. PGP1-iPS-BTHH has the single nucleotides deletion phenotype as the BTHH patient. PGP1-NHEJ has 4 bp deletions that generated frame-shift mutations in a different way. 
         FIG. 19  is a graph showing that cardiomyocyte derived from isogenic PGP1 iPS recapitulated defective ATP production and F1F0 ATPase specific activity as demonstrated in patient specific cells. 
         FIG. 20  shows sequences for re-talen-backbone sequence (SEQ ID NO:20) and re-TALE-TF backbone sequence (SEQ ID NO:21). 
     
    
    
     DETAILED DESCRIPTION 
     Aspects of the present invention are directed to the use of a TALEN that lacks certain repeat sequences, for nucleic acid engineering, for example by cutting double stranded nucleic acid. The use of the TALEN to cut double stranded nucleic acid can result in nonhomologous end joining (NHEJ) or homologous recombination (HR). Aspects of the present disclosure also contemplate the use of a TALEN that lacks repeat sequences for nucleic acid engineering, for example by cutting double stranded nucleic acid, in the presence of a donor nucleic acid and insertion of the donor nucleic acid into the double stranded nucleic acid, such as by nonhomologous end joining (NHEJ) or homologous recombination (HR). 
     Transcription activator-like effector nucleases (TALENs) are known in the art and include artificial restriction enzymes generated by fusing a TAL effector DNA binding domain to a DNA cleavage domain. Restriction enzymes are enzymes that cut DNA strands at a specific sequence. Transcription activator-like effectors (TALEs) can be engineered to bind to a desired DNA sequence. See Boch, Jens (February 2011). “TALEs of genome targeting”.  Nature Biotechnology  29 (2): 135-6 hereby incorporated by reference in its entirety. By combining such an engineered TALE with a DNA cleavage domain (which cuts DNA strands), a TALEN is produced which is a restriction enzyme that is specific for any desired DNA sequence. According to certain aspects, the TALEN is introduced into a cell for target nucleic acid editing in situ, such as genome editing in situ. 
     According to one aspect, the non-specific DNA cleavage domain from the end of the FokI endonuclease can be used to construct hybrid nucleases that are active in yeast cells, plant cells and animal cells. The FokI domain functions as a dimer, requiring two constructs with unique DNA binding domains for sites in the target genome with proper orientation and spacing. Both the number of amino acid residues between the TALE DNA binding domain and the FokI cleavage domain and the number of bases between the two individual TALEN binding sites affect activity. 
     The relationship between amino acid sequence and DNA recognition of the TALE binding domain allows for designable proteins. Software programs such as DNAWorks can be used to design TALE constructs. Other methods of designing TALE constructs are known to those of skill in the art. See Cermak, T ; Doyle, E. L.; Christian, M.; Wang, L.; Zhang, Y.; Schmidt, C.; Baller, J. A.; Somia, N. V. et al. (2011). “Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting”.  Nucleic Acids Research . doi:10.1093/nar/gkr218; Zhang, Feng; et.al. (February 2011). “Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription”,  Nature Biotechnology  29 (2): 149-53; Morbitzer, R.; Elsaesser, J.; Hausner, J.; Lahaye, T. (2011). “Assembly of custom TALE-type DNA binding domains by modular cloning”.  Nucleic Acids Research . doi:10′1093/nar/gkr151; Li, T.; Huang, S.; Zhao, X.; Wright, D. A.; Carpenter, S.; Spalding, M. H.; Weeks, D. P.; Yang, B. (2011). “Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes”.  Nucleic Acids Research . doi″10.1093/nar/gkr188; Geiβler, R.; Scholze, H.; Hahn, S.; Streubel, J.; Bonas, U.; Behrens, S. E.; Boch, J. (2011). “Transcriptional Activators of Human Genes with Programmable DNA-Specificity”. In Shiu, Shin-Han.  PLoS ONE  6 (5): e19509; Weber, E.; Gruetzner, R.; Werner, S.; Engler, C.; Marillonnet, S. (2011). “Assembly of Designer TAL Effectors by Golden Gate Cloning”. In Bendahmane, Mohammed  PLoS ONE  6 (5): e19722 hereby incorporated by reference in their entireties. 
     According to an exemplary aspect, once the TALEN genes have been assembled they may inserted into plasmids according to certain embodiments; the plasmids are then used to transfect the target cell where the gene products are expressed and enter the nucleus to access the genome. According to exemplary aspects, TALENs as described herein can be used to edit target nucleic acids, such as genomes, by inducing double-strand breaks (DSB), which cells respond to with repair mechanisms. Exemplary repair mechanisms include non-homologous end joining (NHEJ) which reconnects DNA from either side of a double-strand break where there is very little or no sequence overlap for annealing. This repair mechanism induces errors in the genome via insertion or deletion (indels), or chromosomal rearrangement; any such errors may render the gene products coded at that location non-functional. See Miller, Jeffrey; et.al. (February 2011). “A TALE nuclease architecture for efficient genome editing”.  Nature Biotechnology  29 (2): 143-8 hereby incorporated by reference in its entirety. Because this activity can vary depending on the species, cell type, target gene, and nuclease used, the activity can be monitored by using a heteroduplex cleavage assay which detects any difference between two alleles amplified by PCR. Cleavage products can be visualized on simple agarose gels or slab gel systems. 
     Alternatively, DNA can be introduced into a genome through NHEJ in the presence of exogenous double-stranded DNA fragments. Homology directed repair can also introduce foreign DNA at the DSB as the transfected double-stranded sequences are used as templates for the repair enzymes. According to certain aspects the TALENs described herein can be used to generate stably modified human embryonic stem cell and induced pluripotent stem cell (IPSCs) clones. According to certain aspects the TALENs described herein can be used to generate knockout species such as C. elegans, knockout rats, knockout mice or knockout zebrafish. 
     According to one aspect of the present disclosure, embodiments are directed to the use of exogenous DNA, nuclease enzymes such as DNA binding proteins and guide RNAs to co-localize to DNA within a stem cell and digest or cut the DNA with insertion of the exogenous DNA. Such DNA binding proteins are readily known to those of skill in the art to bind to DNA for various purposes. Such DNA binding proteins may be naturally occurring. DNA binding proteins included within the scope of the present disclosure include those which may be guided by RNA, referred to herein as guide RNA. According to this aspect, the guide RNA and the RNA guided DNA binding protein form a co-localization complex at the DNA. Such DNA binding proteins having nuclease activity are known to those of skill in the art, and include naturally occurring DNA binding proteins having nuclease activity, such as Cas9 proteins present, for example, in Type II CRISPR systems. Such 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. 
     Exemplary DNA binding proteins having nuclease activity function to nick or cut double stranded DNA. Such nuclease activity may result from the DNA binding protein having one or more polypeptide sequences exhibiting nuclease activity. Such exemplary DNA binding proteins may have two separate nuclease domains with each domain responsible for cutting or nicking a particular strand of the double stranded DNA. Exemplary polypeptide sequences having nuclease activity known to those of skill in the art include the McrA-HNH nuclease related domain and the RuvC-like nuclease domain. Accordingly, exemplary DNA binding proteins are those that in nature contain one or more of the McrA-HNH nuclease related domain and the RuvC-like nuclease domain. 
     An exemplary DNA binding protein is an RNA guided DNA binding protein of a Type II CRISPR System. An exemplary DNA binding protein is a Cas9 protein. 
     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 Jinke 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  BTAil;  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  B510 uid46085;  Rhodospirillum rubrum  ATCC 11170;  Diaphorobacter  TPSY uid29975;  Verminephrobacter eiseniae  EF01-2;  Neisseria meningitides  053442;  Neisseria meningitides  alpha14;  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. Accordingly, aspects of the present disclosure are directed to a Cas9 protein present in a Type II CRISPR system. 
     The Cas9 protein may be referred by one of skill in the art in the literature as Csn1. The  S. pyogenes  Cas9 protein is shown below. See Deltcheva et al.,  Nature  471, 602-607 (2011) hereby incorporated by reference in its entirety. 
     
       
         
           
               
            
               
                 (SEQ ID NO: 22) 
               
               
                 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIG 
               
               
                   
               
               
                 ALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFF 
               
               
                   
               
               
                 HRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTD 
               
               
                   
               
               
                 KADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLF 
               
               
                   
               
               
                 EENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALS 
               
               
                   
               
               
                 LGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAK 
               
               
                   
               
               
                 NLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL 
               
               
                   
               
               
                 PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVK 
               
               
                   
               
               
                 LNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIE 
               
               
                   
               
               
                 KILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQS 
               
               
                   
               
               
                 FIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAF 
               
               
                   
               
               
                 LSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFN 
               
               
                   
               
               
                 ASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLK 
               
               
                   
               
               
                 TYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSD 
               
               
                   
               
               
                 GFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKK 
               
               
                   
               
               
                 GILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRI 
               
               
                   
               
               
                 EEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRL 
               
               
                   
               
               
                 SDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNY 
               
               
                   
               
               
                 WRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV 
               
               
                   
               
               
                 AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINN 
               
               
                   
               
               
                 YHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEI 
               
               
                   
               
               
                 GKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGR 
               
               
                   
               
               
                 DFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWD 
               
               
                   
               
               
                 PKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEK 
               
               
                   
               
               
                 NPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE 
               
               
                   
               
               
                 LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS 
               
               
                   
               
               
                 EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAA 
               
               
                   
               
               
                 FKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD- 
               
            
           
         
       
     
     According to one aspect, the RNA guided DNA binding protein includes homologs and orthologs of Cas9 which retain the ability of the protein to bind to the DNA, be guided by the RNA and cut the DNA. According to one aspect, the Cas9 protein includes the sequence as set forth for naturally occurring Cas9 from  S. pyogenes  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, such as an RNA guided DNA binding protein. 
     According to one aspect, an engineered Cas9-gRNA system is provided which enables RNA-guided genome cutting in a site specific manner in a stem cell, if desired, and modification of the stem cell genome by insertion of exogenous donor nucleic acids. The guide RNAs are complementary to target sites or target loci on the DNA. The guide RNAs can be crRNA-tracrRNA chimeras. The guide RNAs can be introduced from media surrounding the cell. In this manner a method of continuously modifying a cell is provided to the extent that various guide RNAs are provided to surrounding media and with the uptake by the cell of the guide RNAs and with supplementation of the media with additional guide RNAs. Supplementation may be in a continuous manner. The Cas9 binds at or near target genomic DNA. The one or more guide RNAs bind at or near target genomic DNA. The Cas9 cuts the target genomic DNA and exogenous donor DNA is inserted into the DNA at the cut site. 
     Accordingly, methods are directed to the use of a guide RNA with a Cas9 protein and an exogenous donor nucleic acid to multiplex insertions of exogenous donor nucleic acids into DNA within a stem cell expressing Cas9 by cycling the insertion of nucleic acid encoding the RNA (or providing RNA from the surrounding media) and exogenous donor nucleic acid, expressing the RNA (or uptaking the RNA), colocalizing the RNA, Cas9 and DNA in a manner to cut the DNA, and insertion of the exogenous donor nucleic acid. The method steps can be cycled in any desired number to result in any desired number of DNA modifications. Methods of the present disclosure are accordingly directed to editing target genes using the Cas9 proteins and guide RNAs described herein to provide multiplex genetic and epigenetic engineering of stem cells. 
     Further aspects of the present disclosure are directed to the use of DNA binding proteins or systems (such as the modified TALENS or Cas9 described herein) in general for the multiplex insertion of exogenous donor nucleic acids into the DNA, such as genomic DNA, of a stem cell, such as a human stem cell. One of skill in the art will readily identify exemplary DNA binding systems based on the present disclosure. 
     Cells according to the present disclosure unless otherwise specified 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 somatic cells, stem cells, 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, such as human cells. Further, cells include any in which it would be beneficial or desirable to modify DNA. 
     Target nucleic acids include any nucleic acid sequence to which a TALEN or RNA guided DNA binding protein having nuclease activity as described herein can be useful to nick or cut. Target nucleic acids include any nucleic acid sequence to which a co-localization complex as described herein can be useful to nick or cut. Target nucleic acids include genes. For purposes of the present disclosure, DNA, such as double stranded DNA, can include the target nucleic acid and a co-localization complex can bind to or otherwise co-localize with the DNA or a TALEN can otherwise bind with the DNA at or adjacent or near the target nucleic acid and in a manner in which the co-localization complex or the TALEN may have a desired effect on the target nucleic acid. Such target nucleic acids can include endogenous (or naturally occurring) nucleic acids and exogenous (or foreign) nucleic acids. One of skill based on the present disclosure will readily be able to identify or design guide RNAs and Cas9 proteins which co-localize to a DNA or a TALEN which binds to a DNA, including a target nucleic acid. One of skill will further be able to identify transcriptional regulator proteins or domains, such as transcriptional activators or transcriptional repressors, which likewise co-localize to a DNA including a target nucleic acid. DNA includes genomic DNA, mitochondrial DNA, viral DNA or exogenous DNA. According to one aspect, materials and methods useful in the practice of the present disclosure include those described in Di Carlo, et al.,  Nucleic Acids Research,  2013, vol. 41, No. 7 4336-4343 hereby incorporated by reference in its entirety for all purposes including exemplary strains and media, plasmid construction, transformation of plasmids, electroporation of transcient gRNA cassette and donor nucleic acids, transformation of gRNA plasmid with donor DNA into Cas9-expressing cells, galactose induction of Cas9, identification of CRISPR-Cas targets in yeast genome, etc. Additional references including information, materials and methods useful to one of skill in carrying out the invention are provided in Mali, P., Yang, L., Esvelt, K. M., Aach, J., Guell, M., DiCarlo, J. E., Norville, J. E. and Church, G. M. (2013) RNA-Guided human genome engineering via Cas9.  Science,  10.1126fscience.1232033; Storici, F., Durham, C. L., Gordenin, D. A. and Resnick, M. A. (2003) Chromosomal site-specific double-strand breaks are efficiently targeted for repair by oligonucleotides in yeast.  PNAS,  100, 14994-14999 and Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A. and Charpentier, E. (2012) A programmable dual-RNA-Guided DNA endonuclease in adaptive bacterial immunity.  Science,  337, 816-821 each of which are hereby incorporated by reference in their entireties for all purposes. 
     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. 
     Donor nucleic acids include any nucleic acid to be inserted into a nucleic acid sequence as described herein. 
     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 
     Guide RNA Assembly 
     19 bp of the selected target sequence (i.e. 5′-N 19  of 5′-N 19 -NGG-3′) were incorporated into two complementary 100 mer oligonucleotides (TTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGN19GTTTTAGAGCTAGAAATAG CAAGTTAAAATAAGGCTAGTCC) (SEQ ID NO:23). Each 100 mer oligonucleotide was suspended at 100 mM in water, mixed with equal volume and annealed in thermocycle machine (95° C., 5 min; Ramp to 4° C., 0.1° C./sec). To prepare the destination vector, the gRNA cloning vector (Addgene plasmid ID 41824) was linearized using AfIII and the vector was purified. The (10 ul) gRNA assembly reaction was carried out with 10 ng annealed 100 bp fragment, 100 ng destination backbone, 1× Gibson assembly reaction mix (New England Biolabs) at 50° C. for 30 min. The reaction can be processed directly for bacterial transformation to colonize individual assemblies. 
     EXAMPLE II 
     Re-Coded TALEs Design and Assembly 
     re-TALEs were optimized at different levels to facilitate assembly, and improve expression. re-TALE DNA sequences were first co-optimized for a human codon-usage, and low mRNA folding energy at the 5′ end (GeneGA, Bioconductor). The obtained sequence was evolved through several cycles to eliminate repeats (direct or inverted) longer than 11 bp (See  FIG. 12 ). In each cycle, synonymous sequences for each repeat are evaluated. Those with the largest hamming distance to the evolving DNA are selected. The sequence of one of re-TALE possessing 16.5 monomers as follows 
     
       
         
           
               
            
               
                 (SEQ ID NO: 24) 
               
               
                 CTAACCCCTGAAAGGTAGTCGCTATAGCTTCAAATATCGGGGGCAAGCA 
               
               
                   
               
               
                 AGCACTTGAGACCGTTCAACGACTCCTGCCAGTGCTCTGCCAAGCCCAT 
               
               
                   
               
               
                 GGATTGACTCCGGAGCAAGTCGTCGCGATCGCGAGCAACGGCGGGGGGA 
               
               
                   
               
               
                 AGCAGGCGCTGGAAACTGTTCAGAGACTGCTGCCTGTACTTTGTCAGGC 
               
               
                   
               
               
                 GCATGGTCTCACCCCCGAACAGGTTGTCGCAATAGCAAGTAATATAGGC 
               
               
                   
               
               
                 GGTAAGCAAGCCCTAGAGACTGTGCAACGCCTGCTCCCCGTGCTGTGTC 
               
               
                   
               
               
                 AGGCTCACGGTCTGACACCTGAACAAGTTGTCGCGATAGCCAGTCACGA 
               
               
                   
               
               
                 CGGGGGAAAACAAGCTCTAGAAACGGTTCAAAGGTTGTTGCCCGTTCTG 
               
               
                   
               
               
                 TGCCAAGCACATGGGTTAACACCCGAACAAGTAGTAGCGATAGCGTCAA 
               
               
                   
               
               
                 ATAACGGGGGTAAACAGGCTTTGGAGACGGTACAGCGGTTATTGCCGGT 
               
               
                   
               
               
                 CCTCTGCCAGGCCCACGGACTTACGCCAGAACAGGTGGTTGCAATTGCC 
               
               
                   
               
               
                 TCCAACATCGGCGGGAAACAAGCGTTGGAAACTGTGCAGAGACTCCTTC 
               
               
                   
               
               
                 CTGTTTTGTGTCAAGCCCACGGCTTGACGCCTGAGCAGGTTGTGGCCAT 
               
               
                   
               
               
                 CGCTAGCCACGACGGAGGGAAGCAGGCTCTTGAAACCGTACAGCGACTT 
               
               
                   
               
               
                 CTCCCAGTTTTGTGCCAAGCTCACGGGCTAACCCCCGAGCAAGTAGTTG 
               
               
                   
               
               
                 CCATAGCAAGCAACGGAGGAGGAAAACAGGCATTAGAAACAGTTCAGCG 
               
               
                   
               
               
                 CTTGCTCCCGGTACTCTGTCAGGCACACGGTCTAACTCCGGAACAGGTC 
               
               
                   
               
               
                 GTAGCCATTGCTTCCCATGATGGCGGCAAACAGGCGCTAGAGACAGTCC 
               
               
                   
               
               
                 AGAGGCTCTTGCCTGTGTTATGCCAGGCACATGGCCTCACCCCGGAGCA 
               
               
                   
               
               
                 GGTCGTTGCCATCGCCAGTAATATCGGCGGAAAGCAAGCTCTCGAAACA 
               
               
                   
               
               
                 GTACAACGGCTGTTGCCAGTCCTATGTCAAGCTCATGGACTGACGCCCG 
               
               
                   
               
               
                 AGCAGGTAGTGGCAATCGCATCTCACGATGGAGGTAAACAAGCACTCGA 
               
               
                   
               
               
                 GACTGTCCAAAGATTGTTACCCGTACTATGCCAAGCGCATGGTTTAACC 
               
               
                   
               
               
                 CCAGAGCAAGTTGTGGCTATTGCATCTAACGGCGGTGGCAAACAAGCCT 
               
               
                   
               
               
                 TGGAGACAGTGCAACGATTACTGCCTGTCTTATGTCAGGCCCATGGCCT 
               
               
                   
               
               
                 TACTCCTGAGCAAGTCGTAGCTATCGCCAGCAACATAGGTGGGAAACAG 
               
               
                   
               
               
                 GCCCTGGAAACCGTACAACGTCTCCTCCCAGTACTTTGTCAAGCACACG 
               
               
                   
               
               
                 GGTTGACACCGGAACAAGTGGTGGCGATTGCGTCCAACGGCGGAGGCAA 
               
               
                   
               
               
                 GCAGGCACTGGAGACCGTCCAACGGCTTCTTCCGGTTCTTTGCCAGGCT 
               
               
                   
               
               
                 CATGGGCTCACGCCAGAGCAGGTGGTAGCAATAGCGTCGAACATCGGTG 
               
               
                   
               
               
                 GTAAGCAAGCGCTTGAAACGGTCCAGCGTCTTCTGCCGGTGTTGTGCCA 
               
               
                   
               
               
                 GGCGCACGGACTCACACCAGAACAAGTGGTTGCTATTGCTAGTAACAAC 
               
               
                   
               
               
                 GGTGGAAAGCAGGCCCTCGAGACGGTGCAGAGGTTACTTCCCGTCCTCT 
               
               
                   
               
               
                 GTCAAGCGCACGGCCTCACTCCAGAGCAAGTGGTTGCGATCGCTTCAAA 
               
               
                   
               
               
                 CAATGGTGGAAGACCTGCCCTGGAA  
               
            
           
         
       
     
     According to certain aspects, TALEs may be used having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to the above sequence. One of skill will readily understand where the above sequence may vary while still maintaining the DNA binding activity of the TALE. 
     re-TALE dimer blocks encoding two RVDs (see  FIG. 6A ) were generated by two rounds of PCR under standard Kapa HIFI (KPAP) PCR conditions, in which the first round of PCR introduced the RVD coding sequence and the second round of PCR generated the entire dimer blocks with 36 bp overlaps with the adjacent blocks. PCR products were purified using QIAquick 96 PCR Purification Kit (QIAGEN) and the concentrations were measured by Nano-drop. The primer and template sequences are listed in Table 1 and Table 2 below. 
     
       
         
           
               
             
               
                 TABLE 1 
               
               
                   
               
               
                 re-TALE blocks sequences 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                 block0 
                 CGCAATGCGCTCACGGGAGCACCCCTCAACCTAACCCCTGAACAGGTAGTC 
               
               
                   
                 GCTATAGCTTCANNNNNNGGGGGCAAGCAAGCACTTGAGACCGTTCAACG 
               
               
                   
                 ACTCCTGCCAGTGCTCTGCCAAGCCCATGGATTGACTCCGGAGCAAGTCGT 
               
               
                   
                 CGCGATCGCGAGCNNNNNNGGGGGGAAGCAGGCGCTGGAAACTGTTCAGA 
               
               
                   
                 GACTGCTGCCTGTACTTTGTCAGGCGCATGGTCTC (SEQ ID NO: 25) 
               
               
                   
               
               
                 block1 
                 AGACTGCTGCCTGTACTTTGTCAGGCGCATGGTCTCACCCCCGAACAGGTTG 
               
               
                   
                 TCGCAATAGCAAGTNNNNNNGGCGGTAAGCAAGCCCTAGAGACTGTGCAA 
               
               
                   
                 CGCCTGCTCCCCGTGCTGTGTCAGGCTCACGGTCTGACACCTGAACAAGTTG 
               
               
                   
                 TCGCGATAGCCAGTNNNNNNGGGGGAAAACAAGCTCTAGAAACGGTTCAA 
               
               
                   
                 AGGTTGTTGCCCGTTCTGTGCCAAGCACATGGGTTA (SEQ ID NO: 26) 
               
               
                   
               
               
                 block1′ 
                 TGCGCTCACGGGAGCACCCCTCAACCTCACCCCCGAACAGGTTGTCGCAAT 
               
               
                   
                 AGCAAGTNNNNNNGGCGGTAAGCAAGCCCTAGAGACTGTGCAACGCCTGC 
               
               
                   
                 TCCCCGTGCTGTGTCAGGCTCACGGTCTGACACCTGAACAAGTTGTCGCGAT 
               
               
                   
                 AGCCAGTNNNNNNGGGGGAAAACAAGCTCTAGAAACGGTTCAAAGGTTGT 
               
               
                   
                 TGCCCGTTCTGTGCCAAGCACATGGGTTA (SEQ ID NO: 27) 
               
               
                   
               
               
                 block2 
                 AGGTTGTTGCCCGTTCTGTGCCAAGCACATGGGTTAACACCCGAACAAGTA 
               
               
                   
                 GTAGCGATAGCGTCANNNNNNGGGGGTAAACAGGCTTTGGAGACGGTACA 
               
               
                   
                 GCGGTTATTGCCGGTCCTCTGCCAGGCCCACGGACTTACGCCAGAACAGGT 
               
               
                   
                 GGTTGCAATTGCCTCCNNNNNNGGCGGGAAACAAGCGTTGGAAACTGTGCA 
               
               
                   
                 GAGACTCCTTCCTGTTTTGTGTCAAGCCCACGGCTTGACGCCT (SEQ ID 
               
               
                   
                 NO: 28) 
               
               
                   
               
               
                 block3 
                 AGACTCCTTCCTGTTTTGTGTCAAGCCCACGGCTTGACGCCTGAGCAGGTTG 
               
               
                   
                 TGGCCATCGCTAGCNNNNNNGGAGGGAAGCAGGCTCTTGAAACCGTACAG 
               
               
                   
                 CGACTTCTCCCAGTTTTGTGCCAAGCTCACGGGCTAACCCCCGAGCAAGTA 
               
               
                   
                 GTTGCCATAGCAAGCNNNNNNGGAGGAAAACAGGCATTAGAAACAGTTCA 
               
               
                   
                 GCGCTTGCTCCCGGTACTCTGTCAGGCACACGGTCTA (SEQ ID NO: 29) 
               
               
                   
               
               
                 block4 
                 CGCTTGCTCCCGGTACTCTGTCAGGCACACGGTCTAACTCCGGAACAGGTC 
               
               
                   
                 GTAGCCATTGCTTCCNNNNNNGGCGGCAAACAGGCGCTAGAGACCGTCCA 
               
               
                   
                 GAGGCTCTTGCCTGTGTTATGCCAGGCACATGGCCTCACCCCGGAGCAGGT 
               
               
                   
                 CGTTGCCATCGCCAGTNNNNNNGGCGGAAAGCAAGCTCTCGAAACAGTAC 
               
               
                   
                 AACGGCTGTTGCCAGTCCTATGTCAAGCTCATGGACTG (SEQ ID NO: 30) 
               
               
                   
               
               
                 block5 
                 CGGCTGTTGCCAGTCCTATGTCAAGCTCATGGACTGACGCCCGAGCAGGTA 
               
               
                   
                 GTGGCAATCGCATCTNNNNNNGGAGGTAAACAAGCACTCGAGACTGTCCA 
               
               
                   
                 AAGATTGTTACCCGTACTATGCCAAGCGCATGGTTTAACCCCAGAGCAAGT 
               
               
                   
                 TGTGGCTATTGCATCTNNNNNNGGTGGCAAACAAGCCTTGGAGACCGTGCA 
               
               
                   
                 ACGATTACTGCCTGTCTTATGTCAGGCCCATGGCCTT (SEQ ID NO: 31) 
               
               
                   
               
               
                 block6 
                 CGATTACTGCCTGTCTTATGTCAGGCCCATGGCCTTACTCCTGAGCAGGTGG 
               
               
                   
                 TCGCTATCGCCAGCNNNNNNGGGGGCAAGCAAGCACTGGAAACAGTCCAG 
               
               
                   
                 CGTTTGCTTCCAGTACTTTGTCAGGCGCATGGATTGACACCGGAACAAGTG 
               
               
                   
                 GTGGCTATAGCCTCANNNNNNGGAGGAAAGCAGGCGCTGGAAACCGTCCA 
               
               
                   
                 ACGTCTTTTACCGGTGCTTTGCCAGGCGCACGGGCTC (SEQ ID NO: 32) 
               
               
                   
               
               
                 block6′ 
                 CGATTACTGCCTGTCTTATGTCAGGCCCATGGCCTTACTCCTGAGCAAGTCG 
               
               
                   
                 TAGCTATCGCCAGCNNNNNNGGTGGGAAACAGGCCCTGGAAACCGTACAA 
               
               
                   
                 CGTCTCCTCCCAGTACTTTGTCAAGCACACGGGTTGACACCGGAACAAGTG 
               
               
                   
                 GTGGCGATTGCGTCCNNNNNNGGAGGCAAGCAGGCACTGGAGACCGTCCA 
               
               
                   
                 ACGGCTTCTTCCGGTTCTTTGCCAGGCTCATGGGCTC (SEQ ID NO: 33) 
               
               
                   
               
               
                 block7 
                 CGGCTTCTTCCGGTTCTTTGCCAGGCTCATGGGCTCACGCCAGAGCAGGTGG 
               
               
                   
                 TAGCAATAGCGTCGNNNNNNGGTGGTAAGCAAGCGCTTGAAACGGTCCAG 
               
               
                   
                 CGTCTTCTGCCGGTGTTGTGCCAGGCGCACGGACTCACACCAGAACAAGTG 
               
               
                   
                 GTTGCTATTGCTAGTNNNNNNGGTGGAAAGCAGGCCCTCGAGACGGTGCAG 
               
               
                   
                 AGGTTACTTCCCGTCCTCTGTCAAGCGCACGGCCTC (SEQ ID NO: 34) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
               
                   
               
               
                 re-TALE blocks primer sequences 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                 block0-F 
                 CGCAATGCGCTCACGGGAGCACCCCTCAACctAACCCCTGAACAGGT* 
               
               
                   
                 A*G (SEQ ID NO: 35) 
               
               
                   
               
               
                 block0-R 
                 GAGACCATGCGCCTGACAAAGTACAGGCAGCAGTCTCTGAACAG*T* 
               
               
                   
                 T (SEQ ID NO: 36) 
               
               
                   
               
               
                 block1′-F 
                 TGGCGCAATGCGCTCACGGGAGCACCCCTCA*A*C (SEQ ID NO: 37) 
               
               
                   
               
               
                 block1-F 
                 AGACTGCTGCCTGTACTTTGTCAGGCGCATGGTCTCACCCCCGAACA* 
               
               
                   
                 G*G (SEQ ID NO: 38) 
               
               
                   
               
               
                 block1-R/block1′- 
                 TAACCCATGTGCTTGGCACAGAACGGGCAACAACCTTTGAACCG*T*T 
               
               
                 R 
                 (SEQ ID NO: 39) 
               
               
                   
               
               
                 block2-F 
                 AGGTTGTTGCCCGTTCTGTGCCAAGCACATGGGTTAACACCCgaac*a*a 
               
               
                   
                 (SEQ ID NO: 40) 
               
               
                   
               
               
                 blcok2-R 
                 AGGCGTCAAGCCGTGGGCTTGACACAAAACAGGAAGGAGTCTCTGCA 
               
               
                   
                 CAG*T*t (SEQ ID NO: 41) 
               
               
                   
               
               
                 block3-F 
                 AGACTCCTTCCTGTTTTGTGTCAAGCCCACGGCTTGACGCCTG*A*G 
               
               
                   
                 (SEQ ID NO: 42) 
               
               
                   
               
               
                 block3-R 
                 TAGACCGTGTGCCTGACAGAGTACCGGGAGCAAGCGCT*G*A (SEQ ID 
               
               
                   
                 NO: 43) 
               
               
                   
               
               
                 block4-F 
                 CGCTTGCTCCCGGTACTCTGTCAGGCACACGGTCTAA*C*T (SEQ ID 
               
               
                   
                 NO: 44) 
               
               
                   
               
               
                 block4-R 
                 CAGTCCATGAGCTTGACATAGGACTGGCAACAGCCGTT*G*T (SEQ ID 
               
               
                   
                 NO: 45) 
               
               
                   
               
               
                 block5-F 
                 CGGCTGTTGCCAGTCCTATGTCAAGCTCATGGACTGA*C*G (SEQ ID 
               
               
                   
                 NO: 46) 
               
               
                   
               
               
                 block5-R 
                 AAGGCCATGGGCCTGACATAAGACAGGCAGTAATCGTT*G*C (SEQ ID 
               
               
                   
                 NO: 47) 
               
               
                   
               
               
                 block6-F 
                 CGATTACTGCCTGTCTTATGTCAGGCCCATGGCCTTA*C*T (SEQ ID 
               
               
                   
                 NO: 48) 
               
               
                   
               
               
                 block6-R 
                 GAGCCCGTGCGCCTGGCAAAGCACCGGTAAAAGACGTTGGA*C*G 
               
               
                   
                 (SEQ ID NO: 49) 
               
               
                   
               
               
                 block6′-F 
                 CGATTACTGCCTGTCTTATGTCAGGCCCATGGCCTTACTCCTGAGCAA 
               
               
                   
                 *G*T (SEQ ID NO: 50) 
               
               
                   
               
               
                 block6′-R 
                 GAGCCCATGAGCCTGGCAAAGAACCGGAAGAAGCCGTT*G*G (SEQ 
               
               
                   
                 ID NO: 51) 
               
               
                   
               
               
                 block7-F 
                 CGGCTTCTTCCGGTTCTTTGCCAGGCTCATGGGCTCACGCCAGAGCAG 
               
               
                   
                 G*T*G (SEQ ID NO: 52) 
               
               
                   
               
               
                 blcok7-R 
                 GAGGCCGTGCGCTTGACAGAGGACGGGAAGTAACCTCT*G*C (SEQ ID 
               
               
                   
                 NO: 53) 
               
               
                   
               
            
           
         
       
     
     re-TALENs and re-TALE-TF destination vectors were constructed by modifying the TALE-TF and TALEN cloning backbones (see reference 24 hereby incorporated by reference in its entirety). The 0.5 RVD regions on the vectors were re-coded and SapI cutting site was incorporated at the designated re-TALE cloning site. The sequences of re-TALENs and re-TALE-TF backbones are provided in  FIG. 20 . Plasmids can be pre-treated with SapI (New England Biolabs) with manufacturer recommended conditions and purified with QIAquick PCR purification kit (QIAGEN). 
     A (10 ul) one-pot TASA assembly reaction was carried out with 200 ng of each block, 500 ng destination backbone, 1× TASA enzyme mixture (2 U SapI, 100 U Ampligase (Epicentre), 10 mU T5 exonuclease (Epicentre), 2.5 U Phusion DNA polymerase (New England Biolabs)) and 1× isothermal assembly reaction buffer as described before (see reference 25 hereby incorporated by reference in its entirety) (5% PEG-8000, 100 mM Tris-HCl pH 7.5, 10 mM MgCl2, 10 mM DTT, 0.2 mM each of the four dNTPs and 1 mM NAD). Incubations were performed at 37° C. for 5 min and 50° C. for 30 min. TASA assembly reaction can be processed directly for bacterial transformation to colonize individual assemblies. The efficiency of obtaining full length construct is ˜20% with this approach. Alternatively, &gt;90% efficiency can be achieved by a three-step assembly. First, 10 ul re-TALE assembly reactions are performed with 200 ng of each block, 1× re-TALE enzyme mixture (100 U Ampligase, 12.5 mU T5 exonuclease, 2.5 U Phusion DNA polymerase) and 1× isothermal assembly buffer at 50° C. for 30 min, followed by standardized Kapa HIFI PCR reaction, agarose gel electrophoresis, and QIAquick Gel extraction (Qiagen) to enrich the full length re-TALEs. 200 ng re-TALE amplicons can then be mixed with 500 ng Sap1-pre-treated destination backbone, 1× re-TALE assembly mixture and 1× isothermal assembly reaction buffer and incubated at 50° C. for 30 min. The re-TALE final assembly reaction can be processed directly for bacterial transformation to colonize individual assemblies. One of skill in the art will readily be able to select endonucleases, exonucleases, polymerases and ligases from among those known to practice the methods described herein. For example, type IIs endonucleases can be used, such as: Fok 1, Bts I, Ear I, Sap I. Exonucleases which are titralable can be used, such as lamda exonuclease, T5 exonuclease and Exonuclease III. Non-hotstart polymerases can be used, such as phusion DNA polymerase, Taq DNA polymerase and VentR DNA polymerase. Thermostable ligases can be used in this reaction, such as Ampligase, pfu DNA ligase, Taq DNA ligase. In addition, different reaction conditions can be used to activate such endonucleases, exonucleases, polymerases and ligases depending on the particular species used. 
     EXAMPLE III 
     Cell Line and Cell Culture 
     PGP1 iPS cells were maintained on Matrigel (BD Biosciences)-coated plates in mTeSR1 (Stemcell Technologies). Cultures were passaged every 5-7 days with TrypLE Express (Invitrogen). 293T and 293FT cells were grown and maintained in Dulbecco&#39;s modified Eagle&#39;s medium (DMEM, Invitrogen) high glucose supplemented with 10% fetal bovine serum (FBS, Invitrogen), penicillin/streptomycin (pen/strep, Invitrogen), and non-essential amino acids (NEAA, Invitrogen). K562 cells were grown and maintained in RPMI (Invitrogen) supplemented with 10% fetal bovine serum (FBS, Invitrogen 15%) and penicillin/streptomycin (pen/strep, Invitrogen). All cells were maintained at 37° C. and 5% CO2 in a humidified incubator. 
     A stable 293T cell line for detecting HDR efficiency was established as described in reference 26 hereby incorporated by reference in its entirety. Specifically, the reporter cell lines bear genomically integrated GFP coding sequences disrupted by the insertion of a stop codon and a 68 bp genomic fragment derived from the AAVS1 locus. 
     EXAMPLE IV 
     Test of re-TALENs Activity 
     293T reporter cells were seeded at densities of 2×105 cells per well in 24-well plate and transfected them with 1 μg of each re-TALENs plasmid and 2 μg DNA donor plasmid using Lipofectamine 2000 following the manufacturer&#39;s protocols. Cells were harvested using TrypLE Express (Invitrogen) ˜18 h after transfection and resuspended in 200 μl of media for flow cytometry analysis using an LSRFortessa cell analyzer (BD Biosciences). The flow cytometry data were analyzed using FlowJo (FlowJo). At least 25,000 events were analyzed for each transfection sample. For endogenous AAVS1 locus targeting experiment in 293T, the transfection procedures were identical as described above and puromycin selection was conducted with drug concentration at 3 μg/ml 1 week after transfection. 
     EXAMPLE V 
     Functional Lentivirus Generation Assessment 
     The lentiviral vectors were created by standard PCR and cloning techniques. The lentiviral plasmids were transfected by Lipofectamine 2000 with Lentiviral Packaging Mix (Invitrogen) into cultured 293FT cells (Invitrogen) to produce lentivirus. Supernatant was collected 48 and 72 h post-transfection, sterile filtered, and 100 ul filtered supernatant was added to 5×10 5  fresh 293T cells with polybrene. Lentivirus titration was calculated based on the following formula: virus titration=(percentage of GFP+293T cell*initial cell numbers under transduction)/(the volume of original virus collecting supernatant used in the transduction experiment). To test the functionality of lentivirus, 3 days after transduction, lentivirus transduced 293T cells were transfected with 30 ng plasmids carrying mCherry reporter and 500 ng pUC19 plasmids using Lipofectamine 2000 (Invitrogen). Cell images were analyzed using Axio Observer Z.1 (Zeiss) 18 hours after transfection and harvested using TrypLE Express (Invitrogen) and resuspended in 200 μl of media for flow cytometry analysis using a LSRFortessa cell analyzer (BD Biosciences). The flow cytometry data were analyzed using BD FACSDiva (BD Biosciences). 
     EXAMPLE VI 
     Test of re-TALENs and Cas9-gRNA Genome Editing Efficiency 
     PGP1 iPSCs were cultured in Rho kinase (ROCK) inhibitor Y-27632 (Calbiochem) 2 h before nucleofection. Transfections were done using P3 Primary Cell 4D-Nucleofector X Kit (Lonza). Specifically, cells were harvested using TrypLE Express (Invitrogen) and 2×10 6  cells were resuspended in 20 μl nucleofection mixture containing 16.4 μl P3 Nucleofector solution, 3.6 μl supplement, 1 μg of each re-TALENs plasmid or 1 ug Cas9 and 1 ug gRNA construct, 2 μl of 100 μM ssODN. Subsequently, the mixtures were transferred to 20 μl Nucleocuvette strips and nucleofection was conducted using CB150 program. Cells were plated on Matrigel-coated plates in mTeSR1 medium supplemented with ROCK inhibitor for the first 24 hrs. For endogenous AAVS1 locus targeting experiment with dsDNA donor, the same procedure was followed except 2 μg dsDNA donor was used and the mTeSR1 media was supplemented with puromycin at the concentration of 0.5 ug/mL 1 week after transfection. 
     The information of reTALENs, gRNA and ssODNs used in this example are listed in Table 3 and 
     Table 4 below. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Information of re-TALEN pairs/Cas9-gRNA targeting CCR5 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 re- 
                 re- 
                   
                   
                   
                   
               
               
                   
                 TALENs 
                 TALENs 
                   
                   
                   
                   
               
               
                   
                 pair 
                 pair 
                   
                   
                   
                 gRAN 
               
               
                 # 
                 targeting 
                 targeting 
                   
                   
                   
                 targeting 
               
               
                 target- 
                 site 
                 site 
                 re-TALEN-L 
                 re-TALEN-R 
                   
                 sequence 
               
               
                 ing 
                 (start) 
                 (end) 
                 targeting 
                 targeting 
                 gRNA targeting 
                 start 
               
               
                 site 
                 /chr3: 
                 /chr3: 
                 sequence 
                 sequence 
                 sequence 
                 position 
               
               
                   
               
               
                  1 
                 46409942 
                 46409993 
                 TCCCCACTTTCTT 
                 TAACCACTCAGG 
                 CACTTTCTTGTGA 
                 46409946 
               
               
                   
                   
                   
                 GTGAA (SEQ ID 
                 ACAGGG (SEQ ID 
                 ATCCTT (SEQ ID 
                   
               
               
                   
                   
                   
                 NO: 54) 
                 NO: 55) 
                 NO: 56) 
                   
               
               
                   
               
               
                  2 
                 46410227 
                 46410278 
                 TCACACAGCAAG 
                 TAGCGGAGCAG 
                 TGGGCTAGCGGA 
                 46410264 
               
               
                   
                   
                   
                 TCAGCA (SEQ ID 
                 GCTCGGA (SEQ 
                 GCAGGCT (SEQ ID 
                   
               
               
                   
                   
                   
                 NO: 57) 
                 ID NO: 58) 
                 NO: 59) 
                   
               
               
                   
               
               
                  3 
                 46411260 
                 46411311 
                 TACCCAGACGAG 
                 TCAGACTGCCAA 
                 ACCCAGACGAGA 
                 46411261 
               
               
                   
                   
                   
                 AAAGCT (SEQ ID 
                 GCTTGA (SEQ ID 
                 AAGCTGA (SEQ ID 
                   
               
               
                   
                   
                   
                 NO: 60) 
                 NO: 61) 
                 NO: 62) 
                   
               
               
                   
               
               
                  4 
                 46411464 
                 46411515 
                 TCTTGTGGCTCG 
                 TATTGTCAGCAG 
                 AGAGGGCATCTTG 
                 46411456 
               
               
                   
                   
                   
                 GGAGTA (SEQ ID 
                 AGCTGA (SEQ ID 
                 TGGCTC (SEQ ID 
                   
               
               
                   
                   
                   
                 NO: 63) 
                 NO: 64) 
                 NO: 65) 
                   
               
               
                   
               
               
                  5 
                 46411517 
                 46411568 
                 TTGAGATTTTCA 
                 TATACAGTCATA 
                 ATCAAGCTCTCTT 
                 46411538 
               
               
                   
                   
                   
                 GATGTC (SEQ ID 
                 TCAAGC (SEQ ID 
                 GGCGGT (SEQ ID 
                   
               
               
                   
                   
                   
                 NO: 66) 
                 NO: 67) 
                 NO: 68) 
                   
               
               
                   
               
               
                  6 
                 46411634 
                 46411685 
                 TTCAGATAGATT 
                 TGCCAGATACAT 
                 GCTTCAGATAGAT 
                 46411632 
               
               
                   
                   
                   
                 ATATCT (SEQ ID 
                 AGGTGG (SEQ ID 
                 TATATC (SEQ ID 
                   
               
               
                   
                   
                   
                 NO: 69) 
                 NO: 70) 
                 NO: 71) 
                   
               
               
                   
               
               
                  7 
                 46412396 
                 46412447 
                 TTATACTGTCTA 
                 TCAGCTCTTCTG 
                 ACGGATGTCTCAG 
                 46412437 
               
               
                   
                   
                   
                 TATGAT (SEQ ID 
                 GCCAGA (SEQ ID 
                 CTCTTC (SEQ ID 
                   
               
               
                   
                   
                   
                 NO: 72) 
                 NO: 73) 
                 NO: 74) 
                   
               
               
                   
               
               
                  8 
                 46412432 
                 46412483 
                 TGGCCAGAAGA 
                 TTACCGGGGAGA 
                 CCGGGGAGAGTTT 
                 46412461 
               
               
                   
                   
                   
                 GCTGAGA (SEQ 
                 GTTTCT (SEQ ID 
                 CTTGTA (SEQ ID 
                   
               
               
                   
                   
                   
                 ID NO: 75) 
                 NO: 76) 
                 NO: 77) 
                   
               
               
                   
               
               
                  9 
                 46412750 
                 46412801 
                 TTTGCAGAGAGA 
                 TTAGCAGAAGAT 
                 GAAATCTTATCTT 
                 46412782 
               
               
                   
                   
                   
                 TGAGTC (SEQ ID 
                 AAGATT (SEQ ID 
                 CTGCTA (SEQ ID 
                   
               
               
                   
                   
                   
                 NO: 78) 
                 NO: 79) 
                 NO: 80) 
                   
               
               
                   
               
               
                 10 
                 46413152 
                 46413203 
                 TATAAGACTAAA 
                 TCGTCTGCCACC 
                 AATGCATGACATT 
                 46413172 
               
               
                   
                   
                   
                 CTACCC (SEQ ID 
                 ACAGAT (SEQ ID 
                 CATCTG (SEQ ID 
                   
               
               
                   
                   
                   
                 NO: 81) 
                 NO: 82) 
                 NO: 83) 
                   
               
               
                   
               
               
                 11 
                 46414305 
                 46414356 
                 TAAAACAGTTTG 
                 TATAAAGTCCTA 
                 AACAGTTTGCATT 
                 46414308 
               
               
                   
                   
                   
                 CATTCA (SEQ ID 
                 GAATGT (SEQ ID 
                 CATGGA (SEQ ID 
                   
               
               
                   
                   
                   
                 NO: 84) 
                 NO: 85) 
                 NO: 86) 
                   
               
               
                   
               
               
                 12 
                 46414608 
                 46414659 
                 TGGCCATCTCTG 
                 TAGTGAGCCCAG 
                 CCAGAAGGGGAC 
                 46414632 
               
               
                   
                   
                   
                 ACCTGT (SEQ ID 
                 AAGGGG (SEQ ID 
                 AGTAAGA (SEQ ID 
                   
               
               
                   
                   
                   
                 NO: 87) 
                 NO: 88) 
                 NO: 89) 
                   
               
               
                   
               
               
                 13 
                 46414768 
                 46414820 
                 TAGGTACCTGGC 
                 TGACCGTCCTGG 
                 CTGACAATCGATA 
                 46414757 
               
               
                   
                   
                   
                 TGTCGT (SEQ ID 
                 CTTTTA (SEQ ID 
                 GGTACC (SEQ ID 
                   
               
               
                   
                   
                   
                 NO: 90) 
                 NO: 91) 
                 NO: 92) 
                   
               
               
                   
               
               
                 14 
                 46415017 
                 46415068 
                 TGTCATGGTCAT 
                 TCGACACCGAAG 
                 ACACCGAAGCAG 
                 46415046 
               
               
                   
                   
                   
                 CTGCTA (SEQ ID 
                 CAGAGT (SEQ ID 
                 AGTTTTT (SEQ ID 
                   
               
               
                   
                   
                   
                 NO: 93) 
                 NO: 94) 
                 NO: 95) 
                   
               
               
                   
               
               
                 15 
                 46420034 
                 46420084 
                 TGCCCCCGCGAG 
                 TCTGGAAGTTGA 
                 GGAAGTTGAACA 
                 46420062 
               
               
                   
                   
                   
                 GCCACA (SEQ ID 
                 ACACCC (SEQ ID 
                 CCCTTGC (SEQ ID 
                   
               
               
                   
                   
                   
                 NO: 96) 
                 NO: 97) 
                 NO: 98) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
               
                   
               
               
                 ssODN design for studying ssODN-mediated genome editing 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 FIG. 3b 
                 Distance 
                 90-*1 
                 CTACTGTCATTCAGGGCAATACCCAGACGAGAAAGCTGAGGGTATAACA 
               
               
                   
                 between 
                   
                 GGTTTCAAGCTTGGCAGTCTGACTACAGAGGCCACTGGCTT (SEQ ID 
               
               
                   
                 the 
                   
                 NO: 99) 
               
               
                   
                 secondary 
                 90-*2 
                 CTACTGTCATTCAGCCCAATACCCTAACGAGAAAGCTGAGGGTATAACA 
               
               
                   
                 mutation 
                   
                 GGTTTCAAGCTTGGCAGTCTGACTACAGAGGCCACTGGCTT (SEQ ID 
               
               
                   
                 and 
                   
                 NO: 100) 
               
               
                   
                 DSB 
                 90-*3 
                 CTACTGTCATTCAGCCCAATACCCAGACGAGAAAAGTGAGGGTATAACA 
               
               
                   
                   
                   
                 GGTTTCAAGCTTGGCAGTCTGACTACAGAGGCCACTGGCTT (SEQ ID 
               
               
                   
                   
                   
                 NO: 101) 
               
               
                   
                   
                 90M-0 
                 CTACTGTCATTCAGCCCAATACCCAGACGAGAAAGCTGAGGGTATAACA 
               
               
                   
                   
                   
                 GGTTTCAAGCTTGGCAGTCTGACTACAGAGGCCACTGGCTT (SEQ ID 
               
               
                   
                   
                   
                 NO: 102) 
               
               
                   
                   
                 90-*4 
                 CTACTGTCATTCAGCCCAATACCCAGACGAGAAAGCTGAGGGTATAACA 
               
               
                   
                   
                   
                 GGTTTGTAGCTTGGCAGTCTGACTACAGAGGCCACTGGCTT (SEQ ID 
               
               
                   
                   
                   
                 NO: 103) 
               
               
                   
                   
                 90-*5 
                 CTACTGTCATTCAGCCCAATACCCAGACGAGAAAGCTGAGGGTATAACA 
               
               
                   
                   
                   
                 GGTTTCAAGCTTGGCTCTCTGACTACAGAGGCCACTGGCTT (SEQ ID 
               
               
                   
                   
                   
                 NO: 104) 
               
               
                   
                   
                 90-*6 
                 CTACTGTCATTCAGCCCAATACCCAGACGAGAAAGCTGAGGGTATAACA 
               
               
                   
                   
                   
                 GGTTTCAAGCTTGGCAGTCTGACTAGTGAGGCCACTGGCTT (SEQ ID 
               
               
                   
                   
                   
                 NO: 105) 
               
               
                   
               
               
                 FIG. 3c 
                 distance 
                 L670bp_ 
                 CACTTTATATTTCCCTGCTTAAACAGTCCCCCGAGGGTGGGTGCGGAAAA 
               
               
                   
                 between 
                 90M 
                 GGCTCTACACTTGTTATCATTCCCTCTCCACCACAGGCAT (SEQ ID 
               
               
                   
                 ssODN 
                   
                 NO: 106) 
               
               
                   
                 and 
                 L570bp_ 
                 TTTGTATTTGGGTTTTTTTAAAACCTCCACTCTACAGTTAAGAATTCTAAG 
               
               
                   
                 the 
                 90M 
                 GCACAGAGCTTCAATAATTTGGTCAGAGCCAAGTAGCAG (SEQ ID 
               
               
                   
                 DSB 
                   
                 NO: 107) 
               
               
                   
                   
                 L480bp_ 
                 GGAGGTTAAACCCAGCAGCATGACTGCAGTTCTTAATCAATGCCCCTTG 
               
               
                   
                   
                 90M 
                 AATTGCACATATGGGATGAACTAGAACATTTTCTCGATGAT (SEQ ID 
               
               
                   
                   
                   
                 NO: 108) 
               
               
                   
                   
                 L394bp_ 
                 CTCGATGATTCGCTGTCCTTGTTATGATTATGTTACTGAGCTCTACTGTAG 
               
               
                   
                   
                 90M 
                 CACAGACATATGTCCCTATATGGGGCGGGGGTGGGGGTG (SEQ ID 
               
               
                   
                   
                   
                 NO: 109) 
               
               
                   
                   
                 L290bp_ 
                 GGTGTCTTGATCGCTGGGCTATTTCTATACTGTTCTGGCTTTTCGGAAGC 
               
               
                   
                   
                 90M 
                 AGTCATTTCTTTCTATTCTCCAAGCACCAGCAATTAGCTT (SEQ ID 
               
               
                   
                   
                   
                 NO: 110) 
               
               
                   
                   
                 L200bp_ 
                 GCTTCTAGTTTGCTGAAACTAATCTGCTATAGACAGAGACTCCGACGAA 
               
               
                   
                   
                 90M 
                 CCAATTTTATTAGGATTTGATCAAATAAACTCTCTCTGACA (SEQ ID 
               
               
                   
                   
                   
                 NO: 111) 
               
               
                   
                   
                 L114bp_ 
                 GAAAGAGTAACTAAGAGTTTGATGTTTACTGAGTGCATAGTATGCACTA 
               
               
                   
                   
                 90M 
                 GATGCTGGCCGTGGATGCCTCATAGAATCCTCCCAACAACT (SEQ ID 
               
               
                   
                   
                   
                 NO: 112) 
               
               
                   
                   
                 L45bp_ 
                 GCTAGATGCTGGCCGTGGATGCCTCATAGAATCCTCCCAACAACCGATG 
               
               
                   
                   
                 90M 
                 AAATGACTACTGTCATTCAGCCCAATACCCAGACGAGAAAG (SEQ ID 
               
               
                   
                   
                   
                 NO: 113) 
               
               
                   
                   
                 R40bp_ 
                 ACAGGTTTCAAGCTTGGCAGTCTGACTACAGAGGCCACTGGCTTTACCCC 
               
               
                   
                   
                 90M 
                 TGGGTTAGTCTGCCTCTGTAGGATTGGGGGCACGTAATTT (SEQ ID 
               
               
                   
                   
                   
                 NO: 114) 
               
               
                   
                   
                 R100bp_ 
                 TTAGTCTGCCTCTGTAGGATTGGGGGCACGTAATTTTGCTGTTTAAGGTC 
               
               
                   
                   
                 90M 
                 TCATTTGCCTTCTTAGAGATCACAAGCCAAAGCTTTTTAT (SEQ ID 
               
               
                   
                   
                   
                 NO: 115) 
               
               
                   
                   
                 R200bp_ 
                 GGAAGCCCAGAGGGCATCTTGTGGCTCGGGAGTAGCTCTCTGCTACCTT 
               
               
                   
                   
                 90M 
                 CTCAGCTCTGCTGACAATACTTGAGATTTTCAGATGTCACC (SEQ ID 
               
               
                   
                   
                   
                 NO: 116) 
               
               
                   
                   
                 R261bp_ 
                 TCAGCTCTGCTGACAATACTTGAGATTTTCAGATGTCACCAACCAGCAAG 
               
               
                   
                   
                 90M 
                 AGAGCTTGATATGACTGTATATAGTATAGTCATAAAGAAC (SEQ ID 
               
               
                   
                   
                   
                 NO: 117) 
               
               
                   
                   
                 R322bp_ 
                 CATAAAGAACCTGAACTTGACCATATACTTATGTCATGTGGAAATCTTCT 
               
               
                   
                   
                 90M 
                 CATAGCTTCAGATAGATTATATCTGGAGTGAAGAATCCTG (SEQ ID 
               
               
                   
                   
                   
                 NO: 118) 
               
               
                   
                   
                 R375M_ 
                 GTGGAAAATTTCTCATAGCTTCAGATAGATTATATCTGGAGTGAGCAATC 
               
               
                   
                   
                 90M 
                 CTGCCACCTATGTATCTGGCATAGTGTGAGTCCTCATAAA (SEQ ID 
               
               
                   
                   
                   
                 NO: 119) 
               
               
                   
                   
                 R448bp_ 
                 GGTTTGAAGGGCAACAAAATAGTGAACAGAGTGAAAATCCCCACCTAG 
               
               
                   
                   
                 90M 
                 ATCCTGGGTCCAGAAAAAGATGGGAAACCTGTTTAGCTCACC (SEQ ID 
               
               
                   
                   
                   
                 NO: 120) 
               
               
                   
                   
                 Complement- 
                 GGCCACTAGGGACAAAATTGGTGAcagaaa (SEQ ID NO: 121) 
               
               
                   
                   
                 30mer 
                   
               
               
                   
               
               
                 FIG. 3d 
                 ssODN 
                 Complement- 
                 CCCACAGTGGGGCCACTAGGGACAAAATTGGTGAcagaaaagccccatcc (SEQ 
               
               
                   
                 length 
                 50mer 
                 ID NO: 122) 
               
               
                   
                 and 
                 Complement- 
                 TCCCCTCCACCCCACAGTGGGGCCACTAGGGACAAAATTGGTGAcagaaaag 
               
               
                   
                 orientation  
                 70mer 
                 ccccatccttaggcctcc (SEQ ID NO: 123) 
               
               
                   
                 for 
                 Complement- 
                 cttTTATCTGTCCCCTCCACCCCACAGTGGGGCCACTAGGGACAAAATTGG 
               
               
                   
                 Cas9- 
                 90mer 
                 TGAcagaaaagccccatccttaggcctcctccttcctag (SEQ ID NO: 124) 
               
               
                   
                 gRNA 
                 Complement- 
                 gttctgggtacttTTATCTGTCCCCTCCACCCCACAGTGGGGCCACTAGGGACAA 
               
               
                   
                 targeting 
                 110mer  
                 AATTGGTGAcagaaaagccccatccttaggcctcctccttcctagtctcctgata 
               
               
                   
                   
                   
                 (SEQ ID NO: 125) 
               
               
                   
                   
                 Non- 
                 TTTCTGTCACCAATGGTGTCCCTAGTGGCC (SEQ ID NO: 126) 
               
               
                   
                   
                 complement- 
                   
               
               
                   
                   
                 30mer 
                   
               
               
                   
                   
                 Non- 
                 GGATGGGGCTTTTCTGTCACCAATGGTGTCCCTAGTGGCCCCACTGTGGG 
               
               
                   
                   
                 compement- 
                 (SEQ ID NO: 127) 
               
               
                   
                   
                 50mer 
                   
               
               
                   
                   
                 Non- 
                 GGAGGCCTAAGGATGGGGCTTTTCTGTCACCAATGGTGTCCCTAGTGGC 
               
               
                   
                   
                 complement- 
                 CCCACTGTGGGGTGGAGGGGA (SEQ ID NO: 128) 
               
               
                   
                   
                 70mer 
                   
               
               
                   
                   
                 Non- 
                 CTAGGAAGGAGGAGGCCTAAGGATGGGGCTTTTCTGTCACCAATGGTGT 
               
               
                   
                   
                 complement- 
                 CCCTAGTGGCCCCACTGTGGGGTGGAGGGGACAGATAAAAG (SEQ ID 
               
               
                   
                   
                 90mer 
                 NO: 129) 
               
               
                   
                   
                 Non- 
                 TATCAGGAGACTAGGAAGGAGGAGGCCTAAGGATGGGGCTTTTCTGTCA 
               
               
                   
                   
                 complement- 
                 CCAATGGTGTCCCTAGTGGCCCCACTGTGGGGTGGAGGGGACAGATAAA 
               
               
                   
                   
                 110mer 
                 AGTACCCAGAAC (SEQ ID NO: 130) 
               
               
                   
               
               
                 FIG. 2c 
                 ssODN 
                 Cas9- 
                 TTCTAGTAACCACTCAGGACAGGGGGGTTCAGCCCAAAAATTCACAAGA 
               
               
                   
                 donor 
                 gRNA- 
                 AAGTGGGGACCCATGGGAAAT (SEQ ID NO: 131) 
               
               
                   
                 for 
                 CCR5-1 
                   
               
               
                   
                 Cas9- 
                 Cas9- 
                 CAGCAAGTCAGCAGCACAGCGTGTGTGACTCCGAGGGTGCTCCGCTAGC 
               
               
                   
                 gRNA 
                 gRNA- 
                 CCACATTGCCCTCTGGGGGTG (SEQ ID NO: 132) 
               
               
                   
                 targeting 
                 CCR5-2 
                   
               
               
                   
                 CCR5 
                 Cas9- 
                 GTCAGACTGCCAAGCTTGAAACCTGTCTTACCCTCTACTTTCTCGTCTGG 
               
               
                   
                   
                 gRNA- 
                 GTATTGGGCTGAATGACAGT (SEQ ID NO: 133) 
               
               
                   
                   
                 CCR5-3 
                   
               
               
                   
                   
                 Cas9- 
                 CAGAGCTGAGAAGACAGCAGAGAGCTACTCCCGAAGCACAAGATGCCC 
               
               
                   
                   
                 gRNA- 
                 TCTGGGCTTCCGTGACCTTGGC (SEQ ID NO: 134) 
               
               
                   
                   
                 CCR5-4 
                   
               
               
                   
                   
                 Cas9- 
                 CTGACAATACTTGAGATTTTCAGATGTCACCAACGACCAAGAGAGCTTG 
               
               
                   
                   
                 gRNA- 
                 ATATGACTGTATATAGTATAG (SEQ ID NO: 135) 
               
               
                   
                   
                 CCR5-5 
                   
               
               
                   
                   
                 Cas9- 
                 CAGATACATAGGTGGCAGGATTCTTCACTCCAGACTTAATCTATCTGAAG 
               
               
                   
                   
                 gRNA- 
                 CTATGAGAAATTTTCCACAT (SEQ ID NO: 136) 
               
               
                   
                   
                 CCR5-6 
                   
               
               
                   
                   
                 Cas9- 
                 TATATGATTGATTTGCACAGCTCATCTGGCCAGATAAGCTGAGACATCCG 
               
               
                   
                   
                 gRNA- 
                 TTCCCCTACAAGAAACTCTC (SEQ ID NO: 137) 
               
               
                   
                   
                 CCR5-7 
                   
               
               
                   
                   
                 Cas9- 
                 ATCTGGCCAGAAGAGCTGAGACATCCGTTCCCCTTGAAGAAACTCTCCC 
               
               
                   
                   
                 gRNA- 
                 CGGTAAGTAACCTCTCAGCTG (SEQ ID NO: 138) 
               
               
                   
                   
                 CCR5-8 
                   
               
               
                   
                   
                 Cas9- 
                 AGGCATCTCACTGGAGAGGGTTTAGTTCTCCTTAAGAGAAGATAAGATT 
               
               
                   
                   
                 gRNA- 
                 TCAAGAGGGAAGCTAAGACTC (SEQ ID NO: 139) 
               
               
                   
                   
                 CCR5-9 
                   
               
               
                   
                   
                 Cas9- 
                 ATAATATAATAAAAAATGTTTCGTCTGCCACCACTAATGAATGTCATGCA 
               
               
                   
                   
                 gRNA- 
                 TTCTGGGTAGTTTAGTCTTA (SEQ ID NO: 140) 
               
               
                   
                   
                 CCR5-10 
                   
               
               
                   
                   
                 Cas9- 
                 TTTATAAAGTCCTAGAATGTATTTAGTTGCCCTCGTTGAATGCAAACTGT 
               
               
                   
                   
                 gRNA- 
                 TTTATACATCAATAGGTTTT (SEQ ID NO: 141) 
               
               
                   
                   
                 CCR5-11 
                   
               
               
                   
                   
                 Cas9- 
                 GCTCAACCTGGCCATCTCTGACCTGTTTTTCCTTCCCACTGTCCCCTTCTG 
               
               
                   
                   
                 gRNA- 
                 GGCTCACTATGCTGCCGCC (SEQ ID NO: 142) 
               
               
                   
                   
                 CCR5-12 
                   
               
               
                   
                   
                 Cas9- 
                 TTTTAAAGCAAACACAGCATGGACGACAGCCAGGCTCCTATCGATTGTC 
               
               
                   
                   
                 gRNA- 
                 AGGAGGATGATGAAGAAGATT (SEQ ID NO: 143) 
               
               
                   
                   
                 CCR5-13 
                   
               
               
                   
                   
                 Cas9- 
                 GCTTGTCATGGTCATCTGCTACTCGGGAATCCTAATTACTCTGCTTCGGT 
               
               
                   
                   
                 gRNA- 
                 GTCGAAATGAGAAGAAGAGG (SEQ ID NO: 144) 
               
               
                   
                   
                 CCR5-14  
                   
               
               
                   
                   
                 Cas9- 
                 ATACTGCCCCCGCGAGGCCACATTGGCAAACCAGCTTGGGTGTTCAACT 
               
               
                   
                   
                 gRNA- 
                 TCCAGACTTGGCCATGGAGAA (SEQ ID NO: 145) 
               
               
                   
                   
                 CCR5-15 
                   
               
               
                   
                 ssODN 
                 reTALEN- 
                 CTGAAGAATTTCCCATGGGTCCCCACTTTCTTGTGAATCCTTGGAGTGAA 
               
               
                   
                 donor 
                 CCR5-1 
                 CCCCCCTGTCCTGAGTGGTTACTAGAACACACCTCTGGAC (SEQ ID 
               
               
                   
                 for 
                   
                 NO: 146) 
               
               
                   
                 reTALENs 
                 reTALEN- 
                 TGGAAGTATCTTGCCGAGGTCACACAGCAAGTCAGCAGCACAGCCAGTG 
               
               
                   
                 targeting 
                 CCR5-2 
                 TGACTCCGAGCCTGCTCCGCTAGCCCACATTGCCCTCTGGG (SEQ ID 
               
               
                   
                 CCR5 
                   
                 NO: 147) 
               
               
                   
                   
                 reTALEN- 
                 CTACTGTCATTCAGCCCAATACCCAGACGAGAAAGCTGAGGGTATAACA 
               
               
                   
                   
                 CCR5-3 
                 GGTTTCAAGCTTGGCAGTCTGACTACAGAGGCCACTGGCTT (SEQ ID 
               
               
                   
                   
                   
                 NO: 148) 
               
               
                   
                   
                 reTALEN- 
                 GGAAGCCCAGAGGGCATCTTGTGGCTCGGGAGTAGCTCTCTGCTACCTT 
               
               
                   
                   
                 CCR5-4 
                 CTCAGCTCTGCTGACAATACTTGAGATTTTCAGATGTCACC (SEQ ID 
               
               
                   
                   
                   
                 NO: 149) 
               
               
                   
                   
                 reTALEN- 
                 TCAGCTCTGCTGACAATACTTGAGATTTTCAGATGTCACCAACGCCCAAG 
               
               
                   
                   
                 CCR5-5 
                 AGAGCTTGATATGACTGTATATAGTATAGTCATAAAGAAC (SEQ ID 
               
               
                   
                   
                   
                 NO: 150) 
               
               
                   
                   
                 reTALEN- 
                 GTGGAAAATTTCTCATAGCTTCAGATAGATTATATCTGGAGTGAGCAATC 
               
               
                   
                   
                 CCR5-6 
                 CTGCCACCTATGTATCTGGCATAGTGTGAGTCCTCATAAA (SEQ ID 
               
               
                   
                   
                   
                 NO: 151) 
               
               
                   
                   
                 reTALEN- 
                 GAAACAGCATTTCCTACTTTTATACTGTCTATATGATTGATTTGGTCAGC 
               
               
                   
                   
                 CCR5-7 
                 TCATCTGGCCAGAAGAGCTGAGACATCCGTTCCCCTACAA (SEQ ID 
               
               
                   
                   
                   
                 NO: 152) 
               
               
                   
                   
                 reTALEN- 
                 TTGATTTGCACAGCTCATCTGGCCAGAAGAGCTGAGACATCCGTATCCCT 
               
               
                   
                   
                 CCR5-8 
                 ACAAGAAACTCTCCCCGGTAAGTAACCTCTCAGCTGCTTG (SEQ ID 
               
               
                   
                   
                   
                 NO: 153) 
               
               
                   
                   
                 reTALEN- 
                 GGAGAGGGTTTAGTTCTCCTTAGCAGAAGATAAGATTTCAAGATGAGAG 
               
               
                   
                   
                 CCR5-9 
                 CTAAGACTCATCTCTCTGCAAATCTTTCTTTTGAGAGGTAA (SEQ ID 
               
               
                   
                   
                   
                 NO: 154) 
               
               
                   
                   
                 reTALEN- 
                 TAATATAATAAAAAATGTTTCGTCTGCCACCACAGATGAATGTCGAGCA 
               
               
                   
                   
                 CCR5-10 
                 TTCTGGGTAGTTTAGTCTTATAACCAGCTGTCTTGCCTAGT (SEQ ID 
               
               
                   
                   
                   
                 NO: 155) 
               
               
                   
                   
                 reTALEN- 
                 TTAAAAACCTATTGATGTATAAAACAGTTTGCATTCATGGAGGGTGACT 
               
               
                   
                   
                 CCR5-11 
                 AAATACATTCTAGGACTTTATAAAAGATCACTTTTTATTTA (SEQ ID 
               
               
                   
                   
                   
                 NO: 156) 
               
               
                   
                   
                 reTALEN- 
                 GACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTTCCTATTTACT 
               
               
                   
                   
                 CCR5-12 
                 GTCCCCTTCTGGGCTCACTATGCTGCCGCCCAGTGGGAC (SEQ ID NO: 157) 
               
               
                   
                   
                 reTALEN- 
                 TCATCCTCCTGACAATCGATAGGTACCTGGCTGTCGTCCATGCTACGTTT 
               
               
                   
                   
                 CCR5-13 
                 GCTTTAAAAGCCAGGACGGTCACCTTTGGGGTGGTGACAA (SEQ ID 
               
               
                   
                   
                   
                 NO: 158) 
               
               
                   
                   
                 reTALEN- 
                 GGCTGGTCCTGCCGCTGCTTGTCATGGTCATCTGCTACTCGGGAGACCTA 
               
               
                   
                   
                 CCR5-14 
                 AAAACTCTGCTTCGGTGTCGAAATGAGAAGAAGAGGCACA (SEQ ID 
               
               
                   
                   
                   
                 NO: 159) 
               
               
                   
                   
                 reTALEN- 
                 GGCAAGCCTTGGGTCATACTGCCCCCGCGAGGCCACATTGGCAAGTCAG 
               
               
                   
                   
                 CCR5-15 
                 CAAGGGTGTTCAACTTCCAGACTTGGCCATGGAGAAGACAT (SEQ ID 
               
               
                   
                   
                   
                 NO: 160) 
               
               
                   
               
            
           
         
       
     
     EXAMPLE VII 
     Amplicon Library Preparation of the Targeting Regions 
     Cells were harvested 6 days after nucleofection and 0.1 μl prepGEM tissue protease enzyme (ZyGEM) and 1 μl prepGEM gold buffer (ZyGEM) were added to 8.9 μl of the 2˜5×10 5  cells in the medium. 1 ul of the reactions were then added to 9 μl of PCR mix containing 5 ul 2× KAPA Hifi Hotstart Readymix (KAPA Biosystems) and 100 nM corresponding amplification primer pairs. Reactions were incubated at 95° C. for 5 min followed by 15 cycles of 98° C., 20 s; 65° C., 20 s and 72° C., 20 s. To add the Illumina sequence adaptor used, 5 μl reaction products were then added to 20 μl of PCR mix containing 12.5 ul 2× KAPA HIFI Hotstart Readymix (KAPA Biosystems) and 200 nM primers carrying Illumina sequence adaptors. Reactions were incubated at 95° C. for 5 min followed by 25 cycles of 98° C., 20 s; 65° C., 20 s and 72° C., 20 s. PCR products were purified by QIAquick PCR purification kit, mixed at roughly the same concentration, and sequenced with MiSeq Personal Sequencer. The PCR primers are listed in Table 5 below. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 CCR5 targeting site PCR primer sequences 
               
            
           
           
               
               
               
            
               
                 # 
                   
                   
               
               
                 targeting 
                   
                   
               
               
                 in CCR5 
                 name 
                 primer sequence 
               
               
                   
               
               
                  1 
                 site1-F1 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTGATTTTGCA 
               
               
                   
                   
                 GTGTGCGTTACTCC (SEQ ID NO: 161) 
               
               
                   
                 site1-F2 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTACATCGTTTGC 
               
               
                   
                   
                 AGTGTGCGTTACTCC (SEQ ID NO: 162) 
               
               
                   
                 site1-F3 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCCTAATTTGC 
               
               
                   
                   
                 AGTGTGCGTTACTCC (SEQ ID NO: 163) 
               
               
                   
                 site1-F4 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTTGGTCATTTGCA 
               
               
                   
                   
                 GTGTGCGTTACTCC (SEQ ID NO: 164) 
               
               
                   
                 site1-R 
                 CTCGGCATTCCTGCTGAACCGCTCTTCCGATCTCCAAGCAACTA 
               
               
                   
                   
                 AGTCACAGCA (SEQ ID NO: 165) 
               
               
                   
               
               
                  2 
                 Site2-F1 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTGATATGAG 
               
               
                   
                   
                 GAAATGGAAGCTTG (SEQ ID NO: 166) 
               
               
                   
                 Site2-F2 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTACATCGATGAG 
               
               
                   
                   
                 GAAATGGAAGCTTG (SEQ ID NO: 167) 
               
               
                   
                 Site2-F3 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCCTAAATGAG 
               
               
                   
                   
                 GAAATGGAAGCTTG (SEQ ID NO: 168) 
               
               
                   
                 Site2-F4 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTTGGTCAATGAG 
               
               
                   
                   
                 GAAATGGAAGCTTG (SEQ ID NO: 169) 
               
               
                   
                 Site2-R 
                 CTCGGCATTCCTGCTGAACCGCTCTTCCGATCTCATTAGGGTAT 
               
               
                   
                   
                 TGGAGGA (SEQ ID NO: 170) 
               
               
                   
               
               
                  3 
                 site3-F1 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTGATAATCC 
               
               
                   
                   
                 TCCCAACAACTCAT (SEQ ID NO: 171) 
               
               
                   
                 site3-F2 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTACATCGAATCC 
               
               
                   
                   
                 TCCCAACAACTCAT (SEQ ID NO: 172) 
               
               
                   
                 site3-F3 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCCTAAAATCC 
               
               
                   
                   
                 TCCCAACAACTCAT (SEQ ID NO: 173) 
               
               
                   
                 site3-F4 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTTGGTCAAATCC 
               
               
                   
                   
                 TCCCAACAACTCAT (SEQ ID NO: 174) 
               
               
                   
                 site3_R 
                 CTCGGCATTCCTGCTGAACCGCTCTTCCGATCTCCCAATCCTAC 
               
               
                   
                   
                 AGAGGCAG (SEQ ID NO: 175) 
               
               
                   
               
               
                  4 
                 site4-F1 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTGATAAGCC 
               
               
                   
                   
                 AAAGCTTTTTATTC (SEQ ID NO: 176) 
               
               
                   
                 site4-F2 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTACATCGAAGCC 
               
               
                   
                   
                 AAAGCTTTTTATTC (SEQ ID NO: 177) 
               
               
                   
                 site4-F3 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCCTAAAAGCC 
               
               
                   
                   
                 AAAGCTTTTTATTC (SEQ ID NO: 178) 
               
               
                   
                 site4-F4 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTTGGTCAAAGCC 
               
               
                   
                   
                 AAAGCTTTTTATTC (SEQ ID NO: 179) 
               
               
                   
                 site4_R 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTAAGCCAAAGCT 
               
               
                   
                   
                 TTTTATTCT (SEQ ID NO: 180) 
               
               
                   
               
               
                  5 
                 site5-F1 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTGATATCTTG 
               
               
                   
                   
                 TGGCTCGGGAGTAG (SEQ ID NO: 181) 
               
               
                   
                 site5-F2 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTACATCGATCTT 
               
               
                   
                   
                 GTGGCTCGGGAGTAG (SEQ ID NO: 182) 
               
               
                   
                 site5-R 
                 CTCGGCATTCCTGCTGAACCGCTCTTCCGATCTTGGCAGGATTC 
               
               
                   
                   
                 TTCACTCCA (SEQ ID NO: 183) 
               
               
                   
               
               
                  6 
                 site6-F1 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTGATCTATTT 
               
               
                   
                   
                 TGTTGCCCTTCAAA (SEQ ID NO: 184) 
               
               
                   
                 site6-F2 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTACATCGCTATTT 
               
               
                   
                   
                 TGTTGCCCTTCAAA (SEQ ID NO: 185) 
               
               
                   
                 site6-R 
                 CTCGGCATTCCTGCTGAACCGCTCTTCCGATCTAACCTGAACTT 
               
               
                   
                   
                 GACCATATACT (SEQ ID NO: 186) 
               
               
                   
               
               
                  7 
                 site7-F1 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTGATCAGCT 
               
               
                   
                   
                 GAGAGGTTACTTACC (SEQ ID NO: 187) 
               
               
                   
                 site7-F2 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTACATCGCAGCT 
               
               
                   
                   
                 GAGAGGTTACTTACC (SEQ ID NO: 188) 
               
               
                   
                 site7-R 
                 CTCGGCATTCCTGCTGAACCGCTCTTCCGATCTAATGATTTAAC 
               
               
                   
                   
                 TCCACCCTC (SEQ ID NO: 189) 
               
               
                   
               
               
                  8 
                 site8-F1 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTGATACTCC 
               
               
                   
                   
                 ACCCTCCTTCAAAAGA (SEQ ID NO: 190) 
               
               
                   
                 site8-F2 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTACATCGACTCC 
               
               
                   
                   
                 ACCCTCCTTCAAAAGA (SEQ ID NO: 191) 
               
               
                   
                 site8-R 
                 CTCGGCATTCCTGCTGAACCGCTCTTCCGATCTTGGTGTTTGCC 
               
               
                   
                   
                 AAATGTCT (SEQ ID NO: 192) 
               
               
                   
               
               
                  9 
                 site9_F1 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTGATGGGCA 
               
               
                   
                   
                 CATATTCAGAAGGCA (SEQ ID NO: 193) 
               
               
                   
                 site9_F2 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTACATCGGGGCA 
               
               
                   
                   
                 CATATTCAGAAGGCA (SEQ ID NO: 194) 
               
               
                   
                 site9_R 
                 CTCGGCATTCCTGCTGAACCGCTCTTCCGATCTAGTGAAAGACT 
               
               
                   
                   
                 TTAAAGGGAGCA (SEQ ID NO: 195) 
               
               
                   
               
               
                 10 
                 site10-F1 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTGATCACAA 
               
               
                   
                   
                 TTAAGAGTTGTCATA (SEQ ID NO: 196) 
               
               
                   
                 site10-F2 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTACATCGCACAA 
               
               
                   
                   
                 TTAAGAGTTGTCATA (SEQ ID NO: 197) 
               
               
                   
                 site10-R 
                 CTCGGCATTCCTGCTGAACCGCTCTTCCGATCTCTCAGCTAGAG 
               
               
                   
                   
                 CAGCTGAAC (SEQ ID NO: 198) 
               
               
                   
               
               
                 11 
                 site11-F1 
                 CTCGGCATTCCTGCTGAACCGCTCTTCCGATCTGACACTTGATA 
               
               
                   
                   
                 ATCCATC (SEQ ID NO: 199) 
               
               
                   
                 site11-F2 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTACATCGTCAAT 
               
               
                   
                   
                 GTAGACATCTATGTAG (SEQ ID NO: 200) 
               
               
                   
                 site11-R 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTGATTCAAT 
               
               
                   
                   
                 GTAGACATCTATGTAG (SEQ ID NO: 201) 
               
               
                   
               
               
                 12 
                 site12-F1 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTGATACTGC 
               
               
                   
                   
                 AAAAGGCTGAAGAGC (SEQ ID NO: 202) 
               
               
                   
                 site12-F2 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTACATCGACTGC 
               
               
                   
                   
                 AAAAGGCTGAAGAGC (SEQ ID NO: 203) 
               
               
                   
                 site12-F3 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCCTAAACTGC 
               
               
                   
                   
                 AAAAGGCTGAAGAGC (SEQ ID NO: 204) 
               
               
                   
                 site12-F4 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTTGGTCAACTGC 
               
               
                   
                   
                 AAAAGGCTGAAGAGC (SEQ ID NO: 205) 
               
               
                   
                 site12-R 
                 CTCGGCATTCCTGCTGAACCGCTCTTCCGATCTGCCTATAAAAT 
               
               
                   
                   
                 AGAGCCCTGTCAA (SEQ ID NO: 206) 
               
               
                   
               
               
                 13 
                 site13-F1 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTGATCTCTAT 
               
               
                   
                   
                 TTTATAGGCTTCTTC (SEQ ID NO: 207) 
               
               
                   
                 site13-F2 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTACATCGCTCTAT 
               
               
                   
                   
                 TTTATAGGCTTCTTC (SEQ ID NO: 208) 
               
               
                   
                 site13-R 
                 CTCGGCATTCCTGCTGAACCGCTCTTCCGATCTAGCCACCACCC 
               
               
                   
                   
                 AAGTGATC (SEQ ID NO: 209) 
               
               
                   
               
               
                 14 
                 site14-F1 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTACATCGTTCCA 
               
               
                   
                   
                 GACATTAAAGATAGTC (SEQ ID NO: 210) 
               
               
                   
                 site14-F2 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTGATTTCCA 
               
               
                   
                   
                 GACATTAAAGATAGTC (SEQ ID NO: 211) 
               
               
                   
                 site14-R 
                 CTCGGCATTCCTGCTGAACCGCTCTTCCGATCTAATCATGATGG 
               
               
                   
                   
                 TGAAGATAAG (SEQ ID NO: 212) 
               
               
                   
               
               
                 15 
                 site15-F1 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTGATCCGGC 
               
               
                   
                   
                 AGAGACAAACATTAAA (SEQ ID NO: 213) 
               
               
                   
                 site15-F2 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCCGGCAGAGAC 
               
               
                   
                   
                 AAACATTAAA (SEQ ID NO: 214) 
               
               
                   
                 site15-R 
                 CTCGGCATTCCTGCTGAACCGCTCTTCCGATCTAGCTAGGAAGC 
               
               
                   
                   
                 CATGGCAAG (SEQ ID NO: 215) 
               
               
                   
               
               
                 illumina 
                 PE-PCR-F 
                 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACAcgac 
               
               
                 adaptor 
                   
                 *g*c (SEQ ID NO: 216) 
               
               
                   
                 PE-PCR-R 
                 CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCT 
               
               
                   
                   
                 GAACc*g*c (SEQ ID NO: 217) 
               
               
                   
               
            
           
           
               
            
               
                 Multiplex sequencing PCR primer 
               
            
           
           
               
               
               
            
               
                  3 
                 site3-M-F 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTAGTGCATAGTA 
               
               
                   
                   
                 TGTGCTAGATGCTG (SEQ ID NO: 218) 
               
               
                   
                 site3-M-R 
                 GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGATCTCTAA 
               
               
                   
                   
                 GAAGGCAAATGAGAC (SEQ ID NO: 219) 
               
               
                   
               
               
                 illumina 
                 Index-PCR 
                 CAAGCAGAAGACGGCATACGAGATN 1 N 2 N 3 N 4 N 5 N 6 GTGACTGGA 
               
               
                 adaptor 
                   
                 GTTCAGACGTGTGCTCTTCCGATCT (SEQ ID NO: 220) 
               
               
                   
                 universal- 
                 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGA 
               
               
                   
                 PCR 
                 CGCTCTTCCGATCT (SEQ ID NO: 221) 
               
               
                   
               
               
                 *index-PCR primers are purchased from epicentre (ScriptSeq ™ Index PCR Primers) 
               
            
           
         
       
     
     EXAMPLE VIII 
     Genome Editing Assessment System (GEAS) 
     Next generation sequencing has been utilized to detect rare genomic alterations. See references 27-30 hereby incorporated by reference in their entireties. To enable wide use of this approach to quickly assess HDR and NHEJ efficiency in hiPSCS, software was created, referred to as a “pipeline”, to analyze the genome engineering data. This pipeline is integrated in one single Unix module, which uses different tools such as R, BLAT, and FASTX Toolkit. 
     Barcode splitting: Groups of samples were pooled together and sequenced using MiSeq 150 bp paired end (PE150) (Illumina Next Gen Sequencing), and later separated based on DNA barcodes using FASTX Toolkit. 
     Quality filtering: Nucleotides with lower sequence quality (phred score &lt;20) were trimmed. After trimming, reads shorter than 80 nucleotides were discarded. 
     Mapping: BLAT was used to map the paired reads independently to the reference genome and .psl files were generated as output. 
     Indel calling: Indels were defined as the full length reads containing 2 blocks of matches in the alignment. Only reads following this pattern in both paired end reads were considered. As a quality control, the indel reads were required to possess minimal 70 nt matching with the reference genome and both blocks to be at least 20 nt long. Size and position of indels were calculated by the positions of each block to the reference genome. Non-homologous end joining (NHEJ) has been estimated as the percentage of reads containing indels (see equation 1 below). The majority of NHEJ events have been detected at the targeting site vicinity. 
     Homology directed recombination (HDR) efficiency: Pattern matching (grep) within a 12 bp window centering over DSB was used to count specific signatures corresponding to reads containing the reference sequence, modifications of the reference sequence (2 bp intended mismatches), and reads containing only 1 bp mutation within the 2 bp intended mismatches (see equation 1 below). 
     
       
         
           
             
               
                 
                   
                     
                       Estimation 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       of 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       NHEJ 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       and 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       HDR 
                     
                     ⁢ 
                     
                       
 
                     
                     ⁢ 
                     
                       A 
                       = 
                       
                         reads 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         identical 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         to 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         the 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         reference 
                         ⁢ 
                         
                           : 
                         
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         XXXXXABXXXXX 
                       
                     
                     ⁢ 
                     
                       
 
                     
                     ⁢ 
                     
                       B 
                       = 
                       
                         reads 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         containing 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         2 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         bp 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         mismatch 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         programed 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         by 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         ssODN 
                         ⁢ 
                         
                           : 
                         
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         XXXXXabXXXXX 
                       
                     
                     ⁢ 
                     
                       
 
                     
                     ⁢ 
                     C 
                     = 
                     
                       reads 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       containing 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       only 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       1 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       bp 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       mutation 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       in 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       the 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       target 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       site 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       
                         : 
                       
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       such 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       as 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       XXXXXaBXXXXX 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       or 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       XXXXXAbXXXXX 
                     
                   
                   ⁢ 
                   
                     
 
                   
                   ⁢ 
                   
                     D 
                     = 
                     
                       
                         reads 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         containing 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         indels 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         as 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         described 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         above 
                         ⁢ 
                         
                           
 
                         
                         ⁢ 
                         
                           NHEJ 
                           ⁢ 
                           
                               
                           
                           ⁢ 
                           efficiency 
                         
                       
                       = 
                       
                         
                           
                             ( 
                             
                               100 
                               × 
                               
                                 D 
                                 
                                   A 
                                   + 
                                   B 
                                   + 
                                   C 
                                   + 
                                   D 
                                 
                               
                             
                             ) 
                           
                           ⁢ 
                           % 
                           ⁢ 
                           
                             
 
                           
                           ⁢ 
                           HDR 
                           ⁢ 
                           
                               
                           
                           ⁢ 
                           efficiency 
                         
                         = 
                         
                           
                             ( 
                             
                               100 
                               × 
                               
                                 B 
                                 
                                   A 
                                   + 
                                   B 
                                   + 
                                   C 
                                   + 
                                   D 
                                 
                               
                             
                             ) 
                           
                           ⁢ 
                           % 
                         
                       
                     
                   
                 
               
               
                 
                   Equation 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   1 
                 
               
             
           
         
       
     
     EXAMPLE IX 
     Genotype Screening of Colonized hiPSCs 
     Human iPS cells on feeder-free cultures were pre-treated with mTesr-1 media supplemented with SMC4 (5 uM thiazovivin, 1 uM CHIR99021, 0.4 uM PD0325901, 2 uM SB431542) (see reference 23 hereby incorporated by reference in its entirety for at least 2 hrs prior to FACS sorting. Cultures were dissociated using Accutase (Millipore) and resuspended in mTesr-1 media supplemented with SMC4 and the viability dye ToPro-3 (Invitrogen) at concentration of 1˜2×107/mL. Live hiPS cells were single-cell sorted using a BD FACSAria II SORP UV (BD Biosciences) with 100 um nozzle under sterile conditions into 96-well plates coated with irradiated CF-1 mouse embryonic fibroblasts (Global Stem). Each well contained hES cell medium (see reference 31 hereby incorporated by reference in its entirety) with 100 ng/ml recombinant human basic Fibroblast Growth Factor (bFGF) (Millipore) supplemented with SMC4 and 5 ug /ml fibronectin (Sigma). After sorting, plates were centrifuged at 70×g for 3 min. Colony formation was seen 4 days post sorting, and the culture media was replaced with hES cell medium with SMC4. SMC4 can be removed from hES cell medium 8 days after sorting. 
     A few thousand cells were harvested 8 days after Fluorescence-activated cell sorting (FACS) and 0.1 ul prepGEM tissue protease enzyme (ZyGEM) and 1 ul prepGEM gold buffer (ZyGEM) were added to 8.9 μl of cells in the medium. The reactions were then added to 40 μl of PCR mix containing 35.5 ml platinum 1.1× Supermix (Invitrogen), 250 nM of each dNTP and 400 nM primers. Reactions were incubated at 95° C. for 3 min followed by 30 cycles of 95° C., 20 s; 65° C., 30 s and 72° C., 20 s. Products were Sanger sequenced using either one of the PCR primers in Table 5 and sequences were analyzed using DNASTAR (DNASTAR). 
     EXAMPLE X 
     Immunostaining and Teratoma Assays of hiPSCs 
     Cells were incubated in the KnockOut DMEM/F-12 medium at 37° C. for 60 minutes using the following antibody: Anti-SSEA-4 PE (Millipore) (1:500 diluted); Tra-1-60 (BD Pharmingen) (1:100 diluted). After the incubation, cells were washed three times with KnockOut DMEM/F-12 and imaged on the Axio Observer Z.1 (ZIESS). 
     To conduct teratoma formation analysis, human iPSCs were harvested using collagenase type IV (Invitrogen) and the cells were resuspended into 200 μl of Matrigel and injected intramuscularly into the hind limbs of Rag2gamma knockout mice. Teratomas were isolated and fixed in formalin between 4-8 weeks after the injection. The teratomas were subsequently analyzed by hematoxylin and eosin staining. 
     EXAMPLE XI 
     Targeting Genomic Loci in Human Somatic Cells and Human Stem Cells Using reTALENS 
     According to certain aspects, TALEs known to those of skill in the art are modified or re-coded to eliminate repeat sequences. Such TALEs suitable for modification and use in the genome editing methods in viral delivery vehicles and in various cell lines and organisms described herein are disclosed in references 2, 7-12 hereby incorporated by reference herein in their entireties. Several strategies have been developed to assemble the repetitive TALE RVD array sequences (see references 14 and 32-34 hereby incorporated by reference herein in their entireties. However, once assembled, the TALE sequence repeats remain unstable, which limits the wide utility of this tool, especially for viral gene delivery vehicles (see references 13 and 35 hereby incorporated by reference herein in their entireties. Accordingly, one aspect of the present disclosure is directed to TALEs lacking repeats, such as completely lacking repeats. Such a re-coded TALE is advantageous because it enables faster and simpler synthesis of extended TALE RVD arrays. 
     To eliminate repeats, the nucleotide sequences of TALE RVD arrays were computationally evolved to minimize the number of sequence repeats while maintaining the amino acid composition. Re-coded TALE (Re-TALEs) encoding 16 tandem RVD DNA recognition monomers, plus the final half RVD repeat, are devoid of any 12 bp repeats (see  FIG. 5 a   ). Notably, this level of recoding is sufficient to allow PCR amplification of any specific monomer or sub-section from a full-length re-TALE construct (see  FIG. 5 b   ). The improved design of re-TALEs may be synthesized using standard DNA synthesis technology (see reference 36 hereby incorporated by reference in its entirety without incurring the additional costs or procedures associated with repeat-heavy sequences. Furthermore, the recoded sequence design allows efficient assembly of re-TALE constructs using a modified isothermal assembly reaction as described in the methods herein and with reference to  FIG. 6 . 
     Genome editing NGS data was statistically analyzed as follows. For HDR specificity analysis, an exact binomial test was used to compute the probabilities of observing various numbers of sequence reads containing the 2 bp mismatch. Based on the sequencing results of 10 bp windows before and after the targeting site, the maximum base change rates of the two windows (P1 and P2) were estimated. Using the null hypothesis that the changes of each of the two target bp were independent, the expected probability of observing 2 bp mismatch at the targeting site by chance as the product of these two probabilities (P1*P2) was computed. Given a dataset containing N numbers of total reads and n number of HDR reads, we calculated the p-value of the observed HDR efficiency was calculated. For HDR sensitivity analysis, the ssODN DNA donors contained a 2 bp mismatch against the targeting genome, which made likely the co-presence of the base changes in the two target bp if the ssODN was incorporated into the targeting genome. Other non-intended observed sequence changes would not likely change at the same time. Accordingly, non-intended changes were much less interdependent. Based on these assumptions, mutual information (MI) was used to measure the mutual dependence of simultaneous two base pair changes in all other pairs of positions, and the HDR detection limit was estimated as the smallest HDR where MI of the targeting 2 bp site is higher than MI of all the other position pairs. For a given experiment, HDR reads with intended 2 bp mismatch from the original fastq file were identified and a set of fastq files with diluted HDR efficiencies were simulated by systematically removing different numbers of HDR reads from the original data set. Mutual information (MI) was computed between all pairs of positions within a 20 bp window centered on the targeting site. In these calculations, the mutual information of the base composition between any two positions is computed. Unlike the HDR specificity measure described above, this measure does not assess the tendency of position pairs to change to any particular pairs of target bases, only their tendency to change at the same time. (see  FIG. 8A ). Table 6 shows HDR and NHEJ efficiency of re-TALEN/ssODN targeting CCR5 and NHEL efficiency of Cas9-gRNA. We coded our analysis in R and MI was computed using the package infotheo. 
     
       
         
           
               
               
               
               
               
               
               
             
               
                 TABLE 6 
               
               
                   
               
               
                   
                   
                   
                   
                 HDR detection 
                   
                   
               
               
                 # 
                   
                 HDR 
                 NHEJ 
                 limit based on 
                   
                   
               
               
                 targeting 
                   
                 (reTALEN) 
                 (reTALE) 
                 Information 
                 NHEJ 
                 HDR 
               
               
                 site 
                 cell type 
                 (%) 
                 (%) 
                 analysis 
                 (Cas9-gRNA) 
                 (Cas9-gRNA) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 1 
                 PGP1-iPS 
                 0.06% 
                 0.80% 
                 0.04% 
                 0.58% 
                 0.38% 
               
               
                 2 
                 PGP1-iPS 
                 0.48% 
                 0.26% 
                 0.01% 
                 16.02%  
                 3.71% 
               
               
                 3 
                 PGP1-iPS 
                 1.71% 
                 0.07% 
                 0.03% 
                 3.44% 
                 3.20% 
               
               
                 4 
                 PGP1-iPS 
                 0.02% 
                 1.20% 
                 0.02%* 
                 1.50% 
                 0.14% 
               
               
                 5 
                 PGP1-iPS 
                 0.80% 
                 0.04% 
                 0.00% 
                 3.70% 
                 0.39% 
               
               
                 6 
                 PGP1-iPS 
                 0.20% 
                 0.73% 
                 0.00% 
                 1.12% 
                 0.49% 
               
               
                 7 
                 PGP1-iPS 
                 0.01% 
                 0.15% 
                 0.01%* 
                 1.98% 
                 1.78% 
               
               
                 8 
                 PGP1-iPS 
                 0.03% 
                 0.00% 
                 0.00% 
                 1.85% 
                 0.03% 
               
               
                 9 
                 PGP1-iPS 
                 1.60% 
                 0.06% 
                 0.00% 
                 0.50% 
                 0.13% 
               
               
                 10 
                 PGP1-iPS 
                 0.68% 
                 1.25% 
                 0.01% 
                 8.77% 
                 1.32% 
               
               
                 11 
                 PGP1-iPS 
                 0.06% 
                 0.27% 
                 0.00% 
                 0.62% 
                 0.44% 
               
               
                 12 
                 PGP1-iPS 
                 1.60% 
                 0.03% 
                 0.04% 
                 0.18% 
                 0.99% 
               
               
                 13 
                 PGP1-iPS 
                 0.00% 
                 1.47% 
                 0.00% 
                 0.65% 
                 0.02% 
               
               
                 14 
                 PGP1-iPS 
                 0.47% 
                 0.13% 
                 0.02% 
                 2.50% 
                 0.31% 
               
               
                 15 
                 PGP1-iPS 
                  0.8% 
                 0.14% 
                 0.08% 
                 1.50% 
                 1.10% 
               
               
                   
               
               
                 *The group where HDR detection limit exceeds the real HDR detected 
               
            
           
         
       
     
     Correlation betwee genome editing efficiency and epigenetic state was addressed as follows. Person correlation coefficients were computed to study possible associations between epigenetic parameters (DNase I HS or necleosome occupancy) and genome engineering efficiencies (HDR, NHEF). Dataset of DNAaseI Hypersensitivity was downloaded from UCSC genome browser. hiPSCs DNase I HS:/gbdb/hg19/bbi/wgEncodeOpenChromDnaseIpsnihi7Sig.big.Wig 
     To compute P-values, the observed correlation was compared to a simulated distribution which was built by randomizing the position of the epigenetic parameter (N=100000). Observed correlations higher than the 95th percentile, or lower than the 5th percentile of the simulated distribution were considered as potential associations. 
     The function of reTALEN in comparison with the corresponding non-recoded TALEN in human cells was determined. A HEK 293 cell line containing a GFP reporter cassette carrying a frame-shifting insertion was used as described in reference 37 hereby incorporated by reference in its entirety. See also  FIG. 1 a   . Delivery of TALENs or reTALENs targeting the insertion sequence, together with a promoterless GFP donor construct, leads to DSB-induced HDR repair of the GFP cassette, so that GFP repair efficiency can be used to evaluate the nuclease cutting efficiency. See reference 38 hereby incorporated by reference in its entirety. reTALENs induced GFP repair in 1.4% of the transfected cells, similar to that achieved by TALENs (1.2%) (see  FIG. 1 b   ). The activity of reTALENs at the AAVS1 locus in PGP1 hiPSCs was tested (see  FIG. 1 c   ) and successfully recovered cell clones containing specific insertions (see  FIG. 1 d,e   ), confirming that reTALENs are active in both somatic and pluripotent human cells. 
     The elimination of repeats enabled generation of functional lentivirus with a re-TALE cargo. Specifically, lentiviral particles were packaged encoding re-TALE-2A-GFP and were tested for activity of the re-TALE-TF encoded by viral particles by transfecting a mCherry reporter into a pool of lenti-reTALE-2A-GFP infected 293T cells. 293T cells transduced by lenti-re-TALE-TF showed 36× reporter expression activation compared with the reporter only negative (see  FIG. 7   a,b,c ). The sequence integrity of the re-TALE-TF in the lentiviral infected cells was checked and full-length reTALEs in all 10 of the clones tested were detected. (see  FIG. 7 d   ). 
     EXAMPLE XII 
     Comparison of ReTALEs and Cas9-gRNA Efficiency in hiPSCs with Genome Editing Assessment System (GEAS) 
     To compare the editing efficiencies of re-TALENs versus Cas9-gRNA in hiPSCs, a next-generation sequencing platform (Genome Editing Assessment System) was developed to identify and quantify both NHEJ and HDR gene editing events. A re-TALEN pair and a Cas9-gRNA were designed and constructed, both targeting the upstream region of CCR5 (re-TALEN, Cas9-gRNA pair #3 in Table 3), along with a 90 nt ssODN donor identical to the target site except for a 2 bp mismatch (see  FIG. 2 a   ). The nuclease constructs and donor ssODN were transfected into hiPSCs. To quantitate the gene editing efficiency, paired-end deep sequencing on the target genomic region was conducted 3 days after transfection. HDR efficiency was measured by the percentage of reads containing the precise 2 bp mismatch. NHEJ efficiency was measured by the percentage of reads carrying indels. 
     Delivery of the ssODN alone into hiPSCs resulted in minimal HDR and NHEJ rates, while delivery of the re-TALENs and the ssODN led to efficiencies of 1.7% HDR and 1.2% NHEJ (see  FIG. 2 b   ). The introduction of the Cas9-gRNA with the ssODN led to 1.2% HDR and 3.4% NHEJ efficiencies. Notably, the rate of genomic deletions and insertions peaked in the middle of the spacer region between the two reTALENs binding site, but peaked 3-4 bp upstream of the Protospacer Associated Motif (PAM) sequence of Cas9-gRNA targeting site (see  FIG. 2 b   ), as would be expected since double stranded breaks take place in these regions. A median genomic deletion size of 6 bp and insertion size of 3 bp generated by the re-TALENs was observed and a median deletion size of 7 bp and insertion of 1 bp by the Cas9-gRNA was observed (see  FIG. 2 b   ), consistent with DNA lesion patterns usually generated by NHEJ (see reference 4 hereby incorporated by reference in its entirety.) Several analyses of the next-generation sequencing platform revealed that GEAS can detect HDR detection rates as low as 0.007%, which is both highly reproducible (coefficient of variation between replicates=±15%*measured efficiency) and 400× more sensitive than most commonly used mismatch sensitive endonuclease assays (see  FIG. 8 ). 
     re-TALEN pairs and Cas9-gRNAs targeted to fifteen sites at the CCRS genomic locus were built to determine editing efficiency (see  FIG. 2 c   , see Table 3). These sites were selected to represent a wide range of DNaseI sensitivities (see reference 39 hereby incorporated by reference in its entirety. The nuclease constructs were transfected with the corresponding ssODNs donors (see Table 3) into PGP1 hiPSCs. Six days after transfection, the genome editing efficiencies at these sites were profiled (Table 6). For 13 out of 15 re-TALEN pairs with ssODN donors, NHEJ and HDR was detected at levels above statistical detection thresholds, with an average NHEJ efficiency of 0.4% and an average HDR efficiency of 0.6% (see  FIG. 2 c   ). In addition, a statistically significant positive correlation (r 2 =0.81) was found between HR and NHEJ efficiency at the same targeting loci (P&lt;1×10 −4 ) (see  FIG. 9 a   ), suggesting that DSB generation, the common upstream step of both HDR and NHEJ, is a rate-limiting step for reTALEN-mediated genome editing. 
     In contrast, all 15 Cas9-gRNA pairs showed significant levels of NHEJ and HR, with an average NHEJ efficiency of 3% and an average HDR efficiency of 1.0% (see  FIG. 2 c   ). In addition, a positive correlation was also detected between the NHEJ and HDR efficiency introduced by Cas9-gRNA (see  FIG. 9 b   ) (r 2 =0.52, p=0.003), consistent with observations for reTALENs. The NHEJ efficiency achieved by Cas9-gRNA was significantly higher than that achieved by reTALENs (t-test, paired-end, P=0.02). A moderate but statistically significant correlation between NHEJ efficiency and the melting temperature of the gRNA targeting sequence was observed (see  FIG. 9 c   ) (r 2 =0.28, p=0.04), suggesting that the strength of base-pairing between the gRNA and its genomic target could explain as much as 28% of the variation in the efficiency of Cas9-gRNA-mediated DSB generation. Even though Cas9-gRNA produced NHEJ levels at an average of 7 times higher than the corresponding reTALEN, Cas9-gRNA only achieved HDR levels (average=1.0%) similar to that of the corresponding reTALENs (average=0.6%). Without wishing to be bound by scientific theory, these results may suggest either that the ssODN concentration at the DSB is the limiting factor for HDR or that the genomic break structure created by the Cas9-gRNA is not favorable for effective HDR. No correlation between DNaseI HS and the genome targeting efficiencies was observed for either method. (see  FIG. 10 ). 
     EXAMPLE XIII 
     Optimization of ssODN Donor Design for HDR 
     Highly-performing ssODNs in hiPSCs were designed as follows. A set of ssODNs donors of different lengths (50-170 nt), all carrying the same 2 bp mismatch in the middle of the spacer region of the CCR5 re-TALEN pair #3 target sites was designed. HDR efficiency was observed to vary with ssODN length, and an optimal HDR efficiency of ˜1.8% was observed with a 90 nt ssODN, whereas longer ssODNs decreased HDR efficiency (see  FIG. 3 a   ). Since longer homology regions improve HDR rates when dsDNA donors are used with nucleases (see reference 40 hereby incorporated by reference in its entirety), possible reasons for this result may be that ssODNs are used in an alternative genome repair process; longer ssODNs are less available to the genome repair apparatus; or that longer ssODNs incur negative effects that offset any improvements gained by longer homology, compared to dsDNA donors (see reference 41 hereby incorporated by reference in its entirety.) Yet, if either of the first two reasons were the case, then NHEJ rates should either be unaffected or would increase with longer ssODNs because NHEJ repair does not involve the ssODN donor. However, NHEJ rates were observed to decline along with HDR (see  FIG. 3 a   ), suggesting that the longer ssODNs present offsetting effects. Possible hypotheses would be that longer ssODNs are toxic to the cell (see reference 42 hereby incorporated by reference in its entirety), or that transfection of longer ssODNs saturates the DNA processing machinery, thereby causing decreased molar DNA uptake, and reducing the capacity of the cells to take up or express re-TALEN plasmids. 
     How rate of incorporation of a mismatch carried by the ssODN donor varies with its distance to the double stranded break (“DSB”) was examined. A series of 90 nt ssODNs all possessing the same 2 bp mismatch (A) in the center of the spacer region of re-TALEN pair #3 was designed. Each ssODN also contained a second 2 bp mismatch (B) at varying distances from the center (see  FIG. 3 b   ). A ssODN possessing only the center 2 bp mismatch was used as a control. Each of these ssODNs was introduced individually with re-TALEN pair #3 and the outcomes were analyzed with GEAS. We found that overall HDR—as measured by the rate at which the A mismatch was incorporated (A only or A+B)—decreased as the B mismatches became farther from the center (see  FIG. 3 b   , see  FIG. 11 a   ). The higher overall HDR rate observed when B is only 10 bp away from A may reflect a lesser need for annealing of the ssODN against genomic DNA immediately proximal to the dsDNA break. 
     For each distance of B from A, a fraction of HDR events only incorporated the A mismatch, while another fraction incorporated both A and B mismatches (see  FIG. 3 b    (A only and A+B)), These two outcomes may be due to gene conversion tracts (see reference 43 hereby incorporated by reference in its entirety) along the length of the ssDNA oligo, whereby incoporation of A+B mismatches resulted from long conversion tracts that extended beyond the B mismatch, and incorporation of the A-only mismatch resulted from shorter tracts that did not reach B. Under this interpretation, a distribution of gene conversion lengths in both directions along the ssODN were estimated (see  FIG. 11 b   ). The estimated distribution implies that gene conversion tracts progressively become less frequent as their lengths increase, a result very similar to gene conversion tract distributions seen with dsDNA donors, but on a highly compressed distance scale of tens of bases for the ssDNA donor vs. hundreds of bases for dsDNA donors. Consistent with this result, an experiment with a ssODN containing three pairs of 2 bp mismatches spaced at intervals of 10 nt on either side of the central 2 bp mismatch “A” gave rise to a pattern in which A alone was incorporated 86% of the time, with multiple B mismatches incorporated at other times (see  FIG. 11 c   ). Although the numbers of B only incorporation events were too low to estimate a distribution of tract lengths less than 10 bp, it is clear that the short tract region within 10 bp of the nuclease site predominates (see  FIG. 11 b   ). Finally, in all experiments with single B mismatches, a small fraction of B-only incorporation events is seen (0.04%˜1.12%) that is roughly constant across all B distances from A. 
     Furthermore, analysis was carried out of how far the ssODN donor can be placed from the re-TALEN-induced dsDNA break while still observing incorporation. A set of 90 nt ssODNs with central 2 bp mismatches targeting a range of larger distances −600 bp to +400 bp) away from the re-TALEN-induced dsDNA break site were tested. When the ssODNs matched ≥40 bp away, we observed &gt;30× lower HDR efficiencies compared to the control ssODN positioned centrally over the cut region (see  FIG. 3 c   ). The low level of incorporation that was observed may be due to processes unrelated to the dsDNA cut, as seen in experiments in which genomes are altered by a ssDNA donor alone see reference 42 hereby incorporated by reference in its entirety. Meanwhile, the low level of HDR present when the ssODN is ˜40 bp away may be due to a combination of weakened homology on the mismatch-containing side of the dsDNA cut along with insufficient ssODN oligo length on the other side of the dsDNA break. 
     The ssODNs DNA donor design for Cas9-gRNA mediated targeting was tested. Cas9-gRNA (C 2 ) targeting the AAVS1 locus was constructed and ssODN donors of variable orientations (O c : complementary to the gRNA and O n : non-complementary to the gRNA) and lengths (30, 50, 70, 90, 110 nt) were designed. O c  achieved better efficiency than O n , with a 70 mer O c  achieving an optimal HDR rate of 1.5%. (see  FIG. 3 d   ) The same ssODN strand bias was detected using a Cas9-derived nickase (C c : Cas9_D10A), despite the fact that the HDR efficiencies mediated by C c  with ssODN were significantly less than C 2  (t-test, paired-end, P=0.02). (see  FIG. 12 ). 
     EXAMPLE XIV 
     hiPSC Clonal Isolation of Corrected Cells 
     GEAS revealed that re-TALEN pair #3 achieved precise genome editing with an efficiency of ˜1% in hiPSCs, a level at which correctly edited cells can usually be isolated by screening clones. HiPSCs have poor viability as single cells. Optimized protocols described in reference 23 hereby incorporated by reference in its entirety along with a single-cell FACS sorting procedure was used to establish a robust platform for single hiPSCs sorting and maintenance, where hiPSC clones can be recovered with survival rates of &gt;25%. This method was combined with a rapid and efficient genotyping system to conduct chromosomal DNA extraction and targeted genome amplification in 1-hour single tube reactions, enabling large scale genotyping of edited hiPSCs. Together, these methods comprise a pipeline for robustly obtaining genome-edited hiPSCs without selection. 
     To demonstrate this system (see  FIG. 4 a   ), PGP1 hiPSCs were transfected with a pair of re-TALENs and an ssODN targeting CCR5 at site #3 (see Table 3). GEAS was performed with a portion of the transfected cells, finding an HDR frequency of 1.7% (see  FIG. 4 b   ). This information, along with the 25% recovery of sorted single-cell clones, allow estimation of obtaining at least one correctly-edited clone from five 96-well plates with Poisson probability 98% (assuming μ=0.017*0.25*96*5*2). Six days after transfection, hiPSCs were FACS-sorted and eight days after sorting, 100 hiPSC clones were screened. Sanger sequencing revealed that 2 out of 100 of these unselected hiPSC colonies contained a heterozygous genotype possessing the 2 bp mutation introduced by the ssODN donor see ( FIG. 4 c   ). The targeting efficiency of 1% (1%=2/2*100, 2 mono-allelic corrected clones out of 100 cell screened) was consistent with the next-generation sequencing analysis (1.7%) (see  FIG. 4 b   ). The pluripotency of the resulting hiPSCs was confirmed with immunostaining for SSEA4 and TRA-1-60 (see  FIG. 4 d   ). The successfully targeted hiPSCs clones were able to generate mature teratomas with features of all three germ layers (see  FIG. 4 e   ). 
     EXAMPLE XV 
     Method for Continuous Cell Genome Editing 
     According to certain aspects, a method is provided for genome editing in cells, including a human cell, for example a human stem cell, wherein the cell is genetically modified to include a nucleic acid encoding an enzyme that forms a co-localization complex with RNA complementary to the target DNA and that cleaves the target DNA in a site specific manner. Such an enzyme includes an RNA guided DNA binding protein, such as an RNA-guided DNA binding protein of a Type II CRISPR system. An exemplary enzyme is Cas9. According to this aspect, the cell expresses the enzyme and guide RNA is provided to the cell from the media surrounding the cell. The guide RNA and the enzyme form a co-localization complex at target DNA where the enzyme cuts the DNA. Optionally, a donor nucleic acid may be present for insertion into the DNA at the cut site, for example by nonhomologous end joining or homologous recombination. According to one aspect, the nucleic acid encoding an enzyme that forms a co-localization complex with RNA complementary to the target DNA and that cleaves the target DNA in a site specific manner, such as Cas9, is under the influence of a promoter, such as the nucleic acid can be activated and silenced. Such promoters are well known to those of skill in the art. One exemplary promoter is the dox inducible promoter. According to one aspect, the cell is genetically modified by having reversibly inserted into its genome the nucleic acid encoding an enzyme that forms a co-localization complex with RNA complementary to the target DNA and that cleaves the target DNA in a site specific manner. Once inserted, the nucleic acid can be removed by use of a reagent, such as a transposase. In this manner, the nucleic acid can be easily removed after use. 
     According to one aspect, a continuous genome editing system in human induced pluripotent stem cells (hiPSCs) using a CRISPR system is provided. According to an exemplary aspect, the method includes use of a hiPSC line with Cas9 reversibly inserted in the genome (Cas9-hiPSCs); and gRNAs which have been modified from their native form to allow their passage from media surrounding the cells into the cells for use with the Cas9. Such gRNA has been treated with a phosphatase in a manner to remove phosphate groups. Genome editing in the cell is carried out with Cas9 by supplementing phosphatase treated gRNA in the tissue culture media. This approach enables scarless genome editing in HiPSCs with up to 50% efficiencies with single days of treatment, 2-10× times more efficient than the best efficiencies reported so far. Further, the method is easy to use and with significantly lower cellular toxicity. Embodiments of the present disclosure include single editing of hiPSCs for biological research and therapeutic applications, multiplex editing of hiPSCs for biological research and therapeutic applications, directional hiPSCs evolution and phenotype screening of hiPSCs and its derivative cells. 
     According to certain aspects, other cell lines and organisms described herein can be used in addition to stem cells. For example, the method described herein can be used to animal cells such as mouse or rat cells so that stable Cas9 integrated mouse cells and rat cells can be generated and tissue specific genome editing can be conducted by locally introducing phosphatase treated gRNA from media surrounding the cells. Moreover, other Cas9 derivatives can be inserted into many cell lines and organisms, and targeted genomic manipulations, such as sequence specific nicking, gene activation, suppression and epigenetic modification can be conducted. 
     Aspects of the present disclosure are directed to making stable hiPSCs with Cas9 inserted into the genome. Aspects of the present disclosure are directed to modifying RNA to enable entry into a cell through the cell wall and co-localization with Cas9 while avoiding the immune response of the cell. Such modified guide RNA can achieve optimal transfection efficiencies with minimal toxicity. Aspects of the present disclosure are directed to optimzied genome editing in Cas9-hiPSCs using phosphatase treated gRNA. Aspects of the present disclosure include eliminating Cas9 from hiPSCs to achieve scarless genome editing, where the nucleic acid encoding Cas9 has been reversibly placed into the cell genome. Aspects of the present disclosure include biomedical engineering using hiPSCs with Cas9 inserted into the genome to create desired genetic mutations. Such engineered hiPSCs maintain pluripotency and can be successfully differentiated into various cell types, including cardiomyocyte, which fully recapitulate the phenotype of patient cell lines. 
     Aspects of the present disclosure include libraries of phosphatase treated gRNAs for multiplex genome editing. Aspects of the present disclosure include generating a library of PGP cell lines with each one carrying 1 to a few designated mutations in the genome, which can serve as resource for drug screening. Aspects of the present disclosure include generating PGP1 cell lines with all the retrotranselements barcoded with different sequences to track the location and activity of this element. 
     EXAMPLE XVI 
     Generating Stable hiPSCs with Cas9 Inserted Into the Genome 
     Cas9 was encoded under the dox inducible promoter and the construct was placed into a Piggybac vector which can be inserted into and removed out of the genome with the help of Piggybac transposase. PCR reaction validated the stable insertion of the vector (see  FIG. 14 ). The inducible Cas9 expression was determined via RT-QPCR. The mRNA level of Cas9 increased 1000× after 8 hours of 1 ug/mL DOX supplementation in the culture media and the level of Cas9 mRNA dropped to normal level ˜20 hours after withdrawal of the DOX. (See  FIG. 15 ). 
     According to one aspect, the Cas9-hiPSC system based genome editing bypasses the transfection procedure of Cas9 plasmid/RNA, a large construct usually with &lt;1% transfection efficiency in hiPSCs. The present Cas9-hiPSC system can serve as a platform to perform high efficient genomic engineering in human stem cells. In addition, the Cas9 cassette introduced into the hiPSCs using Piggybac system can be removed out from the genome easily upon introducing of transposases. 
     EXAMPLE XVII 
     Phosphatase Treated Guide RNA 
     To enable continuous genome editing on Cas9-hiPSCs, a series of modified RNA encoding gRNA were generated and supplemented into Cas9-iPS culture medium in complex with liposome. Phosphatase treated native RNA without any capping achieved the optimal HDR efficiency of 13%, 30× more than previously reported 5′Cap-Mod RNA (see  FIG. 16 ). 
     According to one aspect, guide RNA is physically attached to the donor DNA. In this manner, a method is provided of coupling Cas9 mediated genomic cutting and ssODN-mediated HDR, thus stimulating sequence specific genomic editing. gRNA linked with DNA ssODN donor with optimized concentration achieved 44% HDR and unspecific NHEJ 2% (see  FIG. 17 ). Of note, this procedure does not incurred visible toxicity as observed with nucleofection or electroporation. 
     According to one aspect, the present disclosure provides an in vitro engineered RNA structure encoding gRNA, which achieved high transfection efficiency, genome editing efficiency in collaboration with genomically inserted Cas9. In addition, the present disclosure provides a gRNA-DNA chimeric construct to couple a genomic cutting event with the homology directed recombination reaction. 
     EXAMPLE XVIII 
     Eliminating Reversibly Engineered Cas9 from hiPSCs to Achieve Scarless Genome Editing 
     According to certain aspects, a Cas9 cassette is inserted into the genome of hiPSC cells using a reversible vector. Accordingly, a Cas9 cassette was reversibly inserted into the genome of hiPSC cells using a PiggyBac vector. The Cas9 cassette was removed from the genome edited hiPSCs by transfecting the cell with transposase-encoding plasmid. Accordingly, aspects of the present disclosure include use of a reversible vector, which is known to those of skill in the art. A reversible vector is one which can be inserted into a genome, for example, and then removed with a corresponding vector removal enzyme. Such vectors and corresponding vector removal enzymes are known to those of skill in the art. A screen was performed on colonized iPS cells and colonies devoid of Cas9-cassette were recovered as confirmed by PCR reaction. Accordingly, the present disclosure provides method of genome editing without affecting the rest of the genome by having a permanent Cas9 cassette present in the cell. 
     EXAMPLE XIX 
     Genome Editing in iPGP1 Cells 
     Research into the pathogenesis of cardiomyopathy has historically been hindered by the lack of suitable model systems. Cardiomyocyte differentiation of patient-derived induced pluripotent stem cells (iPSCs) offers one promising avenue to surmount this barrier, and reports of iPSC modeling of cardiomyopathy have begun to emerge. However, realization of this promise will require approaches to overcome genetic heterogeneity of patient-derived iPSC lines. 
     Cas9-iPGP1 cell lines and phosphatase treated guide RNA bound to DNA were used to generated three iPSC lines that are isogenic except for the sequence at TAZ exon 6, which was identified to carry single nucleotide deletion in Barth syndrome patients. Single round of RNA transfection achieved ˜30% HDR efficiency. Modified Cas9-iPGP1 cells with desired mutations were colonized (see  FIG. 18 ) and the cell lines were differentiated into cardiomyocyte. Cardiomyocyte derived from the engineered Cas9-iPGP1 fully recapitulated the cardiolipin, mitochondrial, and ATP deficits observed in patient-derived iPSCs and in the neonatal rat TAZ knockdown model (see  FIG. 19 ). Accordingly, methods are provided for correcting mutations causing diseases in pluripotent cells followed by differentiation of the cells into desired cell types. 
     EXAMPLE XX 
     Materials and Methods 
     
         
         1. Establishment of PiggyBac Cas9 dox Inducible Stable Human iPS/ES lines
       1. After cells reached 70% confluence pretreat the culture with ROCK inhibitor Y27632 at final concentration of 10 uM for overnight.   2. The next day prepare the nucleofection solution by combine the 82 μl of human stem cell nucleofector solution and 18 ul supplement 1 in a sterile 1.5 ml eppendorf tube. Mix well. Incubate solution at 37° C. for 5 mins.   3. Aspirate mTeSR1; gently rinse the cells with DPBS at 2 mL/well of a six-well plate.   4. Aspirate the DPBS, add 2 mL/well of Versene, and put the culture back to incubator at 37° C. until they become rounded up and loosely adherent, but not detached. This requires 3-7 min.   5. Gently aspirate the Versene and add mTeSR1. Add 1 ml mTeSR1 and dislodge the cells by gently flowing mTeSR1 over them with a 1,000 uL micropipette.   6. Collect the dislodged cells, gently triturate them into a single-cell suspension, and quantitate by hemacytometer and adjust cell density to 1 million cells per ml.   7. Add 1 ml cell suspension to 1.5 ml eppendorf tube and centrifuge at 1100 RPM for 5 min in a bench top centrifuge.   8. Resuspend cells in 100 μl of human stem cell nucleofector solution from Step 2.   9. Transfer cells to a nucleofector cuvette using a 1 ml pipette tip. Add 1 μg of plasmid Transposonase and 5 ug PB Cas9 plasmids into the cell suspension in the cuvette. Mix cells and DNA by gentle swirling.   10. Put the cuvette into the nucleofector. Programs B-016 was selected and nucleofect cells by pressing button X.   11. Add 500 ul mTeSR1 medium with ROCK inhibitor in the cuvette after nucleofection.   12. Asperate the nucleofected cells from the cuvette using the provided Pasteur plastic pipette. And transfer cells drop-wise into matrigel coated well of 6 well plate mTeSR1 mediun with ROCK inhibitor. Incubate the cells at 37° C. overnight.   13. Change the medium to mTesr1 the next day and after 72 hours of transfection; add puromycin at final concentration at 1 ug/ml.And the line will be set up within 7 days.   
     
         2. RNA Preparation
       1. Prepare DNA template with T7 promoter upstream of gRNA coding sequence.   2. Purify the DNA using Mega Clear Purification and normalize the concentration.   3. Prepare Custom NTPS mixtures for different gRNA production.   
     
       
    
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                 #1 Native RNA Mix 
                 [Final] (mM) 
               
               
                   
                   
               
             
            
               
                   
                 GTP 
                 7.5 
               
               
                   
                 ATP 
                 7.5 
               
               
                   
                 CTP 
                 7.5 
               
               
                   
                 UTP 
                 7.5 
               
               
                   
                 Total volume 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                 #2 Capped Native  
                   
               
               
                   
                 RNA Mix 
                 [Final] (mM) 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 3′-O—Me-m7G Cap 
                 6 
               
               
                   
                 structure analog (NEB) 
                   
               
               
                   
                 GTP 
                 1.5 
               
               
                   
                 ATP 
                 7.5 
               
               
                   
                 CTP 
                 7.5 
               
               
                   
                 UTP 
                 7.5 
               
               
                   
                 Total volume 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                 #3 Modified RNA Mix 
                 [Final] (mM) 
               
               
                   
                   
               
             
            
               
                   
                 GTP 
                 7.5 
               
               
                   
                 ATP 
                 7.5 
               
               
                   
                 5-Me-CTP (Tri-Link) 
                 7.5 
               
               
                   
                 Pseudo-UTP (Tri-Link) 
                 7.5 
               
               
                   
                 Total volume 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                 #4 Capped/Modified  
                   
               
               
                   
                 RNA Mix 
                 [Final] (mM) 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 3′-O—Me-m7G Cap 
                 6 
               
               
                   
                 structure analog (NEB) 
                   
               
               
                   
                 GTP 
                 1.5 
               
               
                   
                 ATP 
                 7.5 
               
               
                   
                 5-Me-CTP (Tri-Link) 
                 7.5 
               
               
                   
                 Pseudo-UTP (Tri-Link) 
                 7.5 
               
               
                   
                 Total volume 
               
               
                   
                   
               
            
           
         
       
         
         
           
             4. Prepare the in vitro transcription mix at room temperature. 
           
         
       
    
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                   
                 Amt (ul) 
               
               
                   
                   
               
             
            
               
                   
                 Custom NTPS (*Add 
                 NA 
               
               
                   
                 vol/IVT rxn as indicated 
                   
               
               
                   
                 above) on ice 
                   
               
               
                   
                 PCR product (100 ng/ul) = 
                 16 
               
               
                   
                 1600 ng total 
                   
               
               
                   
                 ([final] = 40 ng/ul) 
                   
               
               
                   
                 Buffer X10 (MEGAscript 
                 4 
               
               
                   
                 kit from Ambion) @ RT 
                   
               
               
                   
                 T7 Enzyme (MEGAscript 
                 4 
               
               
                   
                 kit from Ambion) 
               
               
                   
                   
               
            
           
         
       
         
         
           
             5. Incubate for 4 hours (3-6 hrs ok) at 37° C. (thermocycler). 
             6. Add 2 μl Turbo DNAse (MEGAscript kit from Ambion) to each sample. Mix gently and incubate at 37° C. for 15′. 
             7. Purify DNAse treated reaction using MegaClear from Ambion according to the manufacturer&#39;s instructions. 
             8. Purify RNA using MEGAclear. (Purified RNA can be stored at −80 for several months). 
             9. To remove phosphate groups to avoid Toll 2 immune reaction from the host cell. 
           
         
       
    
     
       
         
           
               
               
               
               
             
               
                   
                   
               
               
                   
                 RNA Phosphatase treatment 
                 1× 
                 12 
               
               
                   
                   
               
             
            
               
                   
                 For each RNA sample 
                 ~100 ul  
                 NA 
               
               
                   
                 10× Antarctic Phosphatase buffer 
                 11 ul 
                 132 
               
               
                   
                 Antarctic Phosphatase 
                  2 ul 
                 24 
               
               
                   
                   
               
               
                   
                 Gently mix sample and incubate at 37° C. for 30′ (30′-1 hr ok) 
               
            
           
         
       
         
         3. RNA Transfection
       1. Plate 10K-20K cells per 48 well without antibiotics. Cells should be 30-50% confluent for transfection.   2. Change the cell media to with B18R (200 ng/ml), DOX (1 ug/ml), Puromycin (2 ug/ml) at least two hours before the transfection.   3. Prepare the transfection reagent containing gRNA (0.5 ug˜2 ug), donor DNA (0.5 ug˜2 ug)and RNAiMax, incubate the mixture in rm temperature for 15 minutes and transfer to the cell.   
     
         4. Single Human iPS Cells Seed and Single Clone Pickup
       1. After 4 days of dox induction and 1 day dox withdraw, asperate the medium, rinse gently with the DPBS. add 2 mL/well of Versene, and put the culture back to incubator at 37° C. until they become rounded up and loosely adherent, but not detached. This requires 3-7 min.   2. Gently aspirate the Versene and add mTeSR1. Add 1 ml mTeSR1 and dislodge the cells by gently flowing mTeSR1 over them with a 1,000 uL micropipette.   3. Collect the dislodged cells, gently triturate them into a single-cell suspension, and quantitate by hemacytometer and adjust cell density to 100K cells per ml.   4. Seeding the cells into matrigel coated 10 cm dishes with mTeSR1 plus ROCK inhibitor at cell density of 50K, 100K and 400K per 10 cm dish.   
     
         5. Single Cell Formed Clones Screening
       1. After 12 days culture in 10 cm dish and clones are big enough to be identified by naked eyes and labeled by colon marker. Do not allow clones become too big and adhere to each other.   2. Put the 10 cm dish to the culture hood and using a P20 pipette (set at 10 ul) with filter tips. Aspirate 10 ul medium for one well of 24 well plates. Pick up clone by scratching the clone into small pieces and transfer to one well of 24 well plate. Each filter tip for each clone.   3. After 4-5 days the clones inside one well of 24 well plate become big enough to split.   4. Aspirate the medium and rinse with 2 mL/well DPBS.   5. Aspirate DPBS, replace with 250 ul/well dispase (0.1 U/mL). and incubate the cells in dispase at 37° C. for 7 min.   6. Replace the dispase with 2 ml DPBS.   7. Add 250 ul mTeSR1. Using a cell scraper to lodge off the cells and collect the cells.   8. Tranfer 125 ul cell suspension into a well of matrigel coated 24 wells plates.   9. Transfer 125 ul cell suspension into 1.5 ml eppendorf tube for genomic DNA extraction.   
     
         6. Clone Screening
       1. Centrifuge the tube from step 7.7   2. Aspirate the medium and add 250 ul lysis buffer per well (10 mM+Tris pH7.5+(or +8.0), 10 mMEDTA, 10 mM.   3. NaCl, +10%SDS, 40 ug/mL+proteinase K (added fresh before using the buffer).   4. Incubate at 55 overnight.   5. Precipitate DNA by adding 250 ul Isopropanol.   6. Spin for 30 minutes at highest speed. Wash with 70% ethanol.   7. Gently remove ethanol. Air dry for 5 min.   8. Resuspend gDNA with100-200 ul dH2O.   9. PCR amplification of the targeted genomic region with specific primers.   10. Sanger sequencing the PCR product with respective primer.   11. Analysis of Sanger sequence data and expansion of targeted clones.   
     
         7. Piggybac Vector Remove
       1. Repeat the step 2.1-2.9   2. Transfer cells to a nucleofector cuvette using a 1 ml pipette tip. Add 2 μg plasmid of Transposonase into the cell suspension in the cuvette. Mix cells and DNA by gentle swirling.   3. Repeat the step 2.10-2.11   4. Asperate the nucleofected cells from the cuvette using the provided Pasteur plastic pipette. And transfer cells drop-wise into matrigel coated well of 10 cm dish with mTeSR1 medium plus ROCK inhibitor. Incubate the cells at 37° C. overnight.   5. The next day change the medium to mTesr 1 and change the medium every day for 4 following days.   6. After the clones became big enough pick up 20-50 clones and seeding into 24 well.   7. Genotype the clones with PB Cas9 PiggyBac vector primers and expansion negative clones.   
     
       
    
     REFERENCES 
     References are designated throughout the specification by their number below and are incorporated into the specification as if fully set forth therein. Each of the following references is hereby incorporated by reference in its entirety.
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