RU2812848C2 - Genome engineering - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a method of changing target DNA in a cell in vitro. This method involves providing the cell with the Cas enzyme of the CRISPR type II system, which forms a colocalization complex with a guide RNA complementary to the target DNA, and which cleaves the target DNA in a site-specific manner, introducing into the cell a guide RNA that is complementary to the target DNA and which directs a CRISPR type II Cas enzyme to a target DNA, where the guide RNA and a CRISPR type II Cas enzyme are members of a colocalization complex for the target DNA, and introducing a donor nucleic acid sequence into the cell, where the guide RNA and the donor nucleic acid sequence are linked.

EFFECT: invention is effective for changing target DNA in a cell in vitro.

18 cl, 20 dwg, 6 tbl, 20 ex

Description

Related ticket data

This application claims priority to U.S. Provisional Patent Application No. 61/858866, filed July 26, 2013 and incorporated herein by reference in its entirety and for all purposes.

Statement of State Interests

This invention was supported by public support under P50 HG003170 from the National Human Genome Research Center for Excellence in Genomics. The government has certain rights to this invention.

Prior Art

Genome editing using sequence-specific nucleases is known. See references 1, 2 and 3, which are incorporated herein by reference in their entirety. Nuclease-mediated double-stranded DNA (dsDNA) breaks in the genome can be repaired by two main mechanisms: non-homologous end joining (NHEJ), which often introduces non-specific insertions and deletions (indels), or homology-directed repair (HDR), which uses homologous chain as a template for repair. See source 4 incorporated by reference in full. When a sequence-specific nuclease is delivered together with a homologous donor DNA construct containing the desired mutations, gene targeting efficiencies are increased 1000-fold compared to delivery of the donor construct alone. See reference 5, which is incorporated herein by reference in its entirety. The use of single-stranded oligodeoxyribonucleotides (“ssODNs”) as DNA donors has been reported. See references 21 and 22, which are incorporated herein by reference in their entirety.

Despite great advances in gene editing technologies, many problems and many questions remain regarding the use of specially designed nucleases in the construction of human induced pluripotent stem cells (“hiPSC”). First, despite the simplicity of their design, TALENs (transcription activator-like effector nucleases) target specific DNA sequences with tandem copies of RVD domains (residue variable domains). See reference 6, which is incorporated herein by reference in its entirety. While the modular nature of RVDs simplifies the design of TALENs, their repetitive sequences complicate methods for synthesizing DNA constructs based on them (see references 2, 9 and 15-19, incorporated herein by reference in their entirety), and also make it virtually impossible their combined use with lentiviral-based gene delivery vehicles. See reference 13, which is incorporated herein by reference in its entirety.

In current practice, NHEJ and HDR are often assessed using separate assays. Non-complementarity-sensitive endonuclease assays (see reference 14, incorporated herein by reference in its entirety) are often used to assess NHEJ, but the quantitative accuracy of this method is variable and sensitivity is limited to NHEJ frequencies above ~3%. See reference 15, which is incorporated herein by reference in its entirety. HDR is often assessed using cloning and sequencing, a completely different and often cumbersome procedure. Sensitivity remains an issue because, despite high editing rates of about 50% often reported for some cell types such as U20S and K562 (see references 12 and 14, incorporated by reference herein in their entirety), in the case of hiPSC the frequencies are generally lower. See Source 10, which is incorporated herein by reference in its entirety. Recently, high editing frequencies have been reported in hiPSCs and hESCs achieved using TALENs (see reference 9, incorporated herein by reference in its entirety), and even higher frequencies have been achieved using the CRISPR Cas9-hydrRNA system (see Sources 16-19, which are incorporated herein by reference in their entirety.However, editing rates appear to vary widely across sites (see Source 17, incorporated herein by reference in their entirety), and in some sites, editing sometimes not detected at all (see reference 20, incorporated herein by reference in its entirety).

Bacterial and archaeal CRISPR-Cas systems rely on short guide RNAs (hydrRNAs) complexed with Cas proteins that direct the degradation of complementary sequences present in invading foreign nucleic acids. 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. & 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. & 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). Recent in vitro reconstitution of the type II CRISPR system from S. pyogenes has shown that crRNA (“CRISPR RNA”) fused typically to trans-encoded tracrRNA (“trans-CRISPR RNA”) is sufficient to target the Cas9 protein to specifically cleave the target DNA sequence corresponding to the crRNA. Expression of guideRNAs homologous to the target site leads to the recruitment of Cas9 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 (Feb, 2008).

The essence of the invention

Aspects of the present disclosure relate to the use of modified TALEN nucleases (transcription activator-like effector nucleases) to genetically modify a cell, such as a somatic cell or stem cell. TALENs are known to include repetitive sequences. Aspects of the present disclosure relate to a method of altering target DNA in a cell, including introducing into the cell TALENs that have lost 100 bp repeat sequences. or longer, where TALEN cleaves target DNA and the cell undergoes non-homologous end joining to produce altered DNA in the cell. In accordance with some aspects, repeated sequences of the desired length were removed from TALEN. In some aspects, TALEN was devoid of repeat sequences of a certain desired length. In accordance with some aspects, the TALEN is provided with repeated sequences of the desired length removed. In accordance with some aspects, TALEN is modified to remove repeated sequences of the desired length. In accordance with some aspects, TALEN is designed to remove repetitive sequences of a desired length.

Aspects of the present invention include methods for altering target DNA in a cell, including incorporating TALENs that have lost 100 bp of repeat sequence into the cell. or longer, and donor nucleic acid sequences, where TALEN cleaves the target DNA and the donor nucleic acid sequence is incorporated into the cell's DNA. Aspects of the present disclosure are directed to a virus comprising a nucleic acid sequence encoding TALEN that has lost 100 bp of repetitive sequences. or longer. Aspects of the present disclosure relate to a cell including a nucleic acid sequence encoding TALEN that has lost 100 bp of repetitive sequences. or longer. In accordance with certain aspects described herein, TALEN has lost 100 bp of repetitive sequences. 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 pairs or longer.

Aspects of the present disclosure are directed to the manufacture of a TALE comprising a combination of an endonuclease, a DNA polymerase, a DNA ligase, an exonuclease, a plurality of nucleotide dimer blocks encoding RVD domains (two residue variable domains) and a TALE-N/TF backbone vector including an endonuclease cleavage site , activating an endonuclease to cut the TALE-N/TF backbone vector at the endonuclease cutting site to produce a first end and a second end, activating an exonuclease to create 3' and 5' overhangs on the TALE-N/TF backbone vector and multiple nucleotide dimer blocks, and connecting the TALE-N/TF framework vector and multiple blocks of nucleotide dimers in the desired order, activating DNA polymerase and DNA ligase to connect the TALE-N/TF framework vector and multiple blocks of nucleotide dimers. One skilled in the art will readily understand from the present disclosure how to identify suitable endonucleases, DNA polymerases, DNA ligases, exonucleases, nucleotide dimer blocks encoding RVD domains, and TALE-N/TF framework vectors.

Aspects of the present disclosure relate to a method of altering target DNA in stem cells expressing an enzyme that forms a colocalization complex with RNA complementary to the target DNA and that cleaves the target DNA in a site-specific manner, comprising (a) introducing into the stem cell a first foreign nucleic acid acid encoding RNA complementary to the target DNA, which directs the enzyme to the target DNA, where the RNA and the enzyme are members of a colocalization complex on the target DNA, introduction into the stem cell of a second foreign nucleic acid encoding the donor nucleic acid sequence, where RNA and donor nucleotide sequence is expressed where the RNA and enzyme colocalize with the target DNA, the enzyme cleaves the target DNA and the donor nucleic acid is incorporated into the target DNA, producing altered DNA in the stem cell.

Aspects of the present disclosure are directed to a stem cell comprising a first foreign nucleic acid encoding an enzyme that forms a colocalization complex with RNA complementary to target DNA and that cleaves target DNA in a site-specific manner.

Aspects of the present disclosure relate to a cell comprising a first foreign nucleic acid encoding an enzyme that forms a colocalization complex with RNA complementary to target DNA and that cleaves target DNA in a site-specific manner, and including an inducible promoter to promote expression of the enzyme. In this way, expression can be regulated, for example it can be started and stopped.

Aspects of the present disclosure are directed to a cell comprising a first foreign nucleic acid encoding an enzyme that forms a colocalization complex with RNA complementary to a target DNA and that cleaves the target DNA in a site-specific manner, wherein the first foreign nucleic acid is excised from the genomic DNA of the cell with using a scavenging enzyme such as transposase.

Aspects of the present disclosure relate to a method of altering target DNA in a cell expressing an enzyme that forms a colocalization complex with RNA complementary to the target DNA and that cleaves target DNA in a site-specific manner, which includes (a) introducing a first foreign nucleic acid into the cell , encoding the donor nucleic acid sequence, introduction into the cell from the environment surrounding the cell, RNA, complementary to the target DNA, which directs the enzyme to the target DNA, where both the RNA and the enzyme are members of a colocalization complex on the target DNA, where the donor nucleic acid sequence expressed where the RNA and enzyme are colocalized on the target DNA, the enzyme cleaves the target DNA and the donor nucleic acid is incorporated into the target DNA to produce altered DNA in the cell.

Aspects of the present disclosure are directed to the use of an RNA-directed DNA-binding protein for the genetic modification of stem cells. In one aspect, the stem cell is genetically modified to include a nucleic acid encoding an RNA-guided DNA-binding protein, and the stem cell expresses the RNA-guided DNA-binding protein. In a particular aspect, donor nucleic acids for introducing specific mutations are optimized for genome editing using either modified TALENs or an RNA-guided DNA binding protein.

Aspects of the present disclosure are directed to DNA modification, e.g., multiplex DNA modification, in a stem cell using one or more guide RNAs (ribonucleic acids) to direct an enzyme having nuclease activity expressed by the stem cell, e.g., a DNA binding protein , having nuclease activity, to a specified position on DNA (deoxyribonucleic acid), where the enzyme cuts the DNA and the exogenous donor nucleic acid is incorporated into the DNA, for example, by homologous recombination. Aspects of the present invention include cyclic or repeated steps of modifying the DNA of a stem cell to create a stem cell having multiple DNA modifications within the cell. Modifications may include the introduction of exogenous donor nucleic acids.

Multiple exogenous nucleic acid insertions can be accomplished by a single step of introducing into a stem cell that expresses the enzyme, nucleic acids encoding multiple RNAs and multiple exogenous donor nucleic acids, for example, by cotransformation, where the RNAs are expressed and where each RNA collectively directs the enzyme to a specific section of DNA, an enzyme cuts the DNA and one of many exogenous nucleic acids is incorporated into the DNA at the site of the cut. According to this aspect, many changes or modifications to DNA in a cell are created in a single cycle.

Multiple exogenous insertions of nucleic acids into a stem cell can be accomplished by repeated steps or cycles of introducing into the stem cell that 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. acids, where RNA is expressed and directs the enzyme to a specific site of DNA, the enzyme cuts the DNA and exogenous nucleic acid is inserted into the DNA at the cut site, resulting in a cell that has several changes or insertions of exogenous DNA into the DNA within the stem cell. In one aspect, a stem cell expressing an enzyme has been genetically modified to express the enzyme, for example, by introducing into the cell a nucleic acid encoding an enzyme that can be expressed by the stem cell. Thus, aspects of the present invention include the cyclic steps of introducing RNA into a stem cell that expresses an enzyme, introducing an exogenous donor nucleic acid into a stem cell that expresses RNA, forming an RNA-enzyme-DNA colocalization complex, enzymatic cutting of the DNA by the enzyme, and inserting a donor nucleic acid acids in DNA. The cycle or repetition of the above stages leads to multiplex genetic modification of stem cells at multiple loci, i.e., leads to the formation of a stem cell having multiple genetic modifications.

In some aspects, DNA-binding proteins or enzymes within the scope of the present invention include a protein that forms a complex with a guide RNA, and with the help of the guide RNA, the complex is directed to a double-stranded DNA sequence, where the complex binds to the DNA sequence. In one aspect, the enzyme may be an RNA-directed DNA-binding protein, such as an RNA-directed DNA-binding protein of the CRISPR type II system, which binds to DNA and is directed by RNA. In one aspect, the RNA-directed DNA binding protein is a Cas9 protein.

This aspect of the present invention can be attributed to the colocalization of RNA and DNA binding protein with double-stranded DNA. Thus, the DNA binding protein-guide RNA complex can be used to cleave multiple sites on double-stranded DNA, thereby producing a stem cell with multiple genetic modifications, such as multiple insertions of exogenous donor DNA.

In accordance with some aspects, a method is provided for making several changes to target DNA in a stem cell expressing an enzyme that forms a colocalization complex with RNA complementary to the target DNA and that cleaves the target DNA in a site-specific manner, which includes (a) administering into the stem cell of a first foreign nucleic acid encoding one or more RNAs complementary to the target DNA, which direct an enzyme to the target DNA, where one or more RNAs and the enzyme are members of a colocalization complex with the target DNA, introduction of a second foreign nucleic acid into the stem cell acid encoding one or more donor nucleic acid sequences, where one or more RNA and one or more donor nucleic acid sequences are expressed, where one or more RNA and an enzyme colocalize with the target DNA, the enzyme cleaves the DNA, and the donor nucleic acid is incorporated into the DNA target, thereby obtaining altered DNA in the stem cell, and repeating step (a) several times to introduce multiple changes into the DNA of the stem cell.

In one aspect, the RNA is between about 10 and about 500 nucleotides in length. In one aspect, the RNA is between about 20 and about 100 nucleotides in length.

In one aspect, the one or more RNAs are guide RNAs. In one aspect, the one or more RNAs are a tracrRNA-crRNA chimera.

In one aspect, the DNA is genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA.

In one aspect, a cell can be genetically modified to reversibly incorporate a nucleic acid encoding a DNA binding enzyme using a vector that can be readily removed by the enzyme. Useful vectors and methods are known to those skilled in the art and include lentiviruses, adeno-associated virus, nuclease- and integrase-mediated targeted insertion methods, and transposon-mediated insertion methods. In one aspect, a nucleic acid encoding a DNA-binding enzyme that has been added, for example, by a cassette or vector, for example, can be removed entirely along with the cassette and vector, without leaving a portion of such nucleic acid, cassette or vector in the genomic DNA. Such removal is referred to in the art as “scarless” removal because the genome becomes the same as it was before the addition of the nucleic acid, cassette or vector.

One exemplary embodiment of scarless insertion and removal is the PiggyBac vector, commercially available from System Biosciences.

Additional features and advantages of certain embodiments of the present invention will become more apparent from the following description of embodiments and drawings, and from the claims.

Brief description of drawings

The above and other features and advantages of the present embodiments will be better understood from the following detailed description of the illustrative embodiments taken in conjunction with the accompanying drawings, wherein:

Fig. 1 relates to functional assays of re-TALENs in human somatic and stem cells.

(a) Schematic representation of an experimental design to test the effectiveness of genome targeting. The GFP coding sequence integrated into the genome is disrupted by the insertion of a stop codon and 68 bp. genomic fragment derived from the AAVS1 locus (bottom). Reconstruction of the GFP sequence by nuclease-mediated homologous recombination with the tGFP donor (top) results in the formation of GFP ⁺ cells that can be quantified by FACS. Re-TALENs and TALENs target identical sequences within AAVS1 fragments.

(b) Bar graph showing the percentage of GFP ⁺ cells generated with tGFP-donor alone, with tGFP-donor TALENs, and tGFP-donor re-TALENs at the target locus, as measured by FACS. (N = 3, error bars = SD). Representative FACS plots are shown below.

(c) Schematic overview depicting the targeting strategy for the native AAVS1 locus. The donor plasmid contains splice acceptor (SA)-2A (self-cleaving peptides), puromycin resistance gene (PURO) and GFP (see reference 10, incorporated herein by reference in its entirety). The locations of PCR primers used to detect successful editing events are depicted as blue arrows.

(d) Successfully targeted PGP1 hiPSCs clones were selected on puromycin (0.5 μg/ml) for 2 weeks. Microscopic images of three representative GFP ⁺ clones are shown. Cells were also stained for pluripotency markers TRA-1-60. Measuring ruler: 200 µm.

(e) PCR assays performed on these monoclonal GFP ⁺ hiPSC clones demonstrated successful insertion of donor cassettes into the AAVS1 site (lanes 1,2,3), while conventional hiPSCs showed no evidence of successful insertion (lane C).

(f) Sequence display SEQ ID NO: 1.

Fig. 2 relates to a comparison of the genome targeting efficiency of reTALENs and Cas9 guideRNAs to CCR5 in iPSCs.

(a) Schematic representation of a genome engineering experiment. A 90-mer ssODN carrying 2 bp was delivered to the target site of the re-TALEN or Cas9-hydrRNA pair in PGP1 hiPSCs. mismatch relative to genomic DNA, together with reTALEN or Cas9 guideRNA constructs. Sites of nuclease cleavage are shown in the figure as red arrows.

(b) Deep sequencing analysis of HDR and NHEJ efficiencies for pairs of re-TALEN (CCR5 #3) and ssODN, or Cas9-hydrRNA and ssODN. Changes in the genome of hiPSCs were analyzed based on high-throughput sequence data using GEAS. Top: HDR was quantified from the fraction of reads containing 2 bp. point mutations inserted into the center of ssODN (blue), and NHEJ activity was quantified from the deletion (gray)/insertion (red) fraction at each specific position in the genome. For the reTALEN and ssODN plots, the green dotted line is drawn to mark the outer boundary of the binding sites of the re-TALENs pairs, which are at the -26 bp position. and + 26 bp relative to the center of the two re-TALEN binding sites. For the Cas9-guideRNA and ssODN plots, the green dotted line marks the outer boundary of the guideRNA targeting region, which is located at positions -20 and -1 bp. in relation to the PAM sequence. Bottom: Deletion/Insertion size distribution in hiPSCs was analyzed across the entire NHEJ population with the treatments indicated above.

(c) Genome editing efficiency of re-TALENs and Cas9 guideRNAs targeting CCR5 in PGP1 hiPSCs.

Top: Schematic representation of regions affected by targeted genome editing in CCR5. The 15 targeted areas are shown with blue arrows below. For each site, cells were cotransfected with a pair of re-TALENs and their corresponding donor ssODN carrying 2 bp. mismatch regarding genomic DNA. The efficiency of genome editing was analyzed 6 days after transfection. Similarly, 15 Cas9 guideRNAs were transfected with the corresponding ssODNs individually into PGP1-hiPSCs to target the same 15 sites and efficiency was analyzed 6 days after transfection. Bottom: genome editing efficiency of re-TALENs and Cas9 guideRNAs when targeting CCR5 in PGP1 hiPSCs. Panels 1 and 2 demonstrate the efficiencies of NHEJ and HDR mediated by reTALENs. Panels 3 and 4 demonstrate the efficiencies of Cas9 guideRNA-mediated NHEJ and HDR. NHEJ scores were calculated from the frequency of genomic alleles carrying deletions or insertions in the targeted region; HDR indices were calculated based on the frequency of genomic alleles carrying 2 bp. inconsistencies. Panel 5, DNAseI HS profile for hiPSC cell line from ENCODE database (Duke DNase HS, iPS NIHi7 DS). It should be noted that the scales of the panels differ.

Fig. 3 aims to explore the functional parameters regulating ssODN-mediated HDR with re-TALENs or Cas9 guideRNAs in PGP1 hiPSCs.

(a) PGP1 hiPSCs were cotransfected with a pair of re-TALENs (#3) and ssODNs of varying lengths (50, 70, 90,110, 130,150,170 nucleotides). All ssODNs shared identical 2 bp. a mismatch with respect to genomic DNA in the middle of its sequence. The 90-mer ssODN achieved optimal HDR levels in the targeted genome. Evaluation of HDR-, NHEJ-mediated deletion and insertion efficiencies is described herein.

(b) 90-mer ssODNs corresponding to re-TALEN pair #3, each containing 2 bp. mismatch (A) in the center and an additional 2 bp. mismatches (B) at different positions offset from A (where offsets varied from -30 bp -> 30 bp) were used to test the consequences of deviation from homology along the ssODN. The genome editing efficiency of each ssODN was assessed in PGP1 hiPSCs. The bottom bar graph shows the frequency of A-only, B-only, and A+B inclusions in the targeted genome. HDR scores decreased as the distance from the center of the homology-biased region increased.

(c) ssODNs targeting sites with varying distances (-620bp ~ 480bp) from the target site of re-TALEN pair #3 were tested to evaluate the maximum distance within which ssODNs could be placed to introduction of mutations. All ssODNs carried 2 bp. inconsistency in the middle of its sequences. Minimal HDR efficiency (<=0.06%) was observed when the mismatch in the ssODN was located 40 bp apart. from the middle of the binding site of the re-TALEN pair.

(d) PGP1 hiPSCs were cotransfected with Cas9-guideRNA (AAVS1) and ssODNs of different orientations (Oc: complementary guideRNA; On: non-complementary guideRNA) and different lengths (30, 50, 70, 90, 110 nucleotides). All ssODNs had identical 2 bp. mismatches with respect to genomic DNA in the middle of their sequences. The 70-mer Oc achieved optimal HDR in the targeted genome.

Fig. 4 aims to use re-TALENs and ssODNs to generate genome-edited monoclonal hiPSCs without selection.

(a) Timeline of the experiment.

(b) The genome editing efficiency of the pair re-TALENs and ssODNs (#3) was assessed using the NGS platform described in Figure 2b.

(c) Sanger sequencing results of monoclonal hiPSC colonies after genome editing. 2 p.o. the heterogeneous genotype (CT/CT->TA/CT) was successfully introduced into the genome of the PGP 1-IPS-3-11, PGPl-IPS-3-13 colonies.

(d) Immunofluorescence staining of targeted PGPl-IPS-3-11. Cells were stained for pluripotency markers TRA-1-60 and SSEA4.

(e) Hematoxylin-eosin staining of teratoma sections obtained from monoclonal cells PGPl-IPS-3-11.

Fig. 5. Design by reTALE. (a) Sequence alignment of the original TALE RVD monomer relative to the monomers in re-TALE-16.5 (re-TALE-M1>re-TALE-M17). Nucleotide changes relative to the original sequence are highlighted in gray, (b) re-TALE repeatability test by PCR. The top panel shows the re-TALE/TALE structure and primer positions for the PCR reaction. The bottom panel shows the PCR bands under the conditions listed below. The electrophoretic pattern of the PCR fragments is shown using the original TALE template (right lane).

Fig. 6. Design and practical implementation of TASA (TALE Single-incubation Assembly), (a) Schematic representation of the re-TALE dimer block library for TASA assembly. A library of 10 re-TALE dimeric blocks encoding two RVDs is presented. Within each block, all 16 dimers have the same sequence, with the exception of the sequences encoding RVD; The dimers in the different blocks have different sequences but are designed to share 32 bp. overlapping with neighboring blocks. The nucleotide and amino acid sequences of one dimer (Block 6_AC) are shown on the right.

(b) Schematic representation of the TASA assembly. The left panel shows the TASA assembly method: a one-pot incubation reaction is carried out with the enzyme mixture/re-TALE block s/re-TALE-N/TF scaffold vectors. The reaction product can be used directly to transform bacteria. The right panel shows the TASA mechanism. The destination vector is linearized by endonuclease at 37°C to cut off the counterselective ccdB cassette; The exonuclease that processes the end of blocks and linearized vectors exposes ssDNA overhangs at the ends of the fragments, allowing the blocks and vector scaffolds to join in the designated order. When the temperature rises to 50°C, polymerases and ligases work together to repair the gap, resulting in final constructs ready for transformation.

(c) TASA assembly efficiency for re-TALEs possessing different monomer lengths. The blocks used for assembly are shown on the left, and the assembly efficiency is shown on the right.

Fig. 7. Functionality and sequence integrity of Lenti-reTALEs. 293T cells transduced with lenti-re-TALE-TF showed 36x upregulation of reporter expression compared to the negative reporter alone.

Fig. 8. Sensitivity and reproducibility of GEAS.

(A) Information analysis of HDR detection limit. Given the re-TALENs (#10)/ssODN dataset, reads containing expected editing (HDR) were identified and these HDR reads were systematically removed to create various 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 obtain artificial sets with HR efficiency in the range of 0-0.67%. For each individual data set, the mutual information (MI) of the background signal (in purple) and the signal received at the target site (in green) was estimated. The MI in the targeted area is noticeably higher than the background, while the HDR efficiency is higher than 0.0014%. The HDR detection limit is estimated to range from 0.0014% to 0.0071%. The calculation of MI is described in this document.

(b) Reproducibility test of the genome editing scoring system. Pairs of graphs (top and bottom) show the results of HDR and NHEJ assessment in duplicate with the pair of re-TALENs and cell type specified above. For each experiment, nucleofection, targeted genome amplification, deep sequencing, and data analysis were performed independently. The spread of repeats when assessing genome editing was calculated as V2 (|HDRl-HDR2|)/((HDR+HDR2)/2) =AHDR/HDR and V2 (|NHEJ1-NHEJ2|) /((NHEJl+NHEJ2)/2) = ANHEJ/NHEJ, and the scatter results are shown below the graphs. The average system spread was (19% + 11% + 4% + 9% + 10% + 35%)/6 = 15%. Factors that may contribute to scatter include cell nucleofection status, nucleofection efficiency, sequencing coverage, and sequencing quality.

Fig. 9. Statistical analysis of NHEJ and HDR efficiencies using reTALENs and Cas9-hydrRNA on CCR5.

(a) Correlation of HR and NHEJ efficiencies using reTALENs at identical sites in iPSCs (i= 0.91, P <IX 10-5).

(b) Correlation of HR and NHEJ efficiencies mediated by Cas9 guideRNA at identical sites in iPSCs (i= 0.74, p = 0.002).

(c) Correlation of Cas9-guideRNA-mediated NHEJ efficiency and guideRNA target site temperature Tm in iPSCs (r = 0.52, p = 0.04)

Fig. 10. Correlation analysis of the efficiency of genome editing and the epigenetic state. Pearson correlations were used to examine possible relationships between DNase I sensitivity and genome engineering efficiency (HR, NHEJ). The observed correlation was compared with a randomized set (N = 100,000). Observed correlations above the 95th percentile or below the 5th percentile of the simulated distribution were considered potential associations. No significant correlation was observed between DNase 1 sensitivity and NHEJ/HR efficiency.

Fig. 11. Effect of homologous pairing in ssODN-mediated genome editing.

(a) In the experiment described in Figure 3b, overall HDR decreased, as measured by the rate at which incorporation of the middle 2 bp occurred. mismatches (A), as the distance at which secondary mismatches B moved away from A increased (the relative position of B in relation to A, k varies from -30 to 30 bp). Higher incorporation rates when B is only 10 bp away. from A (−10 bp and + 10 bp) may reflect a lesser requirement for ssODN pairing against genomic DNA proximal to the DNA double-strand break.

(b) Distribution of gene conversion lengths along the ssODN. At each distance B from A, a fraction of HDR events involve only A, while another fraction involves both A and B. These two events can be interpreted in terms of gene conversion tracts (Elliott et al., 1998), resulting in A+ B events represent long conversion tracts that extend beyond just B and only A events, which represent shorter pathways that do not reach B. According to this interpretation, the distribution of gene conversion tracts in both directions along the oligonucleotide can be estimated (the middle of the ssODN is defined as 0, conversion tracts towards the 5' end of ssODN - as the "-" direction, and towards the 3' end - as the "+" direction). Gene conversion tracts gradually decrease as their lengths increase, a result very similar to the distribution of gene conversion tracts observed with dsDNA donors, but on a highly compressed distance scale of tens of bp. for single-stranded oligodeoxynucleotides compared to hundreds of base pairs for dsDNA donors.

(c) Gene conversion tract assays using a single ssODN that contains a number of mutations and measuring continuous sequences of inclusions. An ssODN donor with three 2 bp pairs was used. mismatches (orange) spaced 10 nucleotides apart from the central 2 bp. mismatches (top). A small number of genomic sequencing reads (see reference 62, incorporated herein by reference in its entirety) were found to carry >=1 mismatch identified by ssODN among >300,000 sequencing reads in this region. All of these reads were plotted (below), and the sequence of reads was color coded. Orange: certain inconsistencies; green: wild-type sequence. Genome editing with these ssODNs produced a pattern in which the average mutation alone was included at 85% (53/62) of time points, with many B mismatches included at other times. Although the number of B-inclusion events was too low to estimate the distribution of path lengths >10 bp, it is clear that a region of short paths from −10 to 10 bp predominates.

Fig. 12. Efficiency of genome editing by Cas9-hydrRNA nuclease and nickases.

PGP1 iPSCs were cotransfected with a combination of nuclease (C2) (Cas9-hydrRNA) or nickase (Cc) (Cas9D10A-hydrRNA) and ssODNs of different orientations (Oc and On). All ssODNs shared identical 2 bp. a mismatch with respect to genomic DNA in the middle of its sequence. HDR evaluation is described in this document.

Fig. 13. Design and optimization of the re-TALE sequence.

The re-TALE sequence was evolved over several design cycles to eliminate redundancies. In each cycle, synonymous sequences from each repeat were evaluated. Those that had the greatest Hamming distance to the evolving DNA were selected. The final sequence had cai = 0.59 ΔG = -9.8 kcal/mol. The R package was used to provide a general framework for synthetic protein design.

Fig. 14 is a gel image showing PCR verification of Cas9 genomic insertion in PGP1 cells. Lanes 3, 6, 9, 12 are PCR products from conventional PGP1 cell lines.

Fig. 15 is a graph of the Cas9 mRNA expression level upon induction.

Fig. 16 is a graph showing the genome targeting efficiency of RNAs with different designs.

Fig. 17 is a graph showing the genome targeting efficiency of 44% homologous recombination achieved using a guide RNA-donor DNA chimera.

Fig. 18 is a diagram showing the genotype of isogenic PGP 1 cell lines produced using the system described herein. PGPl-IPS-BTHH has a single-nucleotide deletion phenotype similar to BTHH patients. PGP 1-NHEJ has 4 bp. deletions that produce frameshift mutations in a different way.

Fig. 19 is a graph showing that cardiomyocytes derived from isogenic PGP1 iPS recapitulate defective ATP production and specific F1F0 ATPase activity as demonstrated in patient-specific cells.

Fig. 20 shows the sequences for the re-TALEN-base sequence and the re-TALE-TF base sequence.

Detailed description

Aspects of the present invention relate to the use of TALENs that have lost certain repetitive sequences for nucleic acid engineering, for example, by cleaving a double-stranded nucleic acid. The use of TALENs to cleave double-stranded nucleic acid can result in non-homologous end joining (NHEJ) or homologous recombination (HR). Aspects of the present disclosure also contemplate the use of TALENs that have lost repetitive sequences for engineering nucleic acids, for example, by cleaving a double-stranded nucleic acid in the presence of a donor nucleic acid and introducing the donor nucleic acid into the double-stranded nucleic acid, for example, by non-homologous end joining (NHEJ) or homologous recombination (HR).

Transcription activator-like effector nucleases (TALENs) are known in the art and include artificial restriction enzymes made by fusing the DNA binding domain of a TAL effector to a DNA cleavage domain. Restriction enzymes are enzymes that cut DNA strands in a specific sequence. Transcription activator-like effectors (TALEs) can be engineered to bind to the DNA sequence of interest. See Boch, Jens (February 2011). "TALEs of genome targeting". Nature Biotechnology 29 (2): 135-6, incorporated herein by reference in its entirety. By combining such an engineered TALE with a DNA cleavage domain (which cuts DNA strands), TALEN is obtained, which is a restriction enzyme specific for any DNA sequence of interest. In some aspects, TALENs are introduced into a cell for targeted in situ nucleic acid editing, such as in situ genome editing.

In one aspect, the nonspecific DNA cleavage domain at the end of the FokI endonuclease can be used to create hybrid nucleases that are active in yeast, plant and animal cells. The FokI domain functions as a dimer, requiring two constructs with unique DNA-binding domains for regions in the target genome with the correct orientation and spacing. Both the number of amino acid residues between the TALE DNA binding domain and the Fokl cleavage domain and the number of bases between the two individual TALEN binding sites influence activity.

The relationship between amino acid sequence and DNA recognition by the TALE binding domain allows the creation of engineered proteins. Computer programs such as DNA Works can be used to develop TALE designs. Other methods for developing TALE structures are known to those skilled in the art. See Cermak, T.; Doyle, E. L.; Christian, M.; Wang, L.; Zhang, Y.; Schmidt, C; Bailer, 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/gkrl51; 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/gkrl88; GeipTer, 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): el9509; Weber, E. ; Graetzner, R. ; Werner, S. ; Engler, C; Marillonnet, S. ( 2011). "Assembly of Designer TAL Effectors by Golden Gate Cloning." In Bendahmane, Mohammed. PLoS One 6 (5): el9722, which are incorporated herein by reference in their entirety.

In accordance with a typical aspect, once assembled, TALEN genes can be inserted into plasmids, in accordance with certain embodiments; the plasmids are then used to transfect the target cell, whereby the gene products are expressed and enter the nucleus to access the genome. In typical aspects, TALENs, as described herein, can be used to edit a target nucleic acid, such as a genome, by inducing double-strand breaks (DSBs), to which cells respond through repair mechanisms. Exemplary repair mechanisms include non-homologous end joining (NHEJ), which repairs DNA on either side of a double-strand break where there is little or no overlap for annealing. This repair mechanism induces errors in the genome, through insertion or deletion (indel), or chromosomal rearrangement; such errors can render the gene products encoded at a given location nonfunctional. See Miller, Jeffrey; et. al. (February 2011). "A TALE nuclease architecture for efficient genome editing". Nature Biotechnology 29 (2): 143-8, incorporated herein by reference in its entirety. Because this activity can vary depending on the species, cell type, target gene, and nuclease used, activity can be monitored using a heteroduplex digestion assay, which detects no difference between two PCR-amplified alleles. Digestion products can be visualized on a plain agarose gel or a block gel system.

Alternatively, DNA may be introduced into the genome via NHEJ in the presence of exogenous double-stranded DNA fragments. Homology-driven repair can also introduce foreign DNA into DSBs because transfected double-stranded sequences are used as templates for repair enzymes. In accordance with certain aspects, the TALENs described herein can be used to produce stably modified human embryonic stem cells and induced pluripotent stem cell clones (iPSCs). In accordance with some 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 colocalize with DNA in a stem cell and hydrolyze or cut the DNA to insert the exogenous DNA. Such DNA-binding proteins are well known to those skilled in the art for binding to DNA for various purposes. Such DNA-binding proteins may be natural. DNA-binding proteins included within the scope of the present invention include those that can be directed by RNA, which are referred to herein as guide RNAs. According to this aspect, the guide RNA and the RNA-guided DNA binding protein form a colocalization complex on DNA. Such DNA-binding proteins having nuclease activity are known to those skilled 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 described in the art. See Makarova et al., Nature Reviews, Microbiology, Vol. 9, June 2011, pp. 467-477 including all additional information, which is incorporated herein by reference in its entirety.

Typical DNA-binding proteins have nuclease activity to introduce single-strand breaks (nicks) or cut double-stranded DNA. Such nuclease activity may result from the action of a DNA binding protein having one or more polypeptide sequences exhibiting nuclease activity. Such typical DNA-binding proteins may have two separate nuclease domains, with each domain responsible for cutting or introducing single-strand breaks on a particular strand of double-stranded DNA. Exemplary polypeptide sequences having nuclease activity known to those skilled in the art include the McrA-HNH nuclease-related domain and the RuvC-like nuclease domain. Accordingly, typical DNA binding proteins are those that inherently contain one or more of a McrA-HNH nuclease-related domain and a RuvC-like nuclease domain.

A typical DNA binding protein is an RNA-driven DNA binding protein of the CRISPR type II system. A typical DNA binding protein is the Cas9 protein.

In S. pyogenes, Cas9Cas9 forms a 3-bp double-stranded break with blunt ends. upstream of the PAM motif (protospacer adjacent motif) by a process mediated by two catalytic domains in the protein: an HNH domain that cleaves the complementary strand of DNA and a RuvC-like domain that cleaves the non-complementary strand. See Jinke et al., Science, 337, 816-821 (2012), incorporated herein by reference in its entirety. Cas9 proteins are known to exist in many type II CRISPR systems, including those defined in supplementary documents 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 Bdl; Bifidobacterium longum DJO10A; Slackia heliotrinireducens DSM 20476; Persephonella marina EX HI; Bacteroides fragilis NCTC 9434; Capnocytophaga ochracea DSM 7271; Flavobacterium psychrophilum JIP02 86; Akkermansia muciniphila ATCC BAA 835; Roseiflexus castenholzii DSM 13941; Roseiflexus RSI; Synechocystis PCC6803; Elusimicrobium minutum Peil91; bacterial phylotype Rs D17 uncultivated group 1 Termite; 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 CHI; Streptococcus mutans NN2025 uid46353; Streptococcus mutans; Streptococcus pyogenes Ml 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 MGAS 10750; 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 rabram ATCC 11170; Diaphorobacter TPSY uid29975; Verminephrobacter eiseniae EF01-2; Neisseria meningitides 053442; Neisseria meningitides alphal4; Neisseria meningitides Z2491; Desulfovibrio salexigens DSM 2638; Campylobacter jejuni doylei 269 97; Campylobacter jejuni 81116; Campylobacter jejuni; Campylobacter lari RM2100; Helicobacter hepaticus; Wolinella succinogenes; Tolumonas auensis DSM 9187; Pseudoalteromonas atlantica T6c; Shewanella pealeana ATCC 700345; Legionella pneumophila Paris; Actinobacillus succinogenes 130Z; Pasteurella multocida; Francisella tularensis novicida U112; Francisella tularensis holarctica; Francisella tularensis FSC 198; Francisella tularensis tularensis; Francisella tularensis WY96-3418; and Treponema denticola ATCC 35405. Accordingly, aspects of the present disclosure are directed to the Cas9 protein present in the type II CRISPR system.

The Cas9 protein may be referred to by one skilled in the art as Csn1. The Cas9 protein from S. pyogenes is shown below. See Deltcheva et al., Nature 471, 602-607 (2011), incorporated herein by reference in its entirety.

In one aspect, the RNA-guided DNA-binding protein includes Cas9 homologs and orthologs that retain the protein's ability to bind to DNA, be guided by RNA, and cut DNA. In one aspect, the Cas9 protein includes a sequence that is present in the natural Cas9 protein from S. pyogenes and protein sequences that are at least 30%, 40%, 50%, 60%, 70%, 80%, 90% homologous. , 95%, 98% or 99% to this protein, and being a DNA-binding protein such as an RNA-guided DNA-binding protein.

In one aspect, an engineered Cas9-hydrRNA system is provided that allows for 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. Guide RNAs are complementary to target regions or target loci on DNA. Guide RNAs can be crRNA-tracrRNA chimeras. Guide RNAs can be introduced from the environment surrounding the cells. Thus, a method is proposed for continuous modification of the cell, which is carried out by introducing guide RNAs into the environment and by absorbing these guide RNAs and by enriching the environment with additional guide RNAs. Enrichment can go on continuously. Cas9 binds to or near target DNA in the genome. One or more guide RNAs bind to or near target DNA in the genome. Cas9 cuts the target DNA in the genome and exogenous donor DNA is incorporated into the DNA at the site of the cut.

Accordingly, the methods are directed to the use of a guide RNA with a Cas9 protein and an exogenous donor nucleic acid for multiplex insertions of exogenous donor nucleic acids into the DNA of a Cas9-expressing stem cell by cyclic insertion of the nucleic acid encoding the RNA (or by providing RNA from the environment) and exogenous donor nucleic acid expressing RNA (or by uptake of RNA), colocalization of RNA, Cas9 and DNA so that DNA is cut, and insertion of exogenous donor nucleic acid. The stages of the method can be cyclically repeated any necessary number of times to obtain the required number of DNA modifications. The methods herein are accordingly directed to editing target genes using the Cas9 proteins and guide RNAs described herein to achieve 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 (e.g., modified TALENs or Cas9 described herein) generally for the multiplex insertion of exogenous donor nucleic acids into DNA, such as the genomic DNA of a stem cell, e.g., a human stem cell . One skilled in the art will readily identify typical DNA binding systems based on the present disclosure.

Cells as used herein, unless otherwise specified, include any cell into which foreign nucleic acids as described herein can be introduced and expressed. It should be understood that the basic principles of the present disclosure described herein are not limited to cell type. The cells of the present invention include somatic cells, stem cells, eukaryotic cells, prokaryotic cells, animal cells, plant cells, fungal cells, cells, archaeal cells, eubacterial cells, etc. Cells include eukaryotic cells such as yeast cells, plant cells and animal cells. Specific cells include mammalian cells, such as human cells. Cells also include any cell in which it would be advantageous or desirable to modify the DNA.

Nucleic acids include any nucleic acid sequence for which a TALEN or an RNA-guided DNA binding protein having nuclease activity as described herein would be useful to introduce single-strand breaks or cuts. Target nucleic acids include any nucleic acid sequence for which a colocalization complex, as described herein, may be useful to introduce single-strand breaks or cuts. Target nucleic acids include genes. For purposes of the present disclosure, DNA, such as double-stranded DNA, may comprise a target nucleic acid, and a colocalization complex may bind or otherwise colocalize with the DNA, or the TALEN may otherwise bind to DNA at or in the vicinity or vicinity of the target nucleic acid such that the colocalization complex or TALEN has the desired effect on the target nucleic acid. Such target nucleic acids may include endogenous (or natural) nucleic acid and exogenous (or foreign) nucleic acid. One skilled in the art will readily identify or design guide RNAs and Cas9 proteins that colocalize with DNA or TALENs that bind to DNA comprising the target nucleic acid based on the present disclosure. One of ordinary skill in the art will also be able to identify transcriptional regulatory proteins or domains, such as transcriptional activators or transcriptional repressors, that similarly colocalize with DNA comprising the target nucleic acid. The DNA includes genomic DNA, mitochondrial DNA, viral DNA or exogenous DNA. In one aspect, materials and methods used in the practice of the present invention include those described in Di Carlo, et al., Nucleic Acids Research, 2013, vol. 41, No. 7 4336-4343, incorporated herein by reference in its entirety and for all purposes, including representative strains and media, plasmid constructs, plasmid transformation, electroporation of the transient guideRNA cassette and donor nucleic acids, transformation of guideRNA plasmid with donor DNA into Cas9- expressing cells, induction of Cas9 by galactose, identification of CRISPR-Cas targets in the yeast genome, etc. Additional references, including information, materials and methods useful to one skilled in the art in practicing 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, l., 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 is incorporated herein by reference in its entirety and for all purposes.

Foreign nucleic acids (ie, those that are not part of the natural nucleic acid composition of the cell) can be introduced into the 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, etc. Such methods are easily understood and adapted by one skilled in the art with the help of easily identifiable literature sources.

Donor nucleic acids include any nucleic acid that will be inserted into a nucleic acid sequence as described herein.

The following examples are provided as representative examples of the present disclosure. These examples should not be construed as limiting the scope of the present specification, as they and other equivalent embodiments will be apparent in light of the present specification, the drawings, and the accompanying claims.

Example I

Guide RNA Assembly

19 p.o. selected target sequence (i.e. 5'-N19 5'-N19-NGG-3') were included in two complementary 100-mer oligonucleotides

Each 100-mer oligonucleotide was suspended at 100 mM in water, mixed in an equal volume, and hybridized in a thermal cycler (95°C, 5 min; ramp down to 4°C, 0.1°C/sec). To prepare the destination vector, a guideRNA cloning vector (Addgene plasmid ID 41824) was linearized with AflII and the vector was purified. The assembly reaction (10 μl) of guideRNA was carried out with 10 ng of the combined 100 bp. fragment, 100 ng of target scaffold, Gibson assembly reaction mixture IX (New England Biolabs) at 50°C for 30 min. The product of the reaction can be immediately transformed into bacteria to produce colonies of individual assemblies.

Example II

Design and Assembly of recoded TALE

re-TALEs have been optimized at different levels to facilitate assembly as well as improve expression. The DNA sequences were first optimized together to approximate the human codon usage frequency and the low folding energy of the mRNA at the 5′ end (GeneGA, Bioconductor). The resulting sequence was subjected to evolution over several cycles to eliminate repeats (direct or inverted) longer than 11 bp. (see Fig. 12). In each cycle, synonymous sequences for each repeat were evaluated. Those with the greatest Hamming distance to the evolving DNA were selected. The sequence of one of the re-TALEs, which has 16.5 monomers, is presented below

CTAACCCCTGAACAGGTAGTCGCTATAGCTTCAAATATCGGGGGCAAGCAAGCACTTGAGACCGTTCAACGACTCCTGCCAGTGCTCTGCCAAGCCCATGGATTGACTCCGGAGCAAGTCGTCGCGATCGCGAGCAACGGCGGGGGGAAGCAGGCGCTGGAAACTGTTCAGAGACTGCTGCCTGTACTTTGTCAGGCGCATGGTCTCACCCCCGAACAGGTTGTCGCAATAGCAAGTAATATA GGCGGTAAGCAAGCCCTAGAGACTGTGCAACGCCTGCTCCCCGTGCTGTGTCAGGCTCACGGTCTGACACCTGAACAAGTTGTCGCGATAGCCAGTCACGACGGGGAAAACAAGCTCTAGAAACGGTTCAAAGGTTGTTGCCCGTTCTGTGCCAAGCACATGGGTTAACACCCGAACAAGTAGTAGCGATAGCGTCAAATAACGGGGGTAAACAGGCTTTGGAGACGGTACAGCGGTT ATTGCCGGTCCTCTGCCAGGCCCACGGACTTACGCCAGAACAGGTGGTTGCAATTGCCTCCAACATCGGCGGGAAACAAGCGTTGGAAACTGTGCAGAGACTCCTTCCTGTTTTGTGTCAAGCCCACGGCTTGACGCCTGAGCAGGTTGTGGCCATCGCTAGCCACGACGGAGGGAAGCAGGCTCTTGAAACCGTACAGCGACTTCTCCCAGTTTTGTGCCAAGCTCACGGGCTAACCCCCGAGCA AGTAGTTGCCATAGCAAGCAACGGAGGAGGAAAACAGGCATTAGAAACAGTTCAGCGCTTGCTCCCGGTACTCTGTCAGGCACACGGTCTAACTCCGGAACAGGTCGTAGCCATTGCTTCCCATGATGGCGGCAAACAGGCGCTAGAGACAGTCCAGAGGCTCTTGCCTGTGTTATGCCAGGCACATGGCCTCACCCCGGAGCAGGTCGTTGCCATCGCCAGTAATATCGGCGGAAAGCAAGCTCTCG CAGTACAACGGCTGTTGCCAGTCCTATGTCAAGCTCATGGACTGACGCCCGAGCAGGTAGTGGCAATCGCATCTCACGATGGAGGTAAACAAGCACTCGAGACTGTCCAAAGATTGTTACCCGTACTATGCCAAGCGCATGGTTTAACCCCAGAGCAAGTTGTGGCTATTGCATCTAACGGCGGTGGCAAACAAGCCTTGGAGACAGTGCAACGATTACTGCCTGTCTTATGTCAGGCCCAT GGCCTTACTCCTGAGCAAGTCGTAGCTATCGCCAGCAACATAGGTGGGAAACAGGCCCTGGAAACCGTACAACGTCTCCTCCCAGTACTTTGTCAAGCACACGGGTTGACACCGGAACAAGTGGTGGCGATTGCGTCCAACGGCGGAGGCAAGCAGGCACTGGAGACCGTCCAACGGCTTCTTCCGGTTCTTTGCCAGGCTCATGGGCTCACGCCAGAGCAGGTGGTAGCAATAGCGTCGAA CATCGGTGGTAAGCAAGCGCTTGAAACGGTCCAGCGTCTTCTGCCGGTGTTGTGCCAGGCGCACGGACTCACACCAGAACAAGTGGTTGCTATTGCTAGTAACAACGGTGGAAAGCAGGCCCTCGAGACGGTGCAGAGGTTACTTCCCGTCCTCTGTCAAGCGCACGGCCTCACTCCAGAGCAAGTGGTTGCGATCGCTTCAAACAATGGTGGAAGACCTGCCCTGGAA

In accordance with some aspects, TALEs having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, may be used. at least 98% sequence identity, or at least 99% sequence identity with the above sequence. One skilled in the art will readily understand where said sequence may vary while maintaining the DNA binding activity of TALE.

The re-TALE dimeric blocks encoding the two RVDs (see Fig. 6A) were generated using two rounds of PCR under standard Kara HIFI (KPAP) conditions, in which the first round of PCR introduced the RVD-coding sequence and the second round of PCR resulted in the formation of intact dimers blocks with 36 bp overlap with neighboring blocks. PCR products were purified using a QIAquick 96 PCR Purification Kit (QIAGEN) and concentrations were measured using a Nano-drop. The primer and template sequences are listed in Table 1 and Table 2 below.

Table 1. re-TALE block sequences

Block 0 CGCAATGCGCTCACGGGAGCACCCCTCAACCTAACCCCTGAACAGGTAGTCGCTATAGCTTCANNNNNNGGGGGCAAGCAAGCACTTGAGACCGTTCAACGACTCCTGCCAGTGCTCTGCCAAGCCCATGGATTGACTCCGGAGCAAGTCGTCGCGATCGCGAGCNNNNNNGGGGGGAAGCAGGCGCTGGAAACTGTTCAGAGACTGCTGCCTGTACTTTGTCAGGCGCATGGTCTC Block 1 AGACTGCTGCCTGTACTTTGTCAGGCGCATGGTCTCACCCCCGAACAGGTTGTCGCAATAGCAAGmNNNNNGGCGGTAAGCAAGCCCTAGAGACTGTGCAACGCCTGCTCCCCGTGCTGTGTCAGGCTCACGGTCTGACACCTGAACAAGTTGTCGCGATAGCCAAGTNNNNNNGGGGGAAAACAAGCTCTAGAAACGGTTCAAAGGTTGTTGCCCGTTCTGTGCCAAGCACATG GGTTA Block 1' TGCGCTCACGGGAGCACCCCTCAACCTCACCCCCGAACAGGTTGTCGCAATAGCAAGTNNNNNNGGCGGTAAGCAAGCCCTAGAGACTGTGCAACGCCTGCTCCCCGTGCTGTGTCAGGCTCACGGTCTGACACCTGAACAAGTTGTCGCGATAGCCAGTNNNNNNGGGGGAAAACAAGCTCTAGAAACGGTTCAAAGGTTGTTGCCCGTTCTGTGCCAAGCACATGGGTTA Block 2 AGGTTGTTGCCCGTTCTGTGCCAAGCACATGGGTTAACACCCGAACAAGTAGTAGCGATAGCGTCANNNNNNGGGGGTAAACAGGCTTTGGAGACGGTACAGCGGTTATTGCCGGTCCTCTGCCAGGCCCACGGACTTACGCCAGAACAGGTGGTTGCAATTGCCTCCNNNNNNGGCGGGAAACAAGCGTTGGAAACTGTGCAGAGACTCCTTCCTGTTTTGTGTCAAGCCCACGGCTTG ACGCCT Block 3 AGACTCCTTCCTGTTTTGTGTCAAGCCCACGGCTTGACGCCTGAGCAGGTTGTGGCCATCGCTAGCNNNNNNGGAGGGAAGCAGGCTCTTGAAACCGTACAGCGACTTCTCCCAGTTTTGTGCCAAGCTCACGGGCTAACCCCCGAGCAAGTAGTTGCCATAGCAAGCNNNNNNGGAGGAAAACAGGCATTAGAAACAGTTCAGCGCTTGCTCCCGGTACTCTGTCAGGCACACGGTCTA Block 4 CGCTTGCTCCCGGTACTCTGTCAGGCACACGGTCTAACTCCGGAACAGGTCGTAGCCATTGCTTCCNNNNNNGGCGGCAAACAGGCGCTAGAGACCGTCCAGAGGCTCTTGCCTGTGTTATGCCAGGCACATGGCCTCACCCCGGAGCAGGTCGTTGCCATCGCCAGTNNNNNNGGCGGAAAGCAAGCTCTCGAAACAGTACAACGGCTGTTGCCAGTCCTATGTCAAGCTCATGGACTG Block 5
CGGCTGTTGCCAGTCCTATGTCAAGCTCATGGACTGACGCCCGAGCAGGTAGTGGCAATCGCATCTNNNNNNNGGAGGTAAACAAGCACTCGAGACTGTCCAAAGATTGTTACCCGTACTATGCCAAGCGCATGGTTTAACCCCAGAGCAAGTTGTGGCTATTGCATCTTSnSINNNNGGTGGCAAACAAGCCTTGGAGACCGTGCAACGATTACTGCCTGTCTTATGTCAGGCCCATG GCCTT Block 6 CGATTACTGCCTGTCTTATGTCAGGCCCATGGCCTTACTCCTGAGCAGGTGGTCGCTATCGCCAGCNNNNNNGGGGGCAAGCAAGCACTGGAAACAGTCCAGCGTTTGCTTCCAGTACTTTGTCAGGCGCATGGATTGACACCGGAACAAGTGGTGGCTATAGCCTCANNNNNNGGAGGAAAGCAGGCGCTGGAAACCGTCCAACGTCTTTTTACCGGTGCTTTGCCAGGCGCACGGGCT C Block 6' CGATTACTGCCTGTCTTATGTCAGGCCCATGGCCTTACTCCTGAGCAAGTCGTAGCTATCGCCAGCNNNNNNGGTGGGAAACAGGCCCTGGAAACCGTACAACGTCTCCTCCCAGTACTTTGTCAAGCACACGGGTTGACACCGGAACAAGTGGTGGCGATTGCGTCCNNNNNNGGAGGCAAGCAGGCACTGGAGACCGTCCAACGGCTTCTTCCGGTTCTTTGCCAGGCTCATGGGCTС Block 7 CGGCTTCTTCCGGTTCTTTGCCAGGCTCATGGGCTCACGCCAGAGCAGGTGGTAGCAATAGCGTCGNNNNNNGGTGGTAAGCAAGCGCTTGAAACGGTCCAGCGTCTTCTGCCGGTGTTGTGCCAGGCGCACGGACTCACACCAGAACAAGTGGTTGCTATTGCTAGTTSnsnsnSINGGTGGAAAGCAGGCCCTCGAGACGGTGCAGAGGTTACTTCCCGTCCTCTGTCAA GCGCACGGCCTC

Table 2. Primer sequences of re-TALE blocks

Block O-F CGCAATGCGCTCACGGGAGCACCCCTCAACctAACCCCTGAACAGGT*A*G O-R block GAGACCATGCGCCTGACAAAAGTACAGGCAGCAGTCTCTGAACAG*T*T Block 1'-F TGGCGCAATGCGCTCACGGGAGCACCCCTCA*A*C Block 1-F AGACTGCTGCCTGTACTTTGTCAGGCGCATGGTCTCACCCCCGAACA*G*G Block -R/Block l'-R TAACCCATGTGCTTGGCACAGAACGGGCAACAACCTTTGAACCG*T*T Block 2-F AGGTTGTTGCCCGTTCTGTGCCAAGCACATGGGTTAACACCCgaac*a*a Block 2-R AGGCGTCAAGCCGTGGGCTTGACACAAAACAGGAAGGAGTCTCTGCACAG*T*T Block 3-F AGACTCCTTCCTGTTTTGTGTCAAGCCCACGGCTTGACGCCTG*A*G Block Z-R TAGACCGTGTGCCTGACAGAGTACCGGGAGCAAGCGCT*G*A Block 4-F CGCTTGCTCCCGGTACTCTGTCAGGCACACGGTCTAA*C*T Block 4-R CAGTCCATGAGCTTGACATAGGACTGGCAACAGCCGTT*G*T Block 5-F CGGCTGTTGCCAGTCCTATGTCAAGCTCATGGACTGA*C*G Block 5-R AAGGCCATGGGCCTGACATAAGACAGGCAGTAATCGTT*G*C Block 6-F CGATTACTGCCTGTCTTATGTCAGGCCCATGGCCTTA*C*T Block 6-R GAGCCCGTGCGCCTGGCAAAGCACCGGTAAAAGACGTTGGA*C*G Block 6'-F CGATTACTGCCTGTCTTATGTCAGGCCCATGGCCTTACTCCTGAGCAA Block 6'-R GAGCCCATGAGCCTGGCAAAGAACCGGAAGAAGCCGTT*G*G Block 7-F CGGCTTCTTCCGGTTCTTTGCCAGGCTCATGGGCTCACGCCAGAGCAGG*T*G Block 7-R GAGGCCGTGCGCTTGACAGAGGACGGGAAGTAACCTCT*G*C

Re-TALENs and re-TALE-TF destination vectors were constructed by modifying TALE-TF and the TALEN cloning framework (see reference 24, incorporated herein by reference in its entirety). The 0.5 RVD regions on the vectors were re-encoded and a SapI cutting site was included in the designated re-TALE cloning site. The sequences of re-TALENs and re-TALE-TF scaffolds are presented in Fig. 20. Plasmids can be predigested with SapI (New England Biolabs) under the manufacturer's recommended conditions, and purified using the QIAquick PCR purification kit (QIAGEN).

A one-pot (10 μl) TASA assembly reaction was performed with 200 ng of each block, 500 ng of target scaffold, TASA enzyme mix IX (2 U SapI, 100 U Ampligase (Epicentre), 2.5 U DNA Polymerase (New England Biolabs )) and isothermal assembly reaction buffer IX as previously described (see reference 25, incorporated herein by reference in its entirety) (5% PEG-8000, 100 mM Tris-HCl, pH 7.5, 10 mM MgC12, 10 mM DTT, 0.2 mM each of the four dNTPs, and 1 mM NAD). Incubation was carried out at 37°C for 5 min and 50°C for 30 min. The reaction can be directly bacterially transformed to produce colonies of individual assemblies. With this approach, the efficiency of obtaining a full-size structure was -20%. Otherwise, >90% efficiency can be achieved with a three-stage assembly. First, a 10 µl re-TALE assembly reaction was carried out with 200 ng of each block, 1X re-TALE enzyme mix, (100 U Ampligase, 12.5 U T5 Exonuclease, 2.5 U Phusion DNA Polymerase) and IX isothermal assembly buffer at 50°C for 30 min, followed by standardized Cara HIFI PCR, agarose gel electrophoresis, and QIAquick Gel extraction (Qiagen) reactions to enrich for full-length re-TALEs. 200 ng of re-TALE amplicons can then be mixed with 500 ng of target scaffold pre-treated with SapI, 1X isothermal re-TALE assembly reaction buffer and incubated at 50°C for 30 min. The final re-TALE assembly reaction can be directly bacterially transformed to produce colonies of individual assemblies. One skilled 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 such as: Fok 1, Bts I, Ear I, Sap I can be used. Titrate exonucleases such as lambda exonuclease, T5 exonuclease and exonuclease III can be used. Non-hot start polymerases such as Phusion DNA polymerase, Taq DNA polymerase and VentR DNA polymerase can be used. Heat-stable ligases such as ampligase, pfu DNA ligase, Taq DNA ligase can be used in this reaction. In addition, depending on the specific type of enzyme used, different reaction conditions may be used to activate such endonucleases, exonucleases, polymerases and ligases.

Example III

Cell Line and Cell Culture

PGP1 iPS cells were maintained in Matrigel (BD Biosciences)-coated plates in mTeSRl (StemCell Technologies). Cultures were subcultured every 5–7 days with TrypLE Express (Invitrogen). 293T and 293FT cells were grown and maintained in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) high glucose supplemented with 10% fetal bovine serum (FBS, Invitrogen), penicillin/streptomycin (pen/strep, Invitrogen), and essential amino acids (NEAA , Invitrogen). K562 cells were grown and maintained in RPMI medium (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 cell line, 293T, was generated to detect the effectiveness of HDR as described in reference 26, which is incorporated herein by reference in its entirety. Specifically, a reporter cell line carrying genome-integrated GFP coding sequences was disrupted by insertion of a stop codon and 68 bp. genomic fragment obtained from the AAVS1 locus.

Example IV

re-TALENs activity test

293T reporter cells were seeded at a density of 2 × 10 cells per well in a 24-well plate and transfected with 1 μg of each re-TALENs plasmid and 2 μg of donor DNA plasmid using Lipofectamine 2000 according to the manufacturer's protocols. Cells were harvested using TrypLE Express (Invitrogen) ∼18 h posttransfection and resuspended in 200 μl of media for flow cytometry analysis using an LSRFortessa Cell Analyzer (BD Biosciences). Flow cytometry data were analyzed using FlowJo (FlowJo). At least 25,000 events were analyzed for each transfected sample. For experiments targeting the endogenous AAVS1 locus in 293T cells, transfection procedures were identical to those described above, and puromycin selection was performed at a drug concentration of 3 μg/ml 1 week after transfection.

Example V

Assessment of the production of functional lentiviruses

Lentiviral vectors were generated using standard PCR and cloning methods. Lentiviral plasmids were transfected with Lipofectamine 2000 using lentiviral packaging mixture (Invitrogen) into cultured 293FT cells (Invitrogen) to produce lentivirus. The supernatant was collected at 48 and 72 h posttransfection, sterile filtered, and 100 μl of the filtered supernatant was added to 5 × 105 fresh 293T cells with polybrene. The lentivirus titer was calculated based on the following formula: virus titer = (percentage of GFP+ 293T cells * initial number of cells during transduction)/(volume of original virus-containing supernatant used in the transduction experiment). To test the performance of the lentiviruses, 3 days after transduction, lentivirus-transduced 293T cells were transfected with 30 ng of plasmid carrying the mCherry reporter and 500 ng of pUC19 plasmid using Lipofectamine 2000 (Invitrogen). Images of cells were analyzed using Axio Observer Zl (Zeiss) 18 hours after transfection, after which cells were collected using TrypLE Express (Invitrogen) and resuspended in 200 μl of medium for flow cytometry analysis using an LSRFortessa Cell Analyzer (BD Biosciences). Flow cytometry data were analyzed using BD FACSDiva (BD Biosciences).

Example VI

Test of genome editing efficiency using re-TALENs and Cas9-hydrRNA

PGP1 iPSCs were cultured with the Rho kinase (ROCK) inhibitor Y-27632 (Calbiochem) 2 h before nucleofection. Transfection was performed using the P3 Primary Cell 4D-Nucleofector X Kit (Lonza). Specifically, cells were harvested using TrypLE Express (Invitrogen) and 2x106 cells were resuspended in 20 μl of nucleofection mixture containing 16.4 μl of P3 Nucleofector solution, 3.6 μl of supplement, 1 μg of each re-TALENs plasmid or 1 μg of Cas9, and 1 µg guideRNA construct, 2 µl 100 µM ssODN. The mixtures were then transferred to 20 μl Nucleocuvette strips and nucleofection was performed using the CB150 program. Cells were seeded onto Matrigel-coated plates in mTeSR1 medium supplemented with ROCK inhibitor for the first 24 hours. For the endogenous AAVS1 locus targeting experiment using donor dsDNA, the same procedure was performed except that 2 μg of donor dsDNA was used and mTeSRl medium was supplemented with 0.5 μg/ml puromycin 1 week after transfection. .

Information about the reTALENs, guideRNAs, and ssODNs used in this example is provided in Table 3 and Table 4 below.

Table 3. Information on re-TALEN/Cas9-hydrRNA pairs targeting CCR5

re-TALENs re-TALENs re-TALEN-L
target sequence re-TALEN-R
target sequence guideRNA target
follower
ness guideRNA starting position of target sequence #
Target area target area of the pair (beginning
lo) target section of the pair (end) /chr3: /chr3: 1 4640
9942 4640
9993 TCCCCACTTTCTTGTGAA TAACCACTCAGGACAGGG CACTTTCTTGTGAATCCTT 4640
9946 2 4641
0227 4641
0278 TCACACAGCAAGTCAGCA TAGCGGAGCAGGCTCGGA TGGGCTAGCGGAGCAGGCT 4641
0264 3 4641
1260 4641
1311 TACCCAGACGAGAAAGCT TCAGACTGCCAAGCTTGA ACCCAGACGAGAAAGCTGA 4641
1261 4 4641
1464 4641
1515 TCTTGTGGCTCGGGAGTA TATTGTCAGCAGAGCTGA AGAGGGCATCTTGTGGCTC 4641
1456 5 4641
1517 4641
1568 TTGAGATTTTCAGATGTC TATACAGTCATATCAAGC ATCAAGCTCTCTTGGCGGT 4641
1538 6 4641
1634 4641
1685 TTCAGATAGATTATATCT TGCCAGATACATAGGTGG GCTTCAGATAGATTATATC 4641
1632 7 4641 4641 TTATACTGTCT TCAGCTCTTCT ACGGATTGTCTCA 4641 2396 2447 ATATGAT GGCCAGA GCTCTTC 2437 8 4641
2432 4641
2483 TGGCCAGAAGAGCTGAGA TTACCGGGGAGAGTTTCT CCGGGGAGAGTTTCTTGTA 4641
2461 9 4641 2750 4641 2801 TTTGCAGAGAG ATGAGTC TTAGCAGAAGA TAAGATT GAAATCTTATCT TCTGCTA 4641 2782 10 4641 3152 4641 3203 TATAAGACTAA ACTACCC TCGTCTGCCAC CACAGAT AATGCATGACAT TCATCTG 4641 3172 eleven 4641 4305 4641 4356 TAAAACAGTTTT GCATTCA TATAAAGTCCT AGAATGT AACAGTTTGCAT TCATGGA 4641 4308 12 4641 4608 4641 4659 TGGCCATCTCT GACCTGT TAGTGAGCCCA GAAGGGG CCAGAAGGGGA CAGTAAGA 4641 4632 13 4641 4768 4641 4820 TAGGTACCTGGCTGTCGT TGACCGTCCTG GCTTTTA CTGACAATCGAT AGGTACC 4641 4757 14 4641 5017 4641 5068 TGTCATGGTCA TCTGCTA TCGACACCGAAA GCAGAGT ACACCGAAGCA 4641 5046 15 4642 0034 4642 0084 TGCCCCCGCGA GGCCACA TCTGGAAGTTG AACACCC GGAAGTTGAAC ACCCTTGC 4642 0062

Table 4. ssODN design for studying ssODN-mediated genome editing

Figure 3b Distance between the second mutation and
DSB 90-
*1 CTACTGTCATTCAGGGCAATACCCAGACGAGAAAGCTGAGGGTATAACAGGTTTCAAGCTTGGCAGTCTGACTACAGAGGGCCACTGGCTT 90-
*2 CTACTGTCATTCAGCCCAATACCCTAACGAGAAAGCTGAGGGTATAACAGGTTTCAAGCTTGGCAGTCTGACTACAGAGGGCCACTGGCTT 90-
*3 CTACTGTCATTCAGCCCAATACCCAGACGAGAAAAGTGAGGGTATAACAGGTTTCAAGCTTGGCAGTCTGACTACAGAGGGCCACTGGCTT 90M-
0 CTACTGTCATTCAGCCCAATACCCAGACGAGAAAGCTGAGGGTATAACAGGTTTCAAGCTTGGCAGTCTGACTACAGAGGGCCACTGGCTT 90-
*4 CTACTGTCATTCAGCCCAATACCCAGACGAGAAAGCTGAGGGTATAACAGGTTTGTAGCTTGGCAGTCTGACTACAGAGGGCCACTGGCTT 90-
*5 CTACTGTCATTCAGCCCAATACCCAGACGAGAAAGCTGAGGGTATAACAGGTTTCAAGCTTGGCTCTCTGACTACAGAGGGCCACTGGCTT 90-
*6 CTACTGTCATTCAGCCCAATACCCAGACGAGAAAGCTGAGGGTATAACAGGTTTCAAGCTTGGCAGTCTGACTAGTGAGGCCACTGGCTT Figure 3c Distance between ssODN and
DSB L670 bp_9 0M CACTTTATATTTCCCCTGCTTAAACAGTCCCCCGAGGGTGGGTGCGGAAAAGGCTCTACACTTGTTATCATTCCCTCTCCACCACAGGCAT L570
bp_9 OM TAAGGCACAGAGCTTCAATAATTTGGTCAGAGCCAAGTAGCAG L480 bp_9 OM GGAGGTTAAACCCAGCAGCATGACTGCAGTTCTTAATCAATGCCCCTTGAATTGCACATATGGGATGAACTAGAACATTTTCTCGATGAT L394 bp_9 OM CTCGATGATTCGCTGTCCTTGTTATGATTATGTTACTGAGCTCTACTGTAGCACAGACATATGTCCCTATATGGGGCGGGGGTGGGGGTG L290 bp_9 OM GGTGTCTTGATCGCTGGGCTATTTCTATACTGTTCTGGCTTTTCGGAAGCAGTCATTTCTTTCTATTCTCCAAGCACCAGCAATTAGCTT L200 bp_9 OM GCTTCTAGTTTGCTGAAACTAATCTGCTATAGACAGAGACTCCGACGAACCAATTTTATTAGGATTTGATCAAATAAACTCTCTCTGACA L114 bp_9 OM GAAAGAGTAACTAAGAGTTTGATGTTTACTGAGTGCATAGTATGCACTAGATGCTGGCCGTGGATGCCTCATAGAATCCTCCCAACAACT L45b p_90 M GCTAGATGCTGGCCGTGGATGCCTCATAGAATCCTCCCAACAACCGATGAAATGACTACTGTCATTCAGCCCAATACCCAGACGAGAAAG R40b p_90 M ACAGGTTTCAAGCTTGGCAGTCTGACTACAGAGGGCCACTGGCTTTACCCCTGGGTTAGTCTGCCTCTGTAGGATTGGGGGCACGTAATTT R100 bp_90 M TTAGTCTGCCTCTGTAGGATTGGGGGCACGTAATTTTGCTGTTTAAG R200 bp_90 M GGAAGCCCAGAGGGCATCTTGTGGCTCGGGAGTAGCTCTCTGCTACCTTCTCAGCTCTGCTGACAATACTTGAGATTTTCAGATGTCACC R261 bp_90 M TCAGCTCTGCTGACAATACTTGAGATTTTCAGATGTCACCAACCAGCAAGAGAGCTTGATATGACTGTATATAGTATAGTCATAAAGAAC R322 bp_90 M CATAAAGAACCTGAACTTGACCATATACTTATGTCATGTGGAAATCTTCTCATAGCTTCAGATAGATTATATCTGGAGTGAAGAATCCTG R375 M_90 M GTGGAAAATTTCTCATAGCTTCAGATAGATTATATCTGGAGTGAGCAATCCTGCCACCTATGTATCTGGCATAGTGTGAGTCCTCATAAA R448 bp_90 M GGTTTGAAGGGCAACAAAATAGTGAACAGAGTGAAAATCCCCACCTAGATCCTGGGTCCAGAAAAAGATGGGAAACCTGTTTAGCTCACC Complementary -
30-mer GGCCACTAGGGACAAAATTGGTGAcagaaa 3d figure Length and
orientation of ssODN for directed action of Cas9-hydrRNA Complementary-
50-mer CCCACAGTGGGGCCACTAGGGACAAAATTGGTGAcagaaaagccccatcc Complementary-
70-mer TCCCCTCCACCCCACAGTGGGGCCACTAGGGACAAAATTGGTGAcag aaaagccccatccttaggcctcc Complementary-
90-mer cttTTATCTGTCCCCTCCACCCCACAGTGGGGCCACTAGGGACAAAAT TGGTGAcagaaaagccccatccttaggcctcctccttcctag Complementary-
110-mer gttctgggtacttTTATCTGTCCCCTCCACCCCACAGTGGGGCCACTAGGGA CAAAATTGGTGAcagaaaagccccatccttaggcctcctccttcctagtctcctgata Non-complementary - 30-
measures TTTCTGTCACCAATGGTGTCCCTAGTGGCC Non-complementary -50-mer GGATGGGGCTTTTCTGTCACCAATGGTGTCCCTAGTGGCCCCACTGTGGG Non-complementary -70-
measures GGAGGCCTAAGGATGGGGCTTTTCTGTCACCAATGGTGTCCCTAGTGGCCCCACTGTGGGGTGGAGGGGA Non-complementary - 90-mer CTAGGAAGGAGGAGGCCTAAGGATGGGGCTTTTCTGTCACCAATGGTGTCCCTAGTGGCCCCACTGTGGGGTGGAGGGGACAGATAAAAG Non-complementary -110-mer TATCAGGAGACTAGGAAGGAGGAGGCCTAAGGATGGGGCTTTTCTGTCACCAATGGTGTCCCTAGTGGCCCCACTGTGGGGTGGAGGGGACAGATAAAAGTACCCAGAAC Figure 2c Donor ssO DN for targeting Cas9-hydrRNA to CC R5 Cas9 guideRNA-CCR
5-1 TTCTAGTAACCACTCAGGACAGGGGGGTTCAGCCCAAAAATTCACA AGAAAGTGGGGACCCATGGGAAAT Cas9 guideRNA-CCR
5-2 CAGCAAGTCAGCAGCACAGCGTGTGTGACTCCGAGGGTGCTCCGCT AGCCCACATTGCCCTCTGGGGGTG Cas9 guideRNA-CCR
5-3 GTCAGACTGCCAAGCTTGAAACCTGTCTTACCCTCTACTTTCTCGTC TGGGTATTGGGCTGAATGACAGT Cas9 guideRNA-CCR
5-4 CAGAGCTGAGAAGACAGCAGAGAGCTACTCCCGAAGCACAAGATG CCCTCTGGGCTTCCGTGACCTTGGC Cas9 guideRNA-CCR
5-5 CTGACAATACTTGAGATTTTCAGATGTCACCAACGACCAAGAGAGC TTGATATGACTGTATATAGTATAG Cas9 guideRNA-CCR
5-6 CAGATACATAGGTGGCAGGATTCTTCACTCCAGACTTAATCTATCT GAAGCTATGAGAAATTTTCCACAT Cas9 guideRNA-CCR
5-7 TATATGATTGATTTGCACAGCTCATCTGGCCAGATAAGCTGAGACA TCCGTTCCCCTACAAGAAACTCTC Cas9 guideRNA-CCR
5-8 ATCTGGCCAGAAGAGCTGAGACATCCGTTCCCCTTGAAGAAACTCTCCCCGGTAAGTAACCTCTCAGCTG Cas9 guideRNA-CCR
5-9 AGGCATCTCACTGGAGAGGGTTTAGTTCTCCTTAAGAGAAGATAAGATTTCAAGAGGGAAGCTAAGACTC Cas9 guideRNA-CCR
5-10 ATAATATAATAAAAAATGTTTCGTCTGCCACCACTAATGAATGTCATGCATTCTGGGTAGTTTAGTCTTA Cas9 guideRNA-CCR
5-11 TTTATAAAGTCCTAGAATGTATTTAGTTGCCCTCGTTGAATGCAAACTGTTTTATACATCAATAGGTTTT Cas9 guideRNA-CCR
5-12 GCTCAACCTGGCCATCTCTGACCTGTTTTTCCTTCCCACTGTCCCCTTCTGGGCTCACTATGCTGCCGCC Cas9 guideRNA-CCR
5-13 TTTTAAAGCAAACACAGCATGGACGACAGCCAGGCTCCTATCGATTGTCAGGAGGATGATGAAGAAGATT Cas9 guideRNA-CCR
5-14 GCTTGTCATGGTCATCTGCTACTCGGGAATCCTAATTACTCTGCTTCGGTGTCGAAATGAGAAGAAGAGG Cas9 guideRNA-CCR
5-15 ATACTGCCCCCGCGAGGCCACATTGGCAAACCAGCTTGGGTGTTCAACTTCCAGACTTGGCCATGGAGAA Donor ssO DN for targeted action of RETALENs on
CC R5 reTALEN-CCR
5-1 CTGAAAGAATTTCCCATGGGTCCCCACTTTCTTGTGAATCCTTGGAGTGAACCCCCCTGTCCTGAGTGGTTACTAGAACACACCTCTGGAC reTALEN-CCR
5-2 TGGAAGTATCTTGCCGAGGTCACACAGCAAGTCAGCAGCACAGCCAGTGTGACTCCGAGCCTGCTCCGCTAGCCCACATTGCCCTCTGGG reTALEN-CCR
5-3 CTACTGTCATTCAGCCCAATACCCAGACGAGAAAGCTGAGGGTATAACAGGTTTCAAGCTTGGCAGTCTGACTACAGAGGGCCACTGGCTT reTALEN-CCR 5-4 GGAAGCCCAGAGGGCATCTTGTGGCTCGGGAGTAGCTCTCTGCTACCTTCTCAGCTCTGCTGACAATACTTGAGATTTTCAGATGTCACC reTALEN-CCR 5-5 TCAGCTCTGCTGACAATACTTGAGATTTTCAGATGTCACCAACGCCCAAGAGAGCTTGATATGACTGTATATAGTATAGTCATAAAGAAC reTALEN-CCR 5-6 GTGGAAAATTTCTCATAGCTTCAGATAGATTATATCTGGAGTGAGCAATCCTGCCACCTATGTATCTGGCATAGTGTGAGTCCTCATAAA reTALEN-CCR
5-7 GAAACAGCATTTCCTACTTTTATACTGTCTATATGATTGATTTGGTCAGCTCATCTGGCCAGAAGAGCTGAGACATCCGTTCCCCTACAA reTALEN-CCR
5-8 TTGATTTGCACAGCTCATCTGGCCAGAAGAGCTGAGACATCCGTATCCCTACAAGAAACTCTCCCCGGTAAGTAACCTCTCAGCTGCTTG reTALEN-CCR 5-9 GGAGAGGGTTTAGTTCTCCTTAGCAGAAGATAAGATTTCAAGATGAGAGCTAAGACTCATCTCTCTGCAAATCTTTCTTTTGAGAGGTAA reTALEN-CCR 5-10 TAATATAATAAAAAATGTTTCGTCTGCCACCAGATGAATGTCGAGCATTCTGGGTAGTTTAGTCTTATAACCAGCTGTCTTGCCTAGT reTALEN-CCR 5-11 TTAAAACCTATTGATGTATAAAACAGTTTGCATTCATGGAGGGTGACTAAATACATTCTAGGACTTTATAAAAGATCACTTTTTATTTA reTALEN-CCR 5-12 GACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTTCCTATTTACTGTCCCCTTCTGGGCTCACTATGCTGCCGCCCAGTGGGAC reTALEN-CCR 5-13 TCATCCTCCTGACAATCGATAGGTACCTGGCTGTCGTCCATGCTACGTTTGCTTTAAAAGCCAGGACGGTCACCTTTGGGGTGGTGACAA reTALEN-CCR 5-14 GGCTGGTCCTGCCGCTGCTTGTCATGGTCATCTGCTACTCGGGAGACCTAAAAACTCTGCTTCGGTGTCGAAATGAGAAGAAGAGGCACA reTALEN-CCR 5-15 GGCAAGCCTTGGGTCATACTGCCCCCGCGAGGCCACATTGGCAAGTCAGCAAGGGTGTTCAACTTCCAGACTTGGCCATGGAGAAGACAT

Example VII

Obtaining a library of amplicons of target 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 2~5 X 105 cells in medium, then 1 µl reactions were added in 9 μl of a PCR mixture containing 5 μl of 2X KAPA Hifi Hotstart Readymix (KAPA Biosystems) and 100 nM of the corresponding primer pairs for amplification. Reaction mixtures 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 adapter sequence, 5 μl of reaction products were then added to a 20 μl PCR mixture containing 12.5 μl of 2X KAPA HIFI Hotstart Readymix (KAPA Biosystems) and 200 nM primers carrying the Illumina adapter sequences. Reaction mixtures 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 using the QIAquick PCR purification kit, mixed at approximately equal concentrations, and sequenced using a MiSeq personal sequencer. PCR primers are listed below in Table 5.

Table 5 PCR primer sequences for the targeting site in CCR5

# of directional impact area in CCR5 Name primer sequence Site1-F1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTGATTTTT GCAGTGTGCGTTACTCC Site1-F2 ACACTCTTTCCCTACACGACGCTCTTCCGATCTACATCGTTT GCAGTGTGCGTTACTCC 1 Site1-F3 ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCCTAATTT GCAGTGTGCGTTACTCC Site1-F4 ACACTCTTTCCCTACACGACGCTCTTCCGATCTTGGTCATTT GCAGTGTGCGTTACTCC
CTCGGCATTCCTGCTGAACCGCTCTTCCGATCTCCAAGCAACTAAGTCACAGCA Site1-R Site2-F1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTGATATG AGGAAATGGAAGCTTG Site2-F2 ACACTCTTTCCCTACACGACGCTCTTCCGATCTACATCGAT GAGGAAATGGAAGCTTG 2 Site2-F3 ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCCTAAAT GAGGAAATGGAAGCTTG Site2-F4 ACACTCTTTCCCTACACGACGCTCTTCCGATCTTGGTCAATG AGGAAATGGAAGCTTG Site2-R CTCGGCATTCCTGCTGAACCGCTCTTCCGATCTCATTAGGG TATTGGAGGA site3-F1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTGATAAT CCTCCCAACAACTCAT site3-F2 ACACTCTTTCCCTACACGACGCTCTTCCGATCTACATCGAA TCCTCCCAACAACTCAT 3 site3-F3 ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCCTAAAA TCCTCCCAACAACTCAT site3-F4 ACACTCTTTCCCTACACGACGCTCTTCCGATCTTGGTCAAAT CCTCCCAACAACTCAT site3_R CTCGGCATTCCTGCTGAACCGCTCTTCCGATCTCCCAATCCT ACAGAGGCAG site4-F1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTGATAA site4-F2 ACACTCTTTCCCTACACGACGCTCTTCCCGATCTACATCGAA 4 site4-F3 ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCCTAAAA site4-F4 ACACTCTTTCCCTACACGACGCTCTTCCGATCTTGGTCAAA site4_R ACACTCTTTCCCTACACGACGCTCTTCCCGATCTAAGCCAAA GCTTTTTATTCT 5 Site5-F1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTGATATC TTGTGGCTCGGGAGTAG Site5-F2 ACACTCTTTCCCTACACGACGCTCTTCGATCTTACATCGATC TTGTGGCTCGGGAGTAG Site5-R CTCGGCATTCCTGCTGAACCGCTCTTCCGATCTTGGCAGGA TTCTTCACTCCA site6-F1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTGATCTA TTTTGTTGCCCTTCAAA 6 site6-F2 ACACTCTTTCCCTACACGACGCTCTTCCGATCTACATCGCTA TTTTGTTGCCCTTCAAA site6-R CTCGGCATTCCTGCTGAACCGCTCTTCCGATCTAACCTGAA CTTGACCATATACT site7-F1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTGATCA GCTGAGAGGTTACTTACC 7 site7-F2 ACACTCTTTCCCTACACGACGCTCTTCCGATCTACATCGCA GCTGAGAGGTTACTTACC site7-R CTCGGCATTCCTGCTGAACCGCTCTTCCGATCTAATGATTTA ACTCCACCCTC site8-F1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTGATACT CCACCCTCCTTCAAAAGA 8 site8-F2 ACACTCTTTCCCTACACGACGCTCTTCCGATCTACATCGACT CCACCCTCCTTCAAAAGA site8-R CTCGGCATTCCTGCTGAACCGCTCTTCCGATCTTGGTGTTTG CCAAATGTCT site9_F1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTGATGG GCACATATTCAGAAGGCA 9 site9_F2 ACACTCTTTCCCTACACGACGCTCTTCCGATCTACATCGGG GCACATATTCAGAAGGCA site9_R CTCGGCATTCCTGCTGAACCGCTCTTCCGATCTAGTGAAAG ACTTTAAAGGGAGCA site10-F1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTGATCAC AATTAAGAGTTGTCATA 10 site10-F2 ACACTCTTTCCCTACACGACGCTCTTCCGATCTACATCGCA CAATTAAGAGTTGTCATA site10-R CTCGGCATTCCTGCTGAACCGCTCTTCTCCGATCTCTCAGCTA GAGCAGCTGAAC eleven site11-Fl CTCGGCATTCCTGCTGAACCGCTCTTCCGATCTGACACTTG ATAATCCATC site11-F2 ACACTCTTTCCCTACACGACGCTCTTCCCGATCTACATCGTCA ATGTAGACATCTATGTAG site11-R ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTGATTCCA ATGTAGACATCTATGTAG 12 site12-F1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTGATACT GCAAAAGGCTGAAGAGC site12-F2 ACACTCTTTCCCTACACGACGCTCTTCCGATCTACATCGACT GCAAAAGGCTGAAGAGC site12-F3 ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCCTAAACT GCAAAAGGCTGAAGAGC site12-F4 ACACTCTTTCCCTACACGACGCTCTTCCGATCTTGGTCAACT GCAAAAGGCTGAAGAGC site12-R CTCGGCATTCCTGCTGAACCGCTCTTCCGATCTGCCTATAA AATAGAGCCCTGTCAA 13 site13-F1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTGATCTC TATTTTATAGGCTTCTTC site13-F2 ACACTCTTTCCCTACACGACGCTCTTCGATCTTACATCGCTC TATTTTATAGGCTTCTTC site13-R CTCGGCATTCCTGCTGAACCGCTCTTCCGATCTAGCCACCA CCCAAGTGATC 14 site14-F1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTACATCGTTC CAGACATTAAAGATAGTC site14-F2 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTGATTTC CAGACATTAAAGATAGTC site14-R CTCGGCATTCCTGCTGAACCGCTCTTCCGATCTAATCATGA TGGTGAAGATAAG 15 site15-Fl ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTGATCCG GCAGAGACAAAACATTAAA site15-F2 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCCGGCAGA GACAAACATTAAA site15-R CTCGGCATTCCTGCTGAACCGCTCTTCCGATCTAGCTAGGA AGCCATGGCAAG Illumina adapter PE-PCR-F AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACA cgac*g*c PE-PCR-R CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCT GCTGAACc*g*c Multiplex sequence primers for PCR 3 site3-MF ACACTCTTTCCCTACACGACGCTCTTCCGATCTAGTGCATA GTATGTGCTAGATGCTG site3-MR GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGATCTC TAAGAAGGCAAATGAGAC Illumina
adapter Index-PCR CAAGCAGAAGACGGCATACGAGATN1N2N3N4N5N6GTGAC TGGAGTTCAGACGTGTGCTCTTCCGATCT universal-PCR AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACA CGACGCTCTTCCGATCT

* index-PCR - primers purchased from Epicentre (ScriptSeq™ Index PCR Primers)

Example VIII

Genome Editing Assessment System (GEAS)

Next generation sequencing was used to detect rare genomic changes. See references 27-30, which are incorporated herein by reference in their entirety. To enable widespread application of this approach to quickly evaluate the performance of HDR and NHEJ in hiPSCS, software called “pipeline” was created for analyzing genome engineering data. "Pipeline" is integrated into a single Unix module that uses various tools such as R, BLAT and FASTX Toolkit.

Barcode breakdown: Sample groups were pooled together and paired-end sequenced with 150 bp read length. on a MiSeq (PE150) sequencer (Illumina Next Gen Sequencing) and were further separated based on DNA barcoding using the FASTX software package.

Filtering quality: Nucleotides with low sequence quality (Phred score <20) were trimmed. After trimming, reads shorter than 80 nucleotides were discarded.

Mapping: BLAT was used to independently map paired-end sequencing reads to the reference genome and output *.psl files.

Definition of Insertions/Deletions: Insertions/deletions were defined as full-length reads containing 2 blocks of matches in the alignment. Only reads matching this pattern in both paired-end reads were taken into account. As quality control, insertion/deletion “reads” were required that had a minimum 70 nucleotide match to the reference genome, and both blocks must be at least 20 nucleotides in length. The size and position of insertions/deletions were calculated from the position of each block in the reference genome. Non-homologous end joining (NHEJ) was estimated as the percentage of “reads” containing insertions/deletions (see Equation 1 below). Most NHEJ events were found in close proximity to the target site.

Homology-directed recombination (HDR) efficiency: Comparison with pattern (grep) in a 12 bp window. DSB-centered were used to count specific signatures corresponding to reads containing the reference sequence, modifications of the reference sequence (putative 2 bp mismatches), and reads containing only 1 bp. mutation within the expected 2 bp. mismatches (2 bp putative mismatches) (see equation 1 below).

Equation 1. Estimation of NHEJ and HDR

A = reads identical to the standard: XXXXXABXXXXXX

B = reads containing 2 bp. mismatches that are encoded by ssODN: XXXXXabXXXXX

C = reads containing only 1 bp. mutation in the target region: for example, XXXXXaBXXXXX or XXXXXAbXXXXX

D = reads containing insertions/deletions as described above

Example IX

Genotype Screening of Colonized hiPSCs

Human iPS cells in feeder-free cultures were pretreated with mTesr-1 medium supplemented with SMC4 (5 μM thiazovivin, 1 μM CHIR99021, 0.4 μM PD0325901, 2 μM SB431542) (see reference 23, incorporated herein by reference in its entirety). at least 2 hours before FACS sorting. Cultures were dissociated using accutase (Millipore) and resuspended in mTesr-1 medium supplemented with SMC4 and ToPro-3 viability dye (Invitrogen) at a concentration of 1-2 X107/ml. Live hiPS cells were sorted one cell at a time using a BD FACSAria II SORP UV (BD Biosciences) with a 100 μm 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, incorporated herein by reference in its entirety) with 100 ng/ml recombinant human basic fibroblast growth factor (bFGF) (Millipore) supplemented with SMC4 and 5 μg/ml fibronectin (Sigma ). After sorting, the plates were centrifuged at 70 x g for 3 min. Colony formation was noted 4 days after sorting, and the culture medium was replaced with hES cell medium with SMC4. SMC4 can be removed from hES cell media 8 days after sorting.

Several thousand cells were collected 8 days after fluorescence-activated cell sorting and (FACS) and 0.1 μl of prepGEM tissue protease enzyme (ZyGEM) and 1 μl of prepGEM gold buffer (ZyGEM) were added to 8.9 μl of cells in the medium. Reactions were then added to 40 μl of PCR mixture containing 35.5 ml of platinum 1.1X Supermix (Invitrogen), 250 nM of each dNTP, and 400 nM of primers. Reaction mixtures were incubated at 95°C for 3 min, followed by 30 cycles of 95°C, 20 s; 65°C, 30 and 72°C, 20 s. Products were Sanger sequenced using any of the PCR primers in Table 5 and sequences analyzed using DNASTAR (DNASTAR).

Example X

Immunostaining and analyzes of teratomas derived from hiPSCs

Cells were incubated in DMEM/F-12 knockout medium at 37°C for 60 minutes using the following antibodies: anti-SSEA-4 PE (Millipore) (diluted 1:500); Tra-1-60 (BD Pharmingen) (diluted 1:100). After incubation, cells were washed three times with knockout DMEM/F-12 and photographed on an Axio Observer Z.1 (ZIESS).

To perform the teratoma formation assay, human iPSCs were harvested using collagenase type IV (Invitrogen), cells were resuspended in 200 μl of Matrigel, and injected intramuscularly into the hindlimb pads of Rag2gamma knockout mice. Teratomas were isolated and fixed in formaldehyde 4-8 weeks after injection. Teratomas were then analyzed after hematoxylin and eosin staining.

Example XI

Orientation of genomic loci in human somatic cells and human stem cells using reTALENS

In some aspects, TALEs known to those skilled in the art are modified or recoded to eliminate repetitive sequences. Such TALEs suitable for modification and use in genome editing methods in viral delivery vehicles and in the various cell lines and microorganisms described herein are described in references 2, 7-12, incorporated herein by reference in their entirety. Several strategies have been developed to assemble the RVD TALE repeat array (see references 14 and 32-34, incorporated herein by reference in its entirety. However, once assembled, the TALE repeat sequences remain unstable, limiting the broad applicability of this tool, especially for viral delivery vehicles gene (see references 13 and 35 incorporated herein by reference in their entirety. Accordingly, one aspect of the present invention relates to TALEs that are devoid of repeats, e.g., completely devoid of repeats. Such recoded TALEs are effective because they allow for faster and easier synthesis of extended arrays TALE RVD.

To eliminate repeats, the nucleotide sequences of the TALE RVD arrays were computationally evolved to minimize the number of repeat sequences while maintaining amino acid composition. Recoded TALEs (Re-TALEs), encoding 16 tandem RVD DNA recognition monomers plus a terminal RVD half-repeat, lack any 12 bp. repetitions (see Figure 5a). Notably, this level of recoding is sufficient to allow PCR amplification of any particular monomer or subsection from the full-length re-TALE construct (see Fig. 5b). The advanced design of re-TALEs can be synthesized using standard DNA synthesis technologies (see reference 36, incorporated herein by reference in its entirety, without any additional costs or procedures associated with re-enriched sequences. In addition, the design of re-TALEs allows for efficient assemble re-TALE constructs using a modified isothermal assembly reaction as described in the methods herein and with reference to Figure 6.

NGS data on genome editing were statistically analyzed as follows. To analyze the specificity of HDR, an exact binomial test was used to calculate the probability of observing different numbers of 2-bp reads. mismatches. Based on the results of sequencing 10 bp. windows before and after the directional exposure section, the maximum replacement rates of the bases of two windows (PI and P2) were assessed. Using the null hypothesis that changes in each of the two target p.o. were independent, the expected probability of detecting 2 bp was calculated. discrepancies in the area of directional influence are random as the product of these two probabilities (P1 * P2). Given a dataset containing N number of total reads and n number of HDR reads, we calculated the p-value of the observed HDR efficiency. For HDR sensitivity assays, donor ssODNs contained 2 bp. mismatch with respect to the target genome, which makes it possible that simultaneous base substitutions in two bp that are targets are possible when ssODN is included in the target genome. Other off-target observed sequence changes will likely not change at the same time. Accordingly, non-target 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 at all other position pairs, and the HDR detection limit was estimated as the smallest HDR, where the MI of the target is 2 bp. site is higher than the MI of all pairs in other positions. For this experiment, HDR reads with the target 2 bp were identified in the original fastq file. mismatch and a set of fastq files with diluted HDR efficiencies was simulated by systematically removing varying numbers of HDR reads from the original dataset. Mutual information (MI) was calculated between all pairs of positions within 20 bp. windows centered on the directional exposure area. These calculations calculate the mutual base composition information between any two positions. Unlike the HDR specificity measure described above, this measure does not assess the tendency of position pairs to change any particular target base pairs, but only assesses their tendency to change at the same time (see Fig. 8A). Table 6 shows the efficiencies of HDR and NHEJ for targeting re-TALEN/ssODN to CCR5 and the efficiencies of NHEL for Cas9-hydrRNA. We programmed our analysis in R and calculated MI using the infotheo package.

Table 6

#
area of directed influence cell type HDR
(ReTALEN) (%) NHEJ (reTALE)
(%) HDR detection limit at
based on information analysis NHEJ (Cas9-hydrRNA) HDR
(Cas9-hydrRNA) 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% eleven 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%

* Group in which the HDR detection limit exceeds the actual HDR detected value

The correlation between genome editing efficiency and epigenetic state was adjusted as follows. Pearson correlation coefficients were calculated to examine possible relationships between epigenetic parameters (DNase I HS occupancy or nucleosomes) and genetic engineering efficiencies (HDR, NHEJ). The DNase I hypersensitivity dataset was downloaded from the UCSC Genome Browser. DNase I HS from hiPSCs:

/gbdb/hgl9/bbi/wgEncodeOpenChromDnaseIpsnihi7Sig. bigWig

To calculate the p value, the observed correlation was compared with the simulated distribution, which was constructed by randomizing the position of the epigenetic parameter (N = 100,000). Observed correlations higher than the 95th percentile or lower than the 5th percentile of the simulated distribution were considered potential associations.

The function of re-TALENs was determined in comparison with the corresponding non-recoded TALENs in human cells. The HEK 293 cell line containing a GFP reporter cassette and a frameshift insert carrier was used as described in reference 37, which is incorporated herein by reference in its entirety. See also Fig. 1a. Delivery of TALENs or reTALENs targeting the insertion sequence together with a promoterless GFP donor construct results in DSB-induced HDR repair of the GFP cassette, and the efficiency of GFP repair can be used to assess the efficiency of nuclease cutting. See reference 38, which is incorporated herein by reference in its entirety. The reTALENs-induced GFP recovery in 1.4% of transfected cells was similar to that obtained with TALENs (1.2%) (see Fig. 1b). The activity of reTALENs was tested at the AAVS1 locus in PGP1 hiPSCs (see Figure 1c), with successfully recovered cell clones containing specific inserts (see Figure 1d,e) confirming that reTALENs are active in both somatic and human pluripotent cells.

Elimination of repeats produces functional re-TALE-loaded lentivirus. Specifically, lentiviral particles into which fragments encoding re-TALE-2A-GFP were packaged were tested for the activity of re-TALE-TFs encoded by the viral particles by transfecting the mCherry reporter into a pool of lenti-reTALE-2A-infected 293T cells -GFP. 293T cells transduced with lenti-re-TALE-TF showed 36x upregulation of reporter expression compared to the negative reporter alone (see Figure 7a,b,c). The sequence integrity of re-TALE-TF in lentivirus-infected cells was checked and full-length reTALEs were detected in all 10 clones tested (see Fig. 1d).

Example XII

Comparison of the efficiency of ReTALEs and Cas9 guideRNAs in hiPSCs using the Genome Editing Assessment System (GEAS)

To compare the editing efficiency of re-TALENs relative to Cas9-hydrRNA in hiPSCs, a next generation sequencing platform (Genome Editing Scoring System) was developed to identify and quantify both NHEJ and HDR gene editing events. The re-TALEN and Cas9-hydrRNA pairs were designed and constructed to target the region upstream of CCR5 (re-TALEN, Cas9-hydrRNA pair #3 in Table 3), together with a 90 nucleotide ssODN donor identical to the targeting site behind with the exception of 2 p.o. mismatches (see Fig. 2a). Nuclease constructs and donor ssODN were transfected into hiPSCs. To quantify the efficiency of gene editing, deep paired-end sequencing was performed on the target genomic region 3 days after transfection. HDR efficiency was measured by the percentage of reads containing the exact 2 bp. mismatch. NHEJ efficiency was measured as the percentage of reads harboring insertions/deletions.

Delivery of ssODN alone into hiPSCs yielded minimal HDR and NHEJ rates, while delivery of re-TALENs and ssODN yielded efficiencies of 1.7% in HDR and 1.2% in NHEJ (see Figure 2b). The introduction of Cas9 guideRNA with ssODN resulted in efficiencies of 1.2% for HDR and 3.4% for NHEJ. Notably, the rate of genomic deletions and insertions peaked in the middle of the spacer region between the two reTALENs binding sites, but peaked at 3–4 bp. upstream of the Protospacer Associated Motif (PAM) sequence of the Cas9-hydrRNA targeting region (see Fig. 2b), as would be expected since double-strand breaks occur in these regions. In the case of re-TALENs, an average genomic deletion size of 6 bp was observed. and the insert size is 3 bp, and in the case of Cas9-hydrRNA, the average size of the deletion is 7 bp, the insertion is 1 bp. (see Fig. 2b), consistent with DNA damage patterns typically generated by NHEJ (see reference 4, incorporated herein by reference in its entirety). Several analyzes on a next-generation sequencing platform have shown that GEAS can detect HDR detection rates of up to 0.007%, which is both highly reproducible (coefficient of variation between replicates = ±15% * measured efficiency) and 400X more sensitive than most commonly used assays with mismatch-sensitive endonuclease (see Fig. 8).

re-TALEN and Cas9 guideRNA pairs targeting fifteen sites in the genomic CCR5 locus were generated to determine editing efficiency (see Figure 2c, Table 3). These regions were chosen to represent the broad range of DNase I sensitivities (see reference 39, incorporated herein by reference in its entirety). Nuclease constructs were transfected along with the corresponding donor ssODNs (see Table 3) into PGP1 hiPSCs. Six days after transfection, genome editing efficiencies at these regions were profiled (Table 6). For 13 of 15 re-TALEN pairs with donor ssODN, NHEJ and HDR were detected at levels above the statistical detection threshold, with an average NHEJ efficiency of 0.4% and an average HDR efficiency of 0.6% (see Figure 2c). Additionally, a statistically significant positive correlation (R2 = 0.81) was found between HR and NHEJ efficiencies at the same target loci (P < 1 x 10-4) (see Fig. 9a), suggesting that DSB formation , a common upstream step for both HDR and NHEJ, is the rate-limiting step for reTALEN-mediated genome editing.

In contrast, all 15 Cas9-hydrRNA pairs showed significant levels of NHEJ and HR, with an average NHEJ efficiency of 3% and an average HDR efficiency of 1.0% (see Figure 2c). In addition, a positive correlation was also found between the efficiency of NHEJ and HDR induced by Cas9 guideRNA (see Fig. 9b) (R2 = 0.52, p = 0.003), which is consistent with the observations for reTALENs. The NHEJ efficiency achieved by Cas9-hydrRNA was significantly higher than that achieved by reTALENs (t test, paired ends, P = 0.02). There was a moderate but statistically significant correlation between NHEJ efficiency and the melting temperature of the guideRNA target sequence (see Figure 9c) (R2 = 0.28, p = 0.04), suggesting that the base pairing strength between the guideRNA and its genomic target can explain up to 28% of the variation in the efficiency of Cas9-hydrRNA-mediated DSB formation. Even though Cas9-hydrRNAs produce levels of NHEJ on average 7 times higher than the corresponding reTALENs, Cas9-hydrRNA achieves HDR levels (mean = 1.0%) similar to those of the corresponding reTALENs (mean = 0.6%). Without being bound by scientific theory, these results suggest either that the concentration of ssODN at DSBs is the limiting factor for HDR, or that the structure of the genomic gap formed by Cas9-hydrRNA is not advantageous for efficient HDR. No correlation was observed between DNase I HS and genome targeting efficiencies for any of the methods (see FIG. 10).

Example XIII

Optimizing the Design of Donor ssODNs for HDR

Highly effective ssODNs in hiPSCs were developed as follows. A set of donor ssODNs of different lengths (50–170 nucleotides) was developed, which all carry the same 2 bp. mismatch in the middle of the spacer region of the sites of directed action of the re-TALEN #3 pair for CCR5. The observed HDR efficiency varied depending on the length of the ssODN, and an optimal HDR efficiency of -1.8% was observed with the 90 nucleotide ssODN, whereas longer ssODNs had reduced HDR efficiency (see Figure 3a). Since longer homology regions improve HDR performance when donor dsDNAs were used with nucleases (see reference 40, 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 accessible to the genome repair machinery; or that longer ssODNs introduce detrimental effects that offset any improvements conferred by longer homology compared to donor dsDNA (see reference 41, incorporated herein by reference in its entirety). However, if either of the first two reasons was the case, then NHEJ rates should either be unchanged or increase with longer ssODNs because NHEJ repair does not involve the donor ssODN. However, a decrease in NHEJ rates together with HDR was observed (see Figure 3 a), suggesting that longer ssODNs exhibit compensatory effects. Possible hypotheses could be that longer ssODNs are toxic to the cell (see reference 42, incorporated herein by reference in its entirety), or that transfection of long ssODNs saturates the DNA processing machinery, thereby causing a decrease in molar uptake DNA and reducing the capacity of cells to take up and express plasmids with re-TALEN.

The variability in the inclusion rate of mismatches carried by donor ssODNs as a function of distance from the double-strand break (“DSB”) was tested. To do this, we developed a series of 90 nucleotide ssODNs, which all have the same 2. bp. mismatch (A) in the center of the spacer region of the re-TALEN #3 pair. Each ssODN also contained a second 2 bp. misalignment (B) at various distances from the center (see Fig. 3b). ssODN, which has only one 2 bp. mismatches were used as controls. Each of these ssODNs was administered individually with a re-TALEN #3 pair and the results were analyzed using GEAS. We found that overall HDR—as measured by the rate at which mismatch A is turned on (A only or A+B)—decreases as B mismatches are located further from the center (see Fig. 3b, see Fig. 11a ). The higher HDR observed when B is only 10 bp away. from A, may reflect a lower requirement for annealing of ssODN against genomic DNA in the vicinity of the double-stranded DNA break.

For each distance B from A, a portion of the HDR events are related to the inclusion of only mismatch A, while another portion is related to the inclusion of both A and B mismatches (see Fig. 3b (A and A+B only)). These two results may be related to gene conversion tracts (see reference 43, incorporated by reference herein in their entirety) along the length of the single-stranded DNA oligonucleotide, resulting in the inclusion of A+B mismatches resulting from long conversion tracts that extend beyond mismatch B, and the inclusion of only the A mismatch results from shorter pathways that do not reach B. Consistent with this interpretation, the distribution of gene conversion tracts in both directions along the ssODN was assessed (see Fig. 11b). The estimated distribution implies that gene conversion tracts gradually become less frequent as their lengths increase, a result very similar to the distributions of gene conversion tracts observed with donor dsDNA, but on a highly compressed distance scale of tens of bases for donor ssDNA relative to hundreds of bases for dsDNA donors. Consistent with this result, an experiment with ssODN comprising three 2 bp pairs. mismatches with an interval of 10 nucleotides on both sides of the central 2 bp. "A" mismatches produced a pattern in which A alone was included 85% of the time, with multiple B mismatches included in other cases (see Fig. 11c). Although the number of B-only events was too low to estimate the distribution of path lengths less than 1 bp, it is clear that the region with a short path within 10 bp. from the nuclease site is predominant (see Fig. 11b). Finally, in all experiments with single B mismatches, a small fraction of B-only inclusion events was observed (0.04% ~ 0.12%), which is approximately constant for all distances from A to B.

In addition, an analysis was made of how far the ssODN donor could be located from a re-TALEN-induced DNA double-strand break while maintaining its incorporation. We tested a set of 90-nucleotide ssODNs with a central 2 bp. mismatches targeting a long distance range (-600 bp to + 400 bp) from the re-TALEN-induced dsDNA break site. When ssODNs match >40 bp of sequence, we observed >30x lower HDR efficiencies compared to control ssODNs centered over the cut region (Figure 3c). The observed low level of incorporation may be the result of processes unrelated to dsDNA cutting, as seen in experiments in which genomes are altered separately by the ssDNA donor, see reference 42, incorporated herein by reference in its entirety. Meanwhile, the low level of HDR that occurs when the ssODN is located at ~40 bp may be due to a combination of weakened homology on the mismatch-containing side of the dsDNA cut, together with insufficient length of the ssDNA oligonucleotide on the other side of the dsDNA break.

The design of ssODNs DNA donor for Cas9-hydrRNA-mediated targeting was tested. We constructed Cas9-hydrRNA (C2), targeting the AAVS 1 locus, and ssODN donors of various orientations (Oc: complementary to guideRNA and On: non-complementary to guideRNA) and lengths (30, 50, 70, 90, 110 nucleotides). Oc were more efficient than On, and the 70-mer Oc achieved an optimal HDR of 1.5% (see Figure 3d.) The same ssODN chain bias was detected using nickase from Cas9 (CC: Cas9_D10A), despite the fact that the HDR efficiencies mediated by Cc with ssODN were significantly less than C2 (t-test, paired ends, P = 0.02). (see Fig. 12).

Example XIV

Clonal Isolation of Corrected hiPSC Cells

GEAS showed that re-TALEN pair #3 achieved accurate genome editing in hiPSCs with an efficiency of ~1%, a level at which correctly edited cells can typically be isolated by lineage screening. HiPSCs have poor viability as single cells. The optimized protocols described in reference 23, incorporated herein by reference in its entirety, together with a single-cell FACS sorting procedure were used to create a robust single-hiPSC sorting and maintenance platform where hiPSC clones can be recovered with survival rates >25%. This method was combined with a fast and efficient genotyping system to perform chromosomal DNA extraction and targeted genome amplification in 1-h reactions in a single tube, allowing genotyping of hiPSCs on a large scale. Together, these methods provide a pipeline for reliably generating genome-edited hiPSCs without selection.

To demonstrate this system (see Fig. 4a), PGP1 hiPSCs were transfected with a pair of reTALENs and ssODNs designed to target CCR5 at site #3 (Table 3). GEAS was performed on a subset of transfected cells and found an HDR frequency of 1.7% (see Figure 4b). This information, along with the 25% yield of sorted single cell clones, allows us to estimate that at least one correctly edited clone is obtained from five 96-well plates with a Poisson probability of 98% (assuming μ = 0.017 * 0.25 * 96 * 5 * 2). Six days after transfection, hiPSCs were sorted by FACS, and eight days after sorting, 100 hiPSC clones were screened. Sanger sequencing revealed that 2 of these 100 unselected hiPSC colonies contained a heterozygous genotype possessing 2 bp. mutation introduced by the donor ssODN (see Fig. 4c). The targeting efficiency of 1% (1% = 2/2 * 100, 2 monoallelic corrected clones per 100 cells screened) was comparable to that obtained from next generation sequencing analysis (1.7%) (see Figure 4b) . The pluripotency of the resulting hiPSCs was confirmed by immunohistochemical staining for SSEA4 and TRA-1-60 (see Figure 4d). Successfully targeted hiPSCs clones were able to form mature teratomas with features of all three germ layers (see Fig. 4E).

Example XV

Method for continuous editing of a cell genome

In some aspects, a method of genome editing in cells is provided, comprising a human cell, such as a human stem cell, wherein the cell is genetically modified to include a nucleic acid encoding an enzyme that forms a colocalization complex with RNA complementary to a target DNA and that cleaves DNA target in a site-specific manner. Such an enzyme includes an RNA-directed DNA-binding protein, such as the RNA-directed DNA-binding protein of the CRISPR type II system. A typical enzyme is Cas9. According to this aspect, the cell expresses the enzyme and the guide RNA is introduced into the cell from the environment surrounding the cell. The guide RNA and enzyme form a colocalization complex on the target DNA, where the enzyme cuts the DNA. Optionally, a donor nucleic acid may be present for insertion into the DNA at the site of the cut, for example, by non-homologous end joining or homologous recombination. In one aspect, a nucleic acid encoding an enzyme that forms a colocalization complex with RNA complementary to target DNA and that cleaves target DNA in a site-specific manner, such as Cas9, is under the control of a promoter such that the nucleic acid can be activated and repressed . Such promoters are well known to those skilled in the art. One exemplary promoter is the DOX-inducible promoter. In one aspect, a cell is genetically modified by the reversible insertion into its genome of a nucleic acid encoding an enzyme that forms a colocalization 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 using a reagent such as a transposase. Thus, the nucleic acid can be easily removed after use.

In one aspect, a system is proposed for continuous genome editing of human induced pluripotent stem cells (hiPSCs) using the CRISPR system. According to a typical aspect, the method includes using a hiPSC line with Cas9 reversibly inserted into the genome (Cas9-hiPSCs); and guideRNAs that have been modified from their native form to allow them to move from the cell environment into the cells for use with Cas9. Such guideRNAs were treated with phosphatase to remove phosphate groups. Genome editing into cells is accomplished using Cas9 by enriching phosphatase-treated guideRNA in tissue culture media. This approach allows scar-free genome editing of HiPSCs with efficiencies of up to 50% in one-day treatments, which are 2-10 times more effective than the best efficiencies known to date. In addition, the method is easy to use and has significantly lower cellular toxicity. Embodiments of the present invention include single editing of hiPSCs for biological research and therapeutic applications, multiplex editing of hiPSCs for biological research and therapeutic applications, directed evolution of hiPSCs, and phenotypic screening of hiPSCs and cells derived from them.

In accordance with some 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 in animal cells, such as mouse or rat cells, to produce mouse and rat cells with stable Cas9 integration, and tissue-specific genome editing can be performed by locally introducing phosphatase-treated guide RNA from the environment surrounding the cells . Moreover, other Cas9 derivatives can be inserted into a variety of cell lines and microorganisms, and targeted genomic manipulations such as sequence-specific single-strand breaks, gene activation, suppression, and epigenetic modification can be performed.

Aspects of the present disclosure are directed toward the generation of stable hiPSCs with Cas9 inserted into the genome. Aspects of the present disclosure are directed to modifying RNA to allow it to enter a cell through the cell wall and colocalize with Cas9 while evading the cell's immune response. Such modified guide RNA can achieve optimal transfection efficiencies with minimal toxicity. Aspects of the present disclosure are directed to optimized genome editing in Cas9-hiPSCs using phosphatase-treated guideRNAs. Aspects of the present invention include eliminating Cas9 from hiPSCs to achieve scar-free genome editing where the nucleic acid encoding Cas9 has been reversibly inserted into the cellular genome. Aspects of the present invention include biomedical engineering using hiPSCs with Cas9 inserted into the genome to create desired genetic mutations. These engineered hiPSCs maintain pluripotency and can successfully differentiate into various cell types, including cardiomyocytes, which fully recapitulate the phenotype of patient cell lines.

Aspects of the present invention include phosphatase-treated guide RNA libraries for multiplex genome editing. Aspects of the present invention include the production of a library of PGP cell lines, in which each cell line carries several designated mutations in the genome that serve as a resource for drug screening. Aspects of the present invention include the generation of PGP1 cell lines with all retroelements barcoded with different sequences to track the locations and activity of this element.

Example XVI

Generation of stable hiPSCs with Cas9 inserted into the genome

The Cas9 construct, expressed under a DOX-inducible promoter, was placed into a PiggyBac vector, which can be inserted into and removed from the genome using the PiggyBac transposase. The PCR reaction confirmed stable introduction of the vector (see Fig. 14). Inducible Cas9 expression was determined by quantitative real-time PCR. Cas9 mRNA levels increased 1000x after 8 hours of 1 μg/ml DOX in culture medium and fell to normal levels ~20 hours after DOX removal (See Figure 15).

In one aspect, genome editing based on the Cas9-hiPSC system bypasses the Cas9/RNA plasmid transfection procedure, a large construct typically with transfection efficiency in hiPSCs of <1%. The present Cas9-hiPSC system can serve as a platform for highly efficient genomic engineering of human stem cells. In addition, the Cas9 cassette introduced into hiPSCs using the PiggyBac system can be easily removed from the genome by introducing a transposase.

Example XVII

Phosphatase-treated guide RNA

To enable continuous genome editing of Cas9-hiPSCs, a series of modified RNAs encoding guide RNAs were prepared and added to the Cas9-iPS culture medium in complex with a liposome. Native RNA treated with phosphatase without any capping achieved an optimal HDR value of 13%, which is more than 30X greater than the efficiency values previously reported for 5'Cap-Mod RNA (see FIG. 16).

In one aspect, the guide RNA is physically attached to the donor DNA. Thus, a method is proposed to combine Cas9-mediated genomic cutting and ssODN-mediated HDR, resulting in stimulation of sequence-specific genome editing. Guide RNA bound to the donor ssODN at an optimized concentration achieves 44% HDR and 2% nonspecific NHEJ (see Figure 17). It should be noted that this procedure does not cause visible toxicity, which is observed in the case of nucleofection and electroporation.

In one aspect, the present invention provides an in vitro engineered RNA structure encoding a guide RNA that achieves high transfection efficiency, genome editing efficiency when combined with inserted Cas9 into the genome. In addition, the present invention provides a chimeric guide RNA-DNA construct for combining a genome cutting event with a homology-directed recombination reaction.

Example XVIII

Removal of reversibly inserted Cas9 from hiPSCs

To achieve scar-free genome editing, in some aspects, a Cas9 cassette was inserted into the genome of hiPSC cells using a reversible vector. Accordingly, the Cas9 cassette is reversibly inserted into the genome of hiPSC cells using the PiggyBac vector. The Cas9 cassette was removed from genome-edited hiPSCs by transfecting the cell with a plasmid encoding the transposase. Accordingly, aspects of the present invention include the use of a reversible vector, which is known to those skilled in the art. A reversible vector is a vector that can, for example, be inserted into a genome and then removed using an appropriate vector removal enzyme. Such vectors and corresponding enzymes for removing the vector are known to those skilled in the art. Screening was carried out on colonized iPS cells, and colonies lacking the Cas9 cassette were isolated, which was confirmed by PCR reaction. Accordingly, the present invention provides a method for genome editing that does not affect the rest of the genome when a Cas9 cassette is permanently inserted into the cell.

Example XIX

Genome editing in iPGP1 cells

Research into the pathogenesis of cardiomyopathy has historically been hampered by the lack of suitable model systems. Differentiation of cardiomyocytes from patient-derived induced pluripotent stem cells (iPSCs) is considered one promising way to overcome this obstacle, and reports of iPSC-based models of cardiomyopathy are now beginning to emerge. However, realizing this promise requires approaches that overcome the genetic heterogeneity of patient-derived iPSC lines.

Cas9-iPGP1 cell lines and phosphatase-treated DNA-linked guide RNA were used to generate three iPSC lines that are isogenic except for the TAZ exon 6 sequence, which was identified as carrying a single-nucleotide deletion in patients with Barth syndrome. Single-round RNA transfection yields ~30% HDR efficiency. Modified Cas9-iPGP1 cells with the desired mutations were grown to the colony level (see Fig. 18), after which the cell lines were differentiated into cardiomyocytes. Cardiomyocytes derived from the engineered Cas9-iPGP1 completely rescued the cardiolipin, mitochondrial, and ATP deficiency observed in iPSCs that were derived from patients and in the neonatal TAZ knockout rat model (see FIG. 19). Accordingly, methods are proposed for correcting disease-causing mutations in pluripotent cells with subsequent differentiation of the cells into the desired cell types.

Example XX

Materials and methods

1. Generation of stable iPS/ES cell lines with DOX-inducible PiggyBacCas9

1. After the cells reached 70% confluency, the culture was pretreated overnight with the ROCK inhibitor Y27632 at a final concentration of 10 μM.

2. The next day, prepare the nucleofection solution by combining 82 μl of human stem cell nucleofection solution and 18 μl of supplement 1 in a sterile 1.5 ml Eppendorf tube. Mixed well. The solution was incubated at 37°C for 5 minutes.

3. mTeSRl was aspirated; gently washed the cells with 2 ml of DPBS per well of a six-well plate.

4. Aspirate DPBS, add 2 ml/well of versene, and leave the culture in an incubator at 37°C until they clump together and lose adhesion, but before detachment. This requires 3-7 minutes.

5. Gently aspirate the versene and add mTeSRl. 1 ml mTeSRl was added and the cells were knocked down using a 1000 µl micropipette with a slow flow of mTeSRl.

6. The detached cells were collected, gently broken into a single-cell suspension, quantified using a hematocytometer, and the cell density was adjusted to 1 million cells per ml.

7. Add 1 ml of cell suspension to a 1.5 ml Eppendorf tube and centrifuge at 1100 rpm for 5 min in a benchtop centrifuge.

8. Resuspend the cells in 100 μl Nucleofector solution for nucleofection of human stem cells from stage 2.

9. Transfer cells to the Nucleofector cuvette using a 1 ml pipette. 1 μg of plasmid with transposase and 5 μg of PB Cas9 plasmids were added to the cell suspension in a cuvette. The cells and DNA were mixed by gentle shaking.

10. Place the cuvette in the Nucleofector. The B-016 program was selected and the cells were subjected to nucleofection by pressing the X button.

11. Add 500ul of mTeSRl medium with ROCK inhibitor to the cuvette after nucleofection. 12. Aspirate the nucleofected cells from the cuvette using the provided plastic Pasteur pipette. The cells were transferred dropwise into Matrigel-coated wells of a 6-well plate with mTeSRl medium and ROCK inhibitor. Cells were incubated at 37°C overnight.

13. The medium in mTesr1 was replaced the next day and 72 hours after transfection, puromycin was added at a final concentration of 1 μg/ml. The line was withdrawn within 7 days.

2. RNA isolation

1. A DNA template was prepared, where the T7 promoter is upstream of the sequence encoding the guide RNA.

2. DNA was purified using Mega Clear Purification and its concentration was normalized.

3. Custom NTPS mixtures were prepared to produce various guide RNAs.

#1 Native RNA Blend [Final] (mM) GTP 7.5 ATP 7.5 CTP 7.5 UTP 7.5 Overall volume ° #2 Capped Native RNA Blend [Final] (mM) structural analogue of 3'-O-Me-m7G Cap (NEB) 6 GTP 1.5 ATP 7.5 CTP 7.5 UTP 7.5 Overall volume #3 Modified RNA mixture [Final] (mM) GTP 7.5 ATP 7.5 5-Me-CTP (Tri-Link) 7.5 Pseudo-UTP (Tri-Link) 7.5 Overall volume ° #4 capped/modified RNA mixture [Final] (mM) structural analogue of 3'-O-Me-m7G Cap (NEB) 6 GTP 1.5 ATP 7.5 5-Me-CTP (Tri-Link) 7.5 Pseudo-UTP (Tri-Link) 7.5 Overall volume °

4. Prepare the mixture for in vitro transcription at room temperature.

Amt (µl)

Custom NTP (*Add
v/IVT rxn as above) on ice N.D. PCR product (100 ng/µl) = 1600 ng total ([Final conc.] = 40 ng/µl) 16 Buffer x10 (MEGAscript set from Ambion) @ RT 4 Enzyme T7 (MEGAscript kit from Ambion) 4

5. Incubate for 4 hours (3-6 hours OK) at 37°C (cycler).

6. Add 2 µl of Turbo DNase (MEGAscript kit from Ambion) to each sample. Mix thoroughly and incubate at 37°C for 15'.

7. Purify the DNase reaction using MegaClear from Ambion according to the manufacturer's instructions.

8. Purify the RNA using MEGAclear (purified RNA can be stored at -80 for several months).

9. Phosphate groups were removed to avoid the Toll2 immune response in the host cell.

Treatment of RNA phosphatase IX 12 For each RNA sample ~ 100 µl ND 10x buffer for Antarctic phosphatase 11 µl 132 Antarctic phosphatase 2 µl 24

Gently mix the sample and incubate at 37 C for 30 seconds (30 seconds - 1 hour approx)

3. RNA transfection

1. Seed 10K-20K cells into 48 wells without antibiotics. For transfection, cells must be 30-50% confluent.

2. Change the cell medium with B18R (200 ng/ml), DOX (1 μg/ml), puromycin (2 μg/ml) at least two hours before transfection.

3. Prepare a transfection reagent containing guide RNA (0.5 μg ~ 2 μg), donor DNA (0.5 μg ~ 2 μg) and RNAiMax, incubate the mixture at rm for 15 minutes and transfer it to the cell chamber.

4. Scattering of single human iPS cells seeded and collection of single clones

1. After 4 days of DOX induction and 1 day of DOX withdrawal, asperate the medium, wash gently with DPBS, add 2 ml/well of versene, and place the culture back in the incubator at 37°C until the cells cluster together and became loosely attached, but not detached. This required 3-7 minutes.

2. Gently aspirate the versene and add mTeSRl. 1 ml mTeSRl was added and cells were detached using a 1000 µl micropipette with a slow flow of mTeSRl.

3. The detached cells were collected, gently broken into a single-cell suspension, quantified using a hematocytometer, and the cell density was adjusted to 100 thousand cells per ml.

4. Cells were seeded onto Matrigel-coated 10 cm dishes with mTeSRl plus ROCK inhibitor at cell densities of 50K, 100K and 400K per 10 cm dish.

5. Screening of clones formed from single cells

1. After 12 days of culture in a 10 cm dish, the clones became large enough to be identified by the naked eye, capable of being marked with a colored marker. The clones were prevented from becoming too large and sticking together.

2. Place a 10 cm dish in laminar and using a P20 pipette (set at 10 µl) with filter tips, aspirate 10 µl of medium from each well of 24 well plates. Clones were selected by scraping the clone and transferring it into each well of a 24-well plate. A separate filter tip was used for each clone.

3. After 4-5 days, clones within one well of a 24-well plate were large enough to separate.

4. Aspirate the medium and wash with 2 ml/well of DPBS.

5. Aspirate DPBS, replace with 250 μl/well of dispase (0.1 U/ml), and incubate cells with dispase at 37°C for 7 minutes.

6. Replaced dispase with 2 ml DPBS.

7. Add 250 µl mTeSRl. A cell scraper was used to remove and collect the cells.

8. Transfer 125 μl of the cell suspension into the well of a 24-well plate coated with Matrigel.

9. Transfer 125 µl of cell suspension into a 1.5 ml Eppendorf tube for genomic DNA extraction.

6. Screening of clones

1. Centrifuge the tube from step 7.7

2. Aspirate the medium and add 250 μl of lysis buffer per well (10 mM + TrispH7.5 + (or + 8.0), 10 mM EDTA, 10 mM

3. NaCl + 10% SDS, 40 µg/ml proteinase K + (added fresh before using buffer).

4. Kept at 55 overnight.

5. DNA was precipitated by adding 250 μl of isopropanol.

6. Centrifuge for 30 minutes at maximum speed. Wash with 70% ethanol.

7. Ethanol was carefully removed. Air dried for 5 minutes.

8. Resuspend gDNA in 100-200 µl dH2O.

9. The target genomic region was amplified by PCR using specific primers.

10. Sanger sequenced the PCR product using the appropriate primer.

11. Sanger sequence data and expansion of target clones were analyzed.

7. Removing the PiggyBac Vector

1. Repeated stages 2.1-2.9

2. Transfer cells to a Nucleofector cuvette using a 1 ml pipette tip. 2 μg of plasmid with transposase was added to the cell suspension in the cuvette. The cells and DNA were mixed by gentle shaking.

3. Repeated stage 2.10-2.11

4. Nucleofified cells were asperated from the cuvette using the provided plastic Pasteur pipette and the cells were transferred dropwise into 10 cm dishes coated with Matrigel with mTeSR1 medium and ROCK inhibitor. Cells were incubated at 37°C overnight.

5. The next day, the mTesr1 medium was replaced and the medium was replaced every day for the next 4 days.

6. After the clones became large enough, 20-50 clones were taken and seeded into a 24-well plate.

7. Clones were genotyped using primers for the PB Cas9 PiggyBac vector and negative clones were propagated.

Bibliography

Literary sources are identified in the description by their number and are included in the description as if they were fully stated therein. Each of the following sources is incorporated herein by reference in its entirety.

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<223>PCR primer

<220>

<221> misc_feature

<222> (34)..(39)

<223> n is a, c, g, or t

<400> 13

ctgacgcccg agcaggtagt ggcaatcgca tctnnnnnng gaggtaaaca agcactcgag 60

actgtccaaa gattgttacc cgtactatgc caagcgcatg gt 102

<210> 14

<211> 102

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<220>

<221> misc_feature

<222> (34)..(39)

<223> n is a, c, g, or t

<400> 14

ttaaccccag agcaagttgt ggctattgca tctnnnnnnng gtggcaaaca agccttggag 60

acagtgcaac gattactgcc tgtcttatgt caggcccatg gc 102

<210> 15

<211> 102

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<220>

<221> misc_feature

<222> (34)..(39)

<223> n is a, c, g, or t

<400> 15

cttactcctg agcaagtcgt agctatcgcc agcnnnnnng gtgggaaaca ggccctggaa 60

accgtacaac gtctcctccc agtactttgt caagcacacg gg 102

<210> 16

<211> 102

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<220>

<221> misc_feature

<222> (34)..(39)

<223> n is a, c, g, or t

<400> 16

ttgacaccgg aacaagtggt ggcgattgcg tccnnnnnng gaggcaagca ggcactggag 60

accgtccaac ggcttcttcc ggttctttgc caggctcatg gg 102

<210> 17

<211> 102

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<220>

<221> misc_feature

<222> (34)..(39)

<223> n is a, c, g, or t

<400> 17

ctcacgccag agcaggtggt agcaatagcg tcgnnnnnng gtggtaagca agcgcttgaa 60

acggtccagc gtcttctgcc ggtgttgtgc caggcgcacg ga 102

<210> 18

<211> 102

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<220>

<221> misc_feature

<222> (34)..(39)

<223> n is a, c, g, or t

<400> 18

ctcacaccag aacaagtggt tgctattgct agtnnnnnng gtggaaagca ggccctcgag 60

acggtgcaga ggttacttcc cgtcctctgt caagcgcacg gc 102

<210> 19

<211> 60

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<220>

<221> misc_feature

<222> (34)..(39)

<223> n is a, c, g, or t

<400> 19

ctcactccag agcaagtggt tgcgatcgct tcannnnnng gtggaagacc tgccctggaa 60

<210> 20

<211> 5714

<212> DNA

<213> Artificial

<220>

<223> Oligonucleotide sequence encoding TALEN backbone

<400> 20

atgtcgcgga cccggctccc ttccccaccc gcacccagcc cagcgttttc ggccgactcg 60

ttctcagacc tgcttaggca gttcgacccc tcactgttta acacatcgtt gttcgactcc 120

cttcctccgt ttggggcgca ccatacggag gcggccaccg gggagtggga tgaggtgcag 180

tcgggattga gagctgcgga tgcaccaccc ccaaccatgc gggtggccgt caccgctgcc 240

cgaccgccga gggcgaagcc cgcaccaagg cggagggcag cgcaaccgtc cgacgcaagc 300

cccgcagcgc aagtagattt gagaactttg ggatattcac agcagcagca ggaaaagatc 360

aagcccaaag tgaggtcgac agtcgcgcag catcacgaag cgctggtggg tcatgggttt 420

acacatgccc acatcgtagc cttgtcgcag caccctgcag cccttggcac ggtcgccgtc 480

aagtaccagg acatgattgc ggcgttgccg gaagccacac atgaggcgat cgtcggtgtg 540

gggaaacagt ggagcggagc ccgagcgctt gaggccctgt tgacggtcgc ggggagagctg 600

agaggcctc cccttcagct ggacacggggc cagttgctga agatcgcgaa gcggggagga 660

gtcacggcgg tcgaggcggt gcacgcgtgg cgcaatgcgc tcacgggagc acccctcaac 720

agttcacgct gacagagacc gcggccgcat taggcacccc aggctttaca ctttatgctt 780

ccggctcgta taatgtgtgg attttgagtt aggatccgtc gagattttca ggagctaagg 840

aagctaaaat ggagaaaaaa atcactggat ataccaccgt tgatatatcc caatggcatc 900

gtaaagaaca ttttgaggca tttcagtcag ttgctcaatg tacctataac cagaccgttc 960

agctggatat tacggccttt ttaaagaccg taaagaaaaa taagcacaag ttttatccgg

1020

cctttattca cattcttgcc cgcctgatga atgctcatcc ggaattccgt atggcaatga

1080

aagacggtga gctggtgata tgggatagtg ttcacccttg ttacaccgtt ttccatgagc

1140

aaactgaaac gttttcatcg ctctggagtg aataccacga cgatttccgg cagtttctac

1200

acatatattc gcaagatgtg gcgtgttacg gtgaaaacct ggcctatttc cctaaagggt

1260

ttattgagaa tatgtttttc gtctcagcca atccctgggt gagtttcacc agttttgatt

1320

taaacgtggc caatatggac aacttcttcg cccccgtttt caccatgggc aaatattata

1380

cgcaaggcga caaggtgctg atgccgctgg cgattcaggt tcatcatgcc gtttgtgatg

1440

gcttccatgt cggcagaatg cttaatgaat tacaacagta ctgcgatgag tggcagggcg

1500

gggcgtaaag atctggatcc ggcttactaa aagccagata acagtatgcg tatttgcgcg

1560

ctgatttttg cggtataaga atatatactg atatgtatac ccgaagtatg tcaaaaagag

1620

gtatgctatg aagcagcgta ttacagtgac agttgacagc gacagctatc agttgctcaa

1680

ggcatatatg atgtcaatat ctccggtctg gtaagcacaa ccatgcagaa tgaagcccgt

1740

cgtctgcgtg ccgaacgctg gaaagcggaa aatcaggaag ggatggctga ggtcgcccgg

1800

tttattgaaa tgaacggctc ttttgctgac gagaacaggg gctggtgaaa tgcagtttaa

1860

ggtttacacc tataaaagag agagccgtta tcgtctgttt gtggatgtac agagtgatat

1920

tattgacacg cccgggcgac ggatggtgat ccccctggcc agtgcacgtc tgctgtcaga

1980

taaagtctcc cgtgaacttt acccggtggt gcatatcggg gatgaaagct ggcgcatgat

2040

gaccaccgat atggccagtg tgccggtctc cgttatcggg gaagaagtgg ctgatctcag

2100

ccaccgcgaa aatgacatca aaaacgccat taacctgatg ttctggggaa tataaatgtc

2160

aggctccctt atacacagcc agtctgcagg tcgacggtct cgctcttcga aggttacttc

2220

ccgtcctctg tcaagcgcac ggcctcactc cagagcaagt ggttgcgatc gcttcaaaca

2280

acggtggaag acctgccctg gaatcaatcg tggcccagct ttcgaggccg gaccccgcgc

2340

tggccgcact cactaatgat catcttgtag cgctggcctg cctcggcgga cgacccgcct

2400

tggatgcggt gaagaagggg ctcccgcacg cgcctgcatt gattaagcgg accaacagaa

2460

ggattcccga gaggacatca catcgagtgg caggttccca actcgtgaag agtgaacttg

2520

aggagaaaaa gtcggagctg cggcacaaat tgaaatacgt accgcatgaa tacatcgaac

2580

ttatcgaaat tgctaggaac tcgactcaag acagaatcct tgagatgaag gtaatggagt

2640

tctttatgaa ggtttatgga taccgaggga agcatctcgg tggatcacga aaacccgacg

2700

gagcaatcta tacggtgggg agcccgattg attacggagt gatcgtcgac acgaaagcct

2760

acagcggtgg gtacaatctt cccatcgggc aggcagatga gatgcaacgt tatgtcgaag

2820

aaaatcagac caggaacaaa cacatcaatc caaatgagtg gtggaaagtg tatccttcat

2880

cagtgaccga gtttaagttt ttgtttgtct ctgggcattt caaaggcaac tataaggccc

2940

agctcacacg gttgaatcac attacgaact gcaatggtgc ggttttgtcc gtagaggaac

3000

tgctcattgg tggagaaatg atcaaagcgg gaactctgac actggaagaa gtcagacgca

3060

agtttaacaa tggcgagatc aatttccgca agctaaaatg gagaaaaaaa tcactggata

3120

taccaccgtt gatatatccc aatggcatcg taaagaacat tttgaggcat ttcagtcagt

3180

tgctcaatgt acctataacc agaccgttca gctggatatt acggcctttt taaagaccgt

3240

aaagaaaaat aagcacaagt tttatccggc ctttattcac attcttgccc gcctgatgaa

3300

tgctcatccg gaattccgta tggcaatgaa agacggtgag ctggtgatat gggatagtgt

3360

tcacccttgt tacaccgttt tccatgagca aactgaaacg ttttcatcgc tctggagtga

3420

ataccacgac gatttccggc agtttctaca catatattcg caagatgtgg cgtgttacgg

3480

tgaaaacctg gcctatttcc ctaaagggtt tattgagaat atgtttttcg tctcagccaa

3540

tccctgggtg agtttcacca gttttgattt aaacgtggcc aatatggaca acttcttcgc

3600

ccccgttttc accatgggca aatattatac gcaaggcgac aaggtgctga tgccgctggc

3660

gattcaggtt catcatgccg tttgtgatgg cttccatgtc ggcagaatgc ttaatgaatt

3720

acaacagtac tgcgatgagt ggcagggcgg ggcgtaaaga tctggatccg gcttactaaa

3780

agccagataa cagtatgcgt atttgcgcgc tgatttttgc ggtataagaa tatatactga

3840

tatgtatacc cgaagtatgt caaaaagagg tatgctatga agcagcgtat tacagtgaca

3900

gttgacagcg acagctatca gttgctcaag gcatatatga tgtcaatatc tccggtctgg

3960

taagcacaac catgcagaat gaagcccgtc gtctgcgtgc cgaacgctgg aaagcggaaa

4020

atcaggaagg gatggctgag gtcgcccggt ttattgaaat gaacggctct tttgctgacg

4080

agaacagggg ctggtgaaat gcagtttaag gtttacacct ataaaagaga gagccgttat

4140

cgtctgtttg tggatgtaca gagtgatatt attgacacgc ccgggcgacg gatggtgatc

4200

cccctggcca gtgcacgtct gctgtcagat aaagtctccc gtgaacttta cccggtggtg

4260

catatcgggg atgaaagctg gcgcatgatg accaccgata tggccagtgt gccggtctcc

4320

gttatcgggg aagaagtggc tgatctcagc caccgcgaaa atgacatcaa aaacgccatt

4380

aacctgatgt tctggggaat ataaatgtca ggctccctta tacacagcca gtctgcaggt

4440

cgacggtctc gctcttcgaa ggttacttcc cgtcctctgt caagcgcacg gcctcactcc

4500

agagcaagtg gttgcgatcg cttcaaacaa cggtggaaga cctgccctgg aatcaatcgt

4560

ggcccagctt tcgaggccgg accccgcgct ggccgcactc actaatgatc atcttgtagc

4620

gctggcctgc ctcggcggac gacccgcctt ggatgcggtg aagaaggggc tcccgcacgc

4680

gcctgcattg attaagcgga ccaacagaag gattcccgag aggacatagc cccaagaaga

4740

agagaaaggt ggaggccagc ggttccggac gggctgacgc attggacgat tttgatctgg

4800

atatgctggg aagtgacgcc ctcgatgatt ttgaccttga catgcttggt tcggatgccc

4860

ttgatgactt tgacctcgac atgctcggca gtgacgccct tgatgatttc gacctggaca

4920

tgctgattaa ctctagaggc agtggagagg gcagaggaag tctgctaaca tgcggtgacg

4980

tcgaggagaa tcctggccca gtgagcaagg gcgaggagct gttcaccggg gtggtgccca

5040

tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgaggcg

5100

agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc

5160

ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct

5220

accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc

5280

aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt

5340

tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg

5400

gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg

5460

ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg

5520

gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc

5580

tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga

5640

agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg

5700

acgagctgta caag 5714

<210> 21

<211> 3465

<212> DNA

<213> Artificial

<220>

<223> Oligonucleotide sequence encoding TALE backbone sequence

<400> 21

atgtcgcgga cccggctccc ttccccaccc gcacccagcc cagcgttttc ggccgactcg 60

ttctcagacc tgcttaggca gttcgacccc tcactgttta acacatcgtt gttcgactcc 120

cttcctccgt ttggggcgca ccatacggag gcggccaccg gggagtggga tgaggtgcag 180

tcgggattga gagctgcgga tgcaccaccc ccaaccatgc gggtggccgt caccgctgcc 240

cgaccgccga gggcgaagcc cgcaccaagg cggagggcag cgcaaccgtc cgacgcaagc 300

cccgcagcgc aagtagattt gagaactttg ggatattcac agcagcagca ggaaaagatc 360

aagcccaaag tgaggtcgac agtcgcgcag catcacgaag cgctggtggg tcatgggttt 420

acacatgccc acatcgtagc cttgtcgcag caccctgcag cccttggcac ggtcgccgtc 480

aagtaccagg acatgattgc ggcgttgccg gaagccacac atgaggcgat cgtcggtgtg 540

gggaaacagt ggagcggagc ccgagcgctt gaggccctgt tgacggtcgc ggggagagctg 600

agaggcctc cccttcagct ggacacggggc cagttgctga agatcgcgaa gcggggagga 660

gtcacggcgg tcgaggcggt gcacgcgtgg cgcaatgcgc tcacgggagc acccctcaac 720

agttcacgct gacagagacc gcggccgcat taggcacccc aggctttaca ctttatgctt 780

ccggctcgta taatgtgtgg attttgagtt aggatccgtc gagattttca ggagctaagg 840

aagctaaaat ggagaaaaaa atcactggat ataccaccgt tgatatatcc caatggcatc 900

gtaaagaaca ttttgaggca tttcagtcag ttgctcaatg tacctataac cagaccgttc 960

agctggatat tacggccttt ttaaagaccg taaagaaaaa taagcacaag ttttatccgg

1020

cctttattca cattcttgcc cgcctgatga atgctcatcc ggaattccgt atggcaatga

1080

aagacggtga gctggtgata tgggatagtg ttcacccttg ttacaccgtt ttccatgagc

1140

aaactgaaac gttttcatcg ctctggagtg aataccacga cgatttccgg cagtttctac

1200

acatatattc gcaagatgtg gcgtgttacg gtgaaaacct ggcctatttc cctaaagggt

1260

ttattgagaa tatgtttttc gtctcagcca atccctgggt gagtttcacc agttttgatt

1320

taaacgtggc caatatggac aacttcttcg cccccgtttt caccatgggc aaatattata

1380

cgcaaggcga caaggtgctg atgccgctgg cgattcaggt tcatcatgcc gtttgtgatg

1440

gcttccatgt cggcagaatg cttaatgaat tacaacagta ctgcgatgag tggcagggcg

1500

gggcgtaaag atctggatcc ggcttactaa aagccagata acagtatgcg tatttgcgcg

1560

ctgatttttg cggtataaga atatatactg atatgtatac ccgaagtatg tcaaaaagag

1620

gtatgctatg aagcagcgta ttacagtgac agttgacagc gacagctatc agttgctcaa

1680

ggcatatatg atgtcaatat ctccggtctg gtaagcacaa ccatgcagaa tgaagcccgt

1740

cgtctgcgtg ccgaacgctg gaaagcggaa aatcaggaag ggatggctga ggtcgcccgg

1800

tttattgaaa tgaacggctc ttttgctgac gagaacaggg gctggtgaaa tgcagtttaa

1860

ggtttacacc tataaaagag agagccgtta tcgtctgttt gtggatgtac agagtgatat

1920

tattgacacg cccgggcgac ggatggtgat ccccctggcc agtgcacgtc tgctgtcaga

1980

taaagtctcc cgtgaacttt acccggtggt gcatatcggg gatgaaagct ggcgcatgat

2040

gaccaccgat atggccagtg tgccggtctc cgttatcggg gaagaagtgg ctgatctcag

2100

ccaccgcgaa aatgacatca aaaacgccat taacctgatg ttctggggaa tataaatgtc

2160

aggctccctt atacacagcc agtctgcagg tcgacggtct cgctcttcga aggttacttc

2220

ccgtcctctg tcaagcgcac ggcctcactc cagagcaagt ggttgcgatc gcttcaaaca

2280

acggtggaag acctgccctg gaatcaatcg tggcccagct ttcgaggccg gaccccgcgc

2340

tggccgcact cactaatgat catcttgtag cgctggcctg cctcggcgga cgacccgcct

2400

tggatgcggt gaagaagggg ctcccgcacg cgcctgcatt gattaagcgg accaacagaa

2460

ggattcccga gaggacatag ccccaagaag aagagaaagg tggaggccag cggttccgga

2520

cgggctgacg cattggacga ttttgatctg gatatgctgg gaagtgacgc cctcgatgat

2580

tttgaccttg acatgcttgg ttcggatgcc cttgatgact ttgacctcga catgctcggc

2640

agtgacgccc ttgatgattt cgacctggac atgctgatta actctagagg cagtggagag

2700

ggcagaggaa gtctgctaac atgcggtgac gtcgaggaga atcctggccc agtgagcaag

2760

ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac

2820

ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc

2880

ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc

2940

ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc

3000

ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac

3060

ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc

3120

gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac

3180

aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg

3240

aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag

3300

cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc

3360

cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc

3420

gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaag 3465

<210> 22

<211> 1368

<212>PRT

<213> Streptococcus pyogenes

<400> 22

Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val

1 5 10 15

Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe

20 25 30

Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile

35 40 45

Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu

50 55 60

Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys

65 70 75 80

Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser

85 90 95

Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys

100 105 110

His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr

115 120 125

His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp

130 135 140

Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His

145 150 155 160

Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro

165 170 175

Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr

180 185 190

Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala

195 200 205

Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn

210 215 220

Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn

225 230 235 240

Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe

245 250 255

Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp

260 265 270

Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp

275 280 285

Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp

290 295 300

Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser

305 310 315 320

Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys

325 330 335

Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe

340 345 350

Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser

355 360 365

Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp

370 375 380

Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg

385 390 395 400

Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu

405 410 415

Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe

420 425 430

Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile

435 440 445

Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp

450 455 460

Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu

465 470 475 480

Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr

485 490 495

Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser

500 505 510

Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys

515 520 525

Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln

530 535 540

Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr

545 550 555 560

Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp

565 570 575

Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly

580 585 590

Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp

595 600 605

Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr

610 615 620

Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala

625 630 635 640

His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr

645 650 655

Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp

660 665 670

Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe

675 680 685

Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe

690 695 700

Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu

705 710 715 720

His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly

725 730 735

Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly

740 745 750

Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln

755 760 765

Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile

770 775 780

Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro

785 790 795 800

Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu

805 810 815

Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg

820 825 830

Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys

835 840 845

Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg

850 855 860

Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys

865 870 875 880

Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys

885 890 895

Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp

900 905 910

Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr

915 920 925

Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp

930 935 940

Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser

945 950 955 960

Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg

965 970 975

Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val

980 985 990

Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe

995 1000 1005

Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala

1010 1015 1020

Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe

1025 1030 1035

Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala

1040 1045 1050

Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu

1055 1060 1065

Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val

1070 1075 1080

Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr

1085 1090 1095

Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys

1100 1105 1110

Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro

1115 1120 1125

Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val

1130 1135 1140

Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys

1145 1150 1155

Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser

1160 1165 1170

Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys

1175 1180 1185

Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu

1190 1195 1200

Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly

1205 1210 1215

Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val

1220 1225 1230

Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser

1235 1240 1245

Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys

1250 1255 1260

His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys

1265 1270 1275

Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala

1280 1285 1290

Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn

1295 1300 1305

Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala

1310 1315 1320

Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser

1325 1330 1335

Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr

1340 1345 1350

Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp

1355 1360 1365

<210> 23

<211> 82

<212> DNA

<213> Artificial

<220>

<223> Oligonucleotide sequence

<220>

<221> misc_feature

<222> (41)..(41)

<223> n is a, c, g, or t

<400> 23

ttcttggctt tatatatctt gtggaaagga cgaaacaccg ngttttagag ctagaaatag 60

caagttaaaa taaggctagt cc 82

<210> 24

<211> 1692

<212> DNA

<213> Artificial

<220>

<223> Oligonucleotide sequence encoding re-TALE

<400> 24

ctaacccctg aacaggtagt cgctatagct tcaaatatcg ggggcaagca agcacttgag 60

accgttcaac gactcctgcc agtgctctgc caagcccatg gattgactcc ggagcaagtc 120

gtcgcgatcg cgagcaacgg cggggggaag caggcgctgg aaactgttca gagactgctg 180

cctgtacttt gtcaggcgca tggtctcacc cccgaacagg ttgtcgcaat agcaagtaat 240

ataggcggta agcaagccct agagactgtg caacgcctgc tccccgtgct gtgtcaggct 300

cacggtctga cacctgaaca agttgtcgcg atagccagtc acgacggggg aaaacaagct 360

ctagaaacgg ttcaaaggtt gttgcccgtt ctgtgccaag cacatgggtt aacacccgaa 420

caagtagtag cgatagcgtc aaataacggg ggtaaacagg ctttggagac ggtacagcgg 480

ttattgccgg tcctctgcca ggcccacgga cttacgccag aacaggtggt tgcaattgcc 540

tccaacatcg gcgggaaaca agcgttggaa actgtgcaga gactccttcc tgttttgtgt 600

caagcccacg gcttgacgcc tgagcaggtt gtggccatcg ctagccacga cggagggaag 660

caggctcttg aaaccgtaca gcgacttctc ccagttttgt gccaagctca cgggctaacc 720

cccgagcaag tagttgccat agcaagcaac ggagggaggaa aacaggcatt agaaacagtt 780

cagcgcttgc tcccggtact ctgtcaggca cacggtctaa ctccggaaca ggtcgtagcc 840

attgcttccc atgatggcgg caaacaggcg ctagagacag tccagaggct cttgcctgtg 900

ttatgccagg cacatggcct caccccggag caggtcgttg ccatcgccag taatatcggc 960

ggaaagcaag ctctcgaaac agtacaacgg ctgttgccag tcctatgtca agctcatgga

1020

ctgacgcccg agcaggtagt ggcaatcgca tctcacgatg gaggtaaaca agcactcgag

1080

actgtccaaa gattgttacc cgtactatgc caagcgcatg gtttaacccc agagcaagtt

1140

gtggctattg catctaacgg cggtggcaaa caagccttgg agacagtgca acgattactg

1200

cctgtcttat gtcaggccca tggccttact cctgagcaag tcgtagctat cgccagcaac

1260

ataggtggga aacaggccct ggaaaccgta caacgtctcc tcccagtact ttgtcaagca

1320

cacgggttga caccggaaca agtggtggcg attgcgtcca acggcggagg caagcaggca

1380

ctggagaccg tccaacggct tcttccggtt ctttgccagg ctcatgggct cacgccagag

1440

caggtggtag caatagcgtc gaacatcggt ggtaagcaag cgcttgaaac ggtccagcgt

1500

cttctgccgg tgttgtgcca ggcgcacgga ctcacaccag aacaagtggt tgctattgct

1560

agtaacaacg gtggaaagca ggccctcgag acggtgcaga ggttacttcc cgtcctctgt

1620

caagcgcacg gcctcactcc agagcaagtg gttgcgatcg cttcaaacaa tggtggaaga

1680

cctgccctgg aa 1692

<210> 25

<211> 237

<212> DNA

<213> Artificial

<220>

<223> Template sequence

<220>

<221> misc_feature

<222> (64)..(69)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (166)..(171)

<223> n is a, c, g, or t

<400> 25

cgcaatgcgc tcacgggagc acccctcaac ctaacccctg aacaggtagt cgctatagct 60

tcannnnnng ggggcaagca agcacttgag accgttcaac gactcctgcc agtgctctgc 120

caagcccatg gattgactcc ggagcaagtc gtcgcgatcg cgagcnnnnn nggggggaag 180

caggcgctgg aaactgttca gagactgctg cctgtacttt gtcaggcgca tggtctc 237

<210> 26

<211> 240

<212> DNA

<213> Artificial

<220>

<223> Template sequence

<220>

<221> misc_feature

<222> (67)..(72)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (169)..(174)

<223> n is a, c, g, or t

<400> 26

agactgctgc ctgtactttg tcaggcgcat ggtctcaccc ccgaacaggt tgtcgcaata 60

gcaagtnnnn nnggcggtaa gcaagcccta gagactgtgc aacgcctgct ccccgtgctg 120

tgtcaggctc acggtctgac acctgaacaa gttgtcgcga tagccagtnn nnnnggggga 180

aaacaagctc tagaaacggt tcaaaggttg ttgcccgttc tgtgccaagc acatgggtta 240

<210> 27

<211> 232

<212> DNA

<213> Artificial

<220>

<223> Template sequence

<220>

<221> misc_feature

<222> (59)..(64)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (161)..(166)

<223> n is a, c, g, or t

<400> 27

tgcgctcacg ggagcacccc tcaacctcac ccccgaacag gttgtcgcaa tagcaagtnn 60

nnnnggcggt aagcaagccc tagagactgt gcaacgcctg ctccccgtgc tgtgtcaggc 120

tcacggtctg acacctgaac aagttgtcgc gatagccagt nnnnnngggg gaaaacaagc 180

tctagaaacg gttcaaaggt tgttgcccgt tctgtgccaa gcacatgggt ta 232

<210> 28

<211> 246

<212> DNA

<213> Artificial

<220>

<223> Template sequence

<220>

<221> misc_feature

<222> (67)..(72)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (169)..(174)

<223> n is a, c, g, or t

<400> 28

aggttgttgc ccgttctgtg ccaagcacat gggttaacac ccgaacaagt agtagcgata 60

gcgtcannnn nngggggtaa acaggctttg gagacggtac agcggttatt gccggtcctc 120

tgccaggccc acggacttac gccagaacag gtggttgcaa ttgcctccnn nnnnggcggg 180

aaacaagcgt tggaaactgt gcagagactc cttcctgttt tgtgtcaagc ccacggcttg 240

acgcct 246

<210> 29

<211> 240

<212> DNA

<213> Artificial

<220>

<223> Template sequence

<220>

<221> misc_feature

<222> (67)..(72)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (169)..(174)

<223> n is a, c, g, or t

<400> 29

agactccttc ctgttttgtg tcaagcccac ggcttgacgc ctgagcaggt tgtggccatc 60

gctagcnnnn nnggagggaa gcaggctctt gaaaccgtac agcgacttct cccagttttg 120

tgccaagctc acgggctaac ccccgagcaa gtagttgcca tagcaagcnn nnnnggagga 180

aaacaggcat tagaaacagt tcagcgcttg ctcccggtac tctgtcaggc acacggtcta 240

<210> 30

<211> 240

<212> DNA

<213> Artificial

<220>

<223> Template sequence

<220>

<221> misc_feature

<222> (67)..(72)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (169)..(174)

<223> n is a, c, g, or t

<400> 30

cgcttgctcc cggtactctg tcaggcacac ggtctaactc cggaacaggt cgtagccatt 60

gcttccnnnn nnggcggcaa acaggcgcta gagaccgtcc agaggctctt gcctgtgtta 120

tgccaggcac atggcctcac cccggagcag gtcgttgcca tcgccagtnn nnnnggcgga 180

aagcaagctc tcgaaacagt acaacggctg ttgccagtcc tatgtcaagc tcatggactg 240

<210> 31

<211> 240

<212> DNA

<213> Artificial

<220>

<223> Template sequence

<220>

<221> misc_feature

<222> (67)..(72)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (169)..(174)

<223> n is a, c, g, or t

<400> 31

cggctgttgc cagtcctatg tcaagctcat ggactgacgc ccgagcaggt agtggcaatc 60

gcatctnnnn nnggaggtaa acaagcactc gagactgtcc aaagattgtt acccgtacta 120

tgccaagcgc atggtttaac cccagagcaa gttgtggcta ttgcatctnn nnnnggtggc 180

aaacaagcct tggagaccgt gcaacgatta ctgcctgtct tatgtcaggc ccatggcctt 240

<210> 32

<211> 240

<212> DNA

<213> Artificial

<220>

<223> Template sequence

<220>

<221> misc_feature

<222> (67)..(72)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (169)..(174)

<223> n is a, c, g, or t

<400> 32

cgattactgc ctgtcttatg tcaggcccat ggccttactc ctgagcaggt ggtcgctatc 60

gccagcnnnn nngggggcaa gcaagcactg gaaacagtcc agcgtttgct tccagtactt 120

tgtcaggcgc atggattgac accggaacaa gtggtggcta tagcctcann nnnnggagga 180

aagcaggcgc tggaaaccgt ccaacgtctt ttaccggtgc tttgccaggc gcacgggctc 240

<210> 33

<211> 240

<212> DNA

<213> Artificial

<220>

<223> Template sequence

<220>

<221> misc_feature

<222> (67)..(72)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (169)..(174)

<223> n is a, c, g, or t

<400> 33

cgattactgc ctgtcttatg tcaggcccat ggccttactc ctgagcaagt cgtagctatc 60

gccagcnnnn nnggtgggaa acaggccctg gaaaccgtac aacgtctcct cccagtactt 120

tgtcaagcac acgggttgac accggaacaa gtggtggcga ttgcgtccnn nnnnggaggc 180

aagcaggcac tggagaccgt ccaacggctt cttccggttc tttgccaggc tcatgggctc 240

<210> 34

<211> 240

<212> DNA

<213> Artificial

<220>

<223> Template sequence

<220>

<221> misc_feature

<222> (67)..(72)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (169)..(174)

<223> n is a, c, g, or t

<400> 34

cggcttcttc cggttctttg ccaggctcat gggctcacgc cagagcaggt ggtagcaata 60

gcgtcgnnnn nnggtggtaa gcaagcgctt gaaacggtcc agcgtcttct gccggtgttg 120

tgccaggcgc acggactcac accagaacaa gtggttgcta ttgctagtnn nnnnggtgga 180

aagcaggccc tcgagacggt gcagaggtta cttcccgtcc tctgtcaagc gcacggcctc 240

<210> 35

<211> 49

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 35

cgcaatgcgc tcacgggagc acccctcaac ctaacccctg aacaggtag 49

<210> 36

<211> 46

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 36

gagaccatgc gcctgacaaa gtacaggcag cagtctctga acagtt 46

<210> 37

<211> 33

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 37

tggcgcaatg cgctcacggg agcacccctc aac 33

<210> 38

<211> 49

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 38

agactgctgc ctgtactttg tcaggcgcat ggtctcaccc ccgaacagg 49

<210> 39

<211> 46

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 39

taacccatgt gcttggcaca gaacgggcaa caacctttga accgtt 46

<210> 40

<211> 48

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 40

aggttgttgc ccgttctgtg ccaagcacat gggttaacac ccgaacaa 48

<210> 41

<211> 52

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 41

aggcgtcaag ccgtgggctt gacacaaaac aggaaggagt ctctgcacag tt 52

<210> 42

<211> 45

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 42

agactccttc ctgttttgtg tcaagcccac ggcttgacgc ctgag 45

<210> 43

<211> 40

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 43

tagaccgtgt gcctgacaga gtaccgggag caagcgctga 40

<210> 44

<211> 39

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 44

cgcttgctcc cggtactctg tcaggcacac ggtctaact 39

<210> 45

<211> 40

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 45

cagtccatga gcttgacata ggactggcaa cagccgttgt 40

<210> 46

<211> 39

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 46

cggctgttgc cagtcctatg tcaagctcat ggactgacg 39

<210> 47

<211> 40

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 47

aaggccatgg gcctgacata agacaggcag taatcgttgc 40

<210> 48

<211> 39

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 48

cgattactgc ctgtcttatg tcaggcccat ggccttact 39

<210> 49

<211> 43

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 49

gagcccgtgc gcctggcaaa gcaccggtaa aagacgttgg acg 43

<210> 50

<211> 50

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 50

cgattactgc ctgtcttatg tcaggcccat ggccttactc ctgagcaagt 50

<210> 51

<211> 40

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 51

gagcccatga gcctggcaaa gaaccggaag aagccgttgg 40

<210> 52

<211> 51

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 52

cggcttcttc cggttctttg ccaggctcat gggctcacgc cagagcaggt g 51

<210> 53

<211> 40

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 53

gaggccgtgc gcttgacaga ggacgggaag taacctctgc 40

<210> 54

<211> 18

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 54

tccccacttt cttgtgaa 18

<210> 55

<211> 18

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 55

taaccactcaggacaggg 18

<210> 56

<211> 19

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 56

cactttcttg tgaatcctt 19

<210> 57

<211> 18

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 57

tcacacagca agtcagca 18

<210> 58

<211> 18

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 58

tagcggagca ggctcgga 18

<210> 59

<211> 19

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 59

tgggctagcg gagcaggct 19

<210> 60

<211> 18

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 60

tacccagacg agaaagct 18

<210> 61

<211> 18

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 61

tcagactgcc aagcttga 18

<210> 62

<211> 19

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 62

acccagacga gaaagctga 19

<210> 63

<211> 18

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 63

tcttgtggct cggggagta 18

<210> 64

<211> 18

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 64

tattgtcagc agagctga 18

<210> 65

<211> 19

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 65

agagggcatc ttgtggctc 19

<210> 66

<211> 18

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 66

ttgagatttt cagatgtc 18

<210> 67

<211> 18

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 67

tatacagtca tatcaagc 18

<210> 68

<211> 19

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 68

atcaagctct cttggcggt 19

<210> 69

<211> 18

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 69

ttcagataga ttatatct 18

<210> 70

<211> 18

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 70

tgccagatac ataggtgg 18

<210> 71

<211> 19

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 71

gcttcagata gattatatc 19

<210> 72

<211> 18

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 72

ttatactgtc tatatgat 18

<210> 73

<211> 18

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 73

tcagctcttc tggccaga 18

<210> 74

<211> 19

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 74

acggatgtct cagctcttc 19

<210> 75

<211> 18

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 75

tggccagaag agctgaga 18

<210> 76

<211> 18

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 76

ttaccgggga gagtttct 18

<210> 77

<211> 19

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 77

ccggggagag tttcttgta 19

<210> 78

<211> 18

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 78

tttgcagaga gatgagtc 18

<210> 79

<211> 18

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 79

ttagcagaag ataagatt 18

<210> 80

<211> 19

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 80

gaaatcttat cttctgcta 19

<210> 81

<211> 18

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 81

tataagacta aactaccc 18

<210> 82

<211> 18

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 82

tcgtctgcca ccacagat 18

<210> 83

<211> 19

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 83

aatgcatgac attcatctg 19

<210> 84

<211> 18

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 84

taaaacagtt tgcattca 18

<210> 85

<211> 18

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 85

tataaagtcc tagaatgt 18

<210> 86

<211> 19

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 86

aacagtttgc attcatgga 19

<210> 87

<211> 18

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 87

tggccatctc tgacctgt 18

<210> 88

<211> 18

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 88

tagtgagccc agaagggg 18

<210> 89

<211> 19

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 89

ccagaagggg acagtaaga 19

<210> 90

<211> 18

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 90

taggtacctg gctgtcgt 18

<210> 91

<211> 18

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 91

tgaccgtcct ggctttta 18

<210> 92

<211> 19

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 92

ctgacaatcg ataggtacc 19

<210> 93

<211> 18

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 93

tgtcatggtc atctgcta 18

<210> 94

<211> 18

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 94

tcgacaccga agcagagt 18

<210> 95

<211> 19

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 95

acaccgaagc agagttttt 19

<210> 96

<211> 18

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 96

tgcccccgcg aggccaca 18

<210> 97

<211> 18

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 97

tctggaagttgaacaccc 18

<210> 98

<211> 19

<212> DNA

<213> Artificial

<220>

<223> Targeting sequence

<400> 98

ggaagttgaa cacccttgc 19

<210> 99

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 99

ctactgtcat tcagggcaat acccagacga gaaagctgag ggtataacag gtttcaagct 60

tggcagtctg actacagagg ccactggctt 90

<210> 100

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 100

ctactgtcat tcagcccaat accctaacga gaaagctgag ggtataacag gtttcaagct 60

tggcagtctg actacagagg ccactggctt 90

<210> 101

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 101

ctactgtcat tcagcccaat acccagacga gaaaagtgag ggtataacag gtttcaagct 60

tggcagtctg actacagagg ccactggctt 90

<210> 102

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 102

ctactgtcat tcagcccaat acccagacga gaaagctgag ggtataacag gtttcaagct 60

tggcagtctg actacagagg ccactggctt 90

<210> 103

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 103

ctactgtcat tcagcccaat acccagacga gaaagctgag ggtataacag gtttgtagct 60

tggcagtctg actacagagg ccactggctt 90

<210> 104

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 104

ctactgtcat tcagcccaat acccagacga gaaagctgag ggtataacag gtttcaagct 60

tggctctctg actacagagg ccactggctt 90

<210> 105

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 105

ctactgtcat tcagcccaat acccagacga gaaagctgag ggtataacag gtttcaagct 60

tggcagtctg actagtgagg ccactggctt 90

<210> 106

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 106

cactttatat ttccctgctt aaacagtccc ccgagggtgg gtgcggaaaa ggctctacac 60

ttgttatcat tccctctcca ccacaggcat 90

<210> 107

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 107

tttgtatttg ggttttttta aaacctccac tctacagtta agaattctaa ggcacagagc 60

ttcaataatt tggtcagagc caagtagcag 90

<210> 108

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 108

ggaggttaaa cccagcagca tgactgcagt tcttaatcaa tgccccttga attgcacata 60

tgggatgaac tagaacattt tctcgatgat 90

<210> 109

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 109

ctcgatgatt cgctgtcctt gttatgatta tgttactgag ctctactgta gcacagacat 60

atgtccctat atggggcggg ggtggggggtg 90

<210> 110

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 110

ggtgtcttga tcgctgggct atttctatac tgttctggct tttcggaagc agtcatttct 60

ttctattctc caagcaccag caattagctt 90

<210> 111

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 111

gcttctagtt tgctgaaact aatctgctat agacagagac tccgacgaac caattttatt 60

aggatttgat caaataaact ctctctgaca 90

<210> 112

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 112

gaaagagtaa ctaagagttt gatgtttact gagtgcatag tatgcactag atgctggccg 60

tggatgcctc atagaatcct cccaacaact 90

<210> 113

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 113

gctagatgct ggccgtggat gcctcataga atcctcccaa caaccgatga aatgactact 60

gtcattcagc ccaataccca gacgagaaag 90

<210> 114

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 114

acaggtttca agcttggcag tctgactaca gaggccactg gctttacccc tgggttagtc 60

tgcctctgta ggattggggg cacgtaattt 90

<210> 115

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 115

ttagtctgcc tctgtaggat tggggggcacg taattttgct gtttaaggtc tcatttgcct 60

tcttagagat cacaagccaa agctttttat 90

<210> 116

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 116

ggaagcccag agggcatctt gtggctcggg agtagctctc tgctaccttc tcagctctgc 60

tgacaatact tgagattttc agatgtcacc 90

<210> 117

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 117

tcagctctgc tgacaatact tgagattttc agatgtcacc aaccagcaag agagcttgat 60

atgactgtat atagtatagt cataaagaac 90

<210> 118

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 118

cataaagaac ctgaacttga ccatatactt atgtcatgtg gaaatcttct catagcttca 60

gatagattat atctggagtg aagaatcctg 90

<210> 119

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 119

gtggaaaatt tctcatagct tcagatagat tatatctgga gtgagcaatc ctgccaccta 60

tgtatctggc atagtgtgag tcctcataaa 90

<210> 120

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 120

ggtttgaagg gcaacaaaat agtgaacaga gtgaaaatcc ccacctagat cctgggtcca 60

gaaaaagatg ggaaacctgt ttagctcacc 90

<210> 121

<211> 30

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 121

ggccactagg gacaaaattg gtgacagaaa 30

<210> 122

<211> 50

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 122

cccacagtgg ggccactagg gacaaaattg gtgacagaaa agccccatcc 50

<210> 123

<211> 70

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 123

tcccctccac cccacagtgg ggccactagg gacaaaattg gtgacagaaa agccccatcc 60

ttaggcctcc 70

<210> 124

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 124

cttttatctg tcccctccac cccacagtgg ggccactagg gacaaaattg gtgacagaaa 60

agccccatcc ttaggcctcc tccttcctag 90

<210> 125

<211> 110

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 125

gttctgggta cttttatctg tcccctccac cccacagtgg ggccactagg gacaaaattg 60

gtgacagaaa agccccatcc ttaggcctcc tccttcctag tctcctgata 110

<210> 126

<211> 30

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 126

tttctgtcac caatggtgtc cctagtggcc 30

<210> 127

<211> 50

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 127

ggatggggct tttctgtcac caatggtgtc cctagtggcc ccactgtggg 50

<210> 128

<211> 70

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 128

ggaggcctaa ggatggggct tttctgtcac caatggtgtc cctagtggcc ccactgtggg 60

gtggagggga 70

<210> 129

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 129

ctaggaagga ggaggcctaa ggatggggct tttctgtcac caatggtgtc cctagtggcc 60

ccactgtggg gtggagggga cagataaaag 90

<210> 130

<211> 110

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 130

tatcaggaga ctaggaagga ggaggcctaa ggatggggct tttctgtcac caatggtgtc 60

cctagtggcc ccactgtggg gtggagggga cagataaaag tacccagaac 110

<210> 131

<211> 70

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 131

ttctagtaac cactcaggac aggggggttc agcccaaaaa ttcacaagaa agtggggacc 60

catgggaaat 70

<210> 132

<211> 70

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 132

cagcaagtca gcagcacagc gtgtgtgact ccgagggtgc tccgctagcc cacattgccc 60

tctggggggtg 70

<210> 133

<211> 70

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 133

gtcagactgc caagcttgaa acctgtctta ccctctactt tctcgtctgg gtattgggct 60

gaatgacagt 70

<210> 134

<211> 70

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 134

cagagctgag aagacagcag agagctactc ccgaagcaca agatgccctc tgggcttccg 60

tgaccttggc 70

<210> 135

<211> 70

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 135

ctgacaatac ttgagatttt cagatgtcac caacgaccaa gagagcttga tatgactgta 60

tatagtatag 70

<210> 136

<211> 70

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 136

cagatacata ggtggcagga ttcttcactc cagacttaat ctatctgaag ctatgagaaa 60

ttttccacat 70

<210> 137

<211> 70

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 137

tatatgattg atttgcacag ctcatctggc cagataagct gagacatccg ttcccctaca 60

agaaactctc 70

<210> 138

<211> 70

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 138

atctggccag aagagctgag acatccgttc cccttgaaga aactctcccc ggtaagtaac 60

ctctcagctg 70

<210> 139

<211> 70

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 139

aggcatctca ctggagaggg tttagttctc cttaagagaa gataagattt caagagggaa 60

gctaagactc 70

<210> 140

<211> 70

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 140

ataatataat aaaaaatgtt tcgtctgcca ccactaatga atgtcatgca ttctgggtag 60

tttagtctta 70

<210> 141

<211> 70

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 141

tttataaagt cctagaatgt atttagttgc cctcgttgaa tgcaaactgt tttatacatc 60

aataggtttt 70

<210> 142

<211> 70

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 142

gctcaacctg gccatctctg acctgttttt ccttcccact gtccccttct gggctcacta 60

tgctgccgcc 70

<210> 143

<211> 70

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 143

ttttaaagca aacacagcat ggacgacagc caggctccta tcgattgtca ggaggatgat 60

gaagaagatt 70

<210> 144

<211> 70

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 144

gcttgtcatg gtcatctgct actcgggaat cctaattact ctgcttcggt gtcgaaatga 60

gaagaagagg 70

<210> 145

<211> 70

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 145

atactgcccc cgcgaggcca cattggcaaa ccagcttggg tgttcaactt ccagacttgg 60

ccatggagaa 70

<210> 146

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 146

ctgaagaatt tcccatgggt ccccactttc ttgtgaatcc ttggagtgaa cccccctgtc 60

ctgagtggtt actagaacac acctctggac 90

<210> 147

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 147

tggaagtatc ttgccgaggt cacacagcaa gtcagcagca cagccagtgt gactccgagc 60

ctgctccgct agcccacatt gccctctggg 90

<210> 148

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 148

ctactgtcat tcagcccaat acccagacga gaaagctgag ggtataacag gtttcaagct 60

tggcagtctg actacagagg ccactggctt 90

<210> 149

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 149

ggaagcccag agggcatctt gtggctcggg agtagctctc tgctaccttc tcagctctgc 60

tgacaatact tgagattttc agatgtcacc 90

<210> 150

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 150

tcagctctgc tgacaatact tgagattttc agatgtcacc aacgcccaag agagcttgat 60

atgactgtat atagtatagt cataaagaac 90

<210> 151

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 151

gtggaaaatt tctcatagct tcagatagat tatatctgga gtgagcaatc ctgccaccta 60

tgtatctggc atagtgtgag tcctcataaa 90

<210> 152

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 152

gaaacagcat ttcctacttt tatactgtct atatgattga tttggtcagc tcatctggcc 60

agaagagctg agacatccgt tcccctacaa 90

<210> 153

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 153

ttgatttgca cagctcatct ggccagaaga gctgagacat ccgtatccct acaagaaact 60

ctccccggta agtaacctct cagctgcttg 90

<210> 154

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 154

ggagagggtt tagttctcct tagcagaaga taagatttca agatgagagc taagactcat 60

ctctctgcaa atctttcttt tgagaggtaa 90

<210> 155

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 155

taatataata aaaaatgttt cgtctgccac cacagatgaa tgtcgagcat tctgggtagt 60

ttagtcttat aaccagctgt cttgcctagt 90

<210> 156

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 156

ttaaaaacct attgatgtat aaaacagttt gcattcatgg agggtgacta aatacattct 60

aggactttat aaaagatcac tttttattta 90

<210> 157

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 157

gacatctacc tgctcaacct ggccatctct gacctgtttt tcctatttac tgtccccttc 60

tgggctcact atgctgccgc ccagtgggac 90

<210> 158

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 158

tcatcctcct gacaatcgat aggtacctgg ctgtcgtcca tgctacgttt gctttaaaag 60

ccaggacggt cacctttggg gtggtgacaa 90

<210> 159

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 159

ggctggtcct gccgctgctt gtcatggtca tctgctactc gggagaccta aaaactctgc 60

ttcggtgtcg aaatgagaag aagaggcaca 90

<210> 160

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Target sequence

<400> 160

ggcaagcctt gggtcatact gcccccgcga ggccacattg gcaagtcagc aagggtgttc 60

aacttccaga cttggccatg gagaagacat 90

<210> 161

<211> 59

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 161

acactctttc cctacacgac gctcttccga tctcgtgatt ttgcagtgtg cgttactcc 59

<210> 162

<211> 59

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 162

acactctttc cctacacgac gctcttccga tctacatcgt ttgcagtgtg cgttactcc 59

<210> 163

<211> 59

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 163

acactctttc cctacacgac gctcttccga tctgcctaat ttgcagtgtg cgttactcc 59

<210> 164

<211> 59

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 164

acactctttc cctacacgac gctcttccga tcttggtcat ttgcagtgtg cgttactcc 59

<210> 165

<211> 54

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 165

ctcggcattc ctgctgaacc gctcttccga tctccaagca actaagtcac agca 54

<210> 166

<211> 58

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 166

acactctttc cctacacgac gctcttccga tctcgtgata tgaggaaatg gaagcttg 58

<210> 167

<211> 58

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 167

acactctttc cctacacgac gctcttccga tctacatcga tgaggaaatg gaagcttg 58

<210> 168

<211> 58

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 168

acactctttc cctacacgac gctcttccga tctgcctaaa tgaggaaatg gaagcttg 58

<210> 169

<211> 58

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 169

acactctttc cctacacgac gctcttccga tcttggtcaa tgaggaaatg gaagcttg 58

<210> 170

<211> 51

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 170

ctcggcattc ctgctgaacc gctcttccga tctcattagg gtattggagg a 51

<210> 171

<211> 58

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 171

acactctttc cctacacgac gctcttccga tctcgtgata atcctcccaa caactcat 58

<210> 172

<211> 58

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 172

acactctttc cctacacgac gctcttccga tctacatcga atcctcccaa caactcat 58

<210> 173

<211> 58

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 173

acactctttc cctacacgac gctcttccga tctgcctaaa atcctcccaa caactcat 58

<210> 174

<211> 58

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 174

acactctttc cctacacgac gctcttccga tcttggtcaa atcctcccaa caactcat 58

<210> 175

<211> 52

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 175

ctcggcattc ctgctgaacc gctcttccga tctcccaatc ctacagaggc ag 52

<210> 176

<211> 58

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 176

acactctttc cctacacgac gctcttccga tctcgtgata agccaaagct ttttattc 58

<210> 177

<211> 58

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 177

acactctttc cctacacgac gctcttccga tctacatcga agccaaagct ttttattc 58

<210> 178

<211> 58

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 178

acactctttc cctacacgac gctcttccga tctgcctaaa agccaaagct ttttattc 58

<210> 179

<211> 58

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 179

acactctttc cctacacgac gctcttccga tcttggtcaa agccaaagct ttttattc 58

<210> 180

<211> 53

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 180

acactctttc cctacacgac gctcttccga tctaagccaa agctttttat tct 53

<210> 181

<211> 59

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 181

acactctttc cctacacgac gctcttccga tctcgtgata tcttgtggct cgggagtag 59

<210> 182

<211> 59

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 182

acactctttc cctacacgac gctcttccga tctacatcga tcttgtggct cgggagtag 59

<210> 183

<211> 53

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 183

ctcggcattc ctgctgaacc gctcttccga tcttggcagg attcttcact cca 53

<210> 184

<211> 59

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 184

acactctttc cctacacgac gctcttccga tctcgtgatc tattttgttg cccttcaaa 59

<210> 185

<211> 59

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 185

acactctttc cctacacgac gctcttccga tctacatcgc tattttgttg cccttcaaa 59

<210> 186

<211> 55

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 186

ctcggcattc ctgctgaacc gctcttccga tctaacctga acttgaccat atact 55

<210> 187

<211> 59

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 187

acactctttc cctacacgac gctcttccga tctcgtgatc agctgagagg ttacttacc 59

<210> 188

<211> 59

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 188

acactctttc cctacacgac gctcttccga tctacatcgc agctgagagg ttacttacc 59

<210> 189

<211> 53

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 189

ctcggcattc ctgctgaacc gctcttccga tctaatgatt taactccacc ctc 53

<210> 190

<211> 60

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 190

acactctttc cctacacgac gctcttccga tctcgtgata ctccaccctc cttcaaaaga 60

<210> 191

<211> 60

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 191

acactctttc cctacacgac gctcttccga tctacatcga ctccaccctc cttcaaaaga 60

<210> 192

<211> 52

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 192

ctcggcattc ctgctgaacc gctcttccga tcttggtgtt tgccaaatgt ct 52

<210> 193

<211> 59

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 193

acactctttc cctacacgac gctcttccga tctcgtgatg ggcacatatt cagaaggca 59

<210> 194

<211> 59

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 194

acactctttc cctacacgac gctcttccga tctacatcgg ggcacatatt cagaaggca 59

<210> 195

<211> 56

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 195

ctcggcattc ctgctgaacc gctcttccga tctagtgaaa gactttaaag ggagca 56

<210> 196

<211> 59

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 196

acactctttc cctacacgac gctcttccga tctcgtgatc acaattaaga gttgtcata 59

<210> 197

<211> 59

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 197

acactctttc cctacacgac gctcttccga tctacatcgc acaattaaga gttgtcata 59

<210> 198

<211> 53

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 198

ctcggcattc ctgctgaacc gctcttccga tctctcagct agagcagctg aac 53

<210> 199

<211> 51

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 199

ctcggcattc ctgctgaacc gctcttccga tctgacactt gataatccat c 51

<210> 200

<211> 60

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 200

acactctttc cctacacgac gctcttccga tctacatcgt caatgtagac atctatgtag 60

<210> 201

<211> 60

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 201

acactctttc cctacacgac gctcttccga tctcgtgatt caatgtagac atctatgtag 60

<210> 202

<211> 59

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 202

acactctttc cctacacgac gctcttccga tctcgtgata ctgcaaaagg ctgaagagc 59

<210> 203

<211> 59

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 203

acactctttc cctacacgac gctcttccga tctacatcga ctgcaaaagg ctgaagagc 59

<210> 204

<211> 59

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 204

acactctttc cctacacgac gctcttccga tctgcctaaa ctgcaaaagg ctgaagagc 59

<210> 205

<211> 59

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 205

acactctttc cctacacgac gctcttccga tcttggtcaa ctgcaaaagg ctgaagagc 59

<210> 206

<211> 57

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 206

ctcggcattc ctgctgaacc gctcttccga tctgcctata aaatagagcc ctgtcaa 57

<210> 207

<211> 60

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 207

acactctttc cctacacgac gctcttccga tctcgtgatc tctattttat aggcttcttc 60

<210> 208

<211> 60

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 208

acactctttc cctacacgac gctcttccga tctacatcgc tctattttat aggcttcttc 60

<210> 209

<211> 52

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 209

ctcggcattc ctgctgaacc gctcttccga tctagccacc acccaagtga tc 52

<210> 210

<211> 60

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 210

acactctttc cctacacgac gctcttccga tctacatcgt tccagacatt aaagatagtc 60

<210> 211

<211> 60

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 211

acactctttc cctacacgac gctcttccga tctcgtgatt tccagacatt aaagatagtc 60

<210> 212

<211> 54

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 212

ctcggcattc ctgctgaacc gctcttccga tctaatcatg atggtgaaga taag 54

<210> 213

<211> 60

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 213

acactctttc cctacacgac gctcttccga tctcgtgatc cggcagagac aaacattaaa 60

<210> 214

<211> 54

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 214

acactctttc cctacacgac gctcttccga tctccggcag agacaaacat taaa 54

<210> 215

<211> 53

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 215

ctcggcattc ctgctgaacc gctcttccga tctagctagg aagccatggc aag 53

<210> 216

<211> 47

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 216

aatgatacgg cgaccaccga gatctacact ctttccctac acgacgc 47

<210> 217

<211> 50

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 217

caagcagaag acggcatacg agatcggtct cggcattcct gctgaaccgc 50

<210> 218

<211> 58

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 218

acactctttc cctacacgac gctcttccga tctagtgcat agtatgtgct agatgctg 58

<210> 219

<211> 59

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 219

gtgactggag ttcagacgtg tgctcttccg atcttgatct ctaagaaggc aaatgagac 59

<210> 220

<211> 64

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<220>

<221> misc_feature

<222> (25)..(30)

<223> n is a, c, g, or t

<400> 220

caagcagaag acggcatacg agatnnnnnn gtgactggag ttcagacgtg tgctcttccg 60

atct 64

<210> 221

<211> 58

<212> DNA

<213> Artificial

<220>

<223>PCR primer

<400> 221

aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatct 58

<210> 222

<211> 34

<212>PRT

<213> Artificial

<220>

<223> Deduced TALE template amino acid sequence

<220>

<221> misc_feature

<222> (12)..(13)

<223> Xaa can be any naturally occurring amino acid

<400> 222

Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Xaa Xaa Gly Gly Lys

1 5 10 15

Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala

20 25 30

His Gly

<210> 223

<211> 246

<212> DNA

<213> Artificial

<220>

<223> TALE dimer nucleotide sequence

<400> 223

cgattactgc ctgtcttatg tcaggcccat ggccttactc ctgagcaagt cgtagctatc 60

gccagcaaca taggtgggaa acaggccctg gaaaccgtac aacgtctcct cccagtactt 120

tgtcaagcac acgggttgac accggaacaa gtggtggcga ttgcgtccca cgatggaggc 180

aagcaggcac tggagaccgt ccaacggctt cttccggttc tttgccaggc tcatgggctc 240

acgcca 246

<210> 224

<211> 82

<212>PRT

<213> Artificial

<220>

<223> Deduced TALE dimer amino acid sequence

<400> 224

Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln

1 5 10 15

Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr

20 25 30

Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro

35 40 45

Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu

50 55 60

Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu

65 70 75 80

Thr Pro

<---

Claims (23)

1. A method for changing target DNA in a cell in vitro, including providing the cell with the Cas enzyme of the CRISPR type II system, which forms a colocalization complex with guide RNA complementary to the target DNA, and which cleaves the target DNA in a site-specific manner, introducing into the cell a guide RNA that is complementary to the target DNA and that directs the CRISPR type II Cas enzyme to the target DNA, where the guide RNA and the CRISPR type II Cas enzyme are members of a colocalization complex for the target DNA, introduction into a cell of a donor nucleic acid sequence, where the guide RNA and the donor nucleic acid sequence are linked, wherein the guide RNA and the CRISPR Type II Cas enzyme colocalize on the target DNA, and the CRISPR Type II Cas enzyme cleaves the target DNA and a donor nucleic acid sequence is incorporated into the target DNA to produce altered DNA in the cell, and where the cell is not a human germline cell or an embryonic cell. 2. The method of claim 1, wherein the guide RNA contains from about 10 to about 500 nucleotides. 3. The method of claim 1, wherein the guide RNA contains from about 20 to about 100 nucleotides. 4. The method of claim 1, wherein the guide RNA contains approximately 100 nucleotides. 5. The method of claim 1, wherein the guide RNA is a crRNA-tracrRNA fusion. 6. The method according to claim 1, wherein the donor nucleic acid sequence is inserted through homologous recombination. 7. The method according to claim 1, wherein the donor nucleic acid sequence is inserted through non-homologous end joining. 8. The method of claim 1, wherein the guide RNA is inserted into the cell as the first foreign nucleic acid that is expressed in the cell. 9. The method of claim 1 further comprising introducing a plurality of donor nucleic acids and a plurality of guide RNAs to produce a plurality of DNA changes in the cell. 10. The method according to claim 1, wherein the Cas enzyme of the type II CRISPR system is a Cas 9 enzyme. 11. The method according to claim 1, wherein the Cas enzyme of the CRISPR type II system is a Cas 9 enzyme from S. pyogenes. 12. The method of claim 1, wherein the target DNA is genomic DNA, mitochondrial DNA, viral DNA or exogenous DNA. 13. The method of claim 1, wherein the cell is a eukaryotic cell. 14. The method of claim 1, wherein the cell is a yeast cell, a plant cell or an animal cell. 15. The method of claim 1, wherein the cell is a human cell. 16. The method according to claim 1, where the cell is a stem cell. 17. The method according to claim 1, where the cell is a human stem cell. 18. The method according to claim 1, where the cell is a human induced pluripotent stem cell.