COMPOSITIONS AND METHODS FOR IMPROVED DETECTION OF GENOMIC EDITING EVENTS

Abstract

This invention pertains to compositions and methods for detecting single and/or multiple nucleotide mismatches on DNA fragments in vitro by enzymatic cleavage using a mixture of mismatch endonucleases. Additionally, this invention pertains to the ability to detect successful genome editing events by programmable nucleases, e.g., TALENS, RGENs, or ZFNs.

Claims

1. A method for detecting genomic editing events comprising: a) obtaining an edited genomic sequences; b) PCR amplifying the edited genomic sequences around the expected edited region to generate a plurality of PCR amplicons; c) reacting the plurality of PCR amplicons with a mixture of mismatch endonucleases to generate a plurality of cleaved fragments; d) detecting the cleaved fragments indicating a genomic editing event.

2. The method of claim 1 wherein the edited genomic sequence is edited with a programmable nuclease.

3. The method of claim 2 wherein the programmable nuclease is a TALEN, a RGEN, or a ZFN.

4. The method of claim 3 wherein the RGEN is a Cas9 enzyme or a Cpf1 enzyme.

5. The method of claim 1 wherein the mixture of mismatch endonucleases comprises T7EI and T4E7.

6. An enzyme composition for detecting edited genomic DNA comprising a mixture of mismatch endonucleases.

7. The composition of claim 6 wherein the mixture of mismatch endonucleases comprises T7E1 and T4E7.

8. The composition of claim 7 wherein the mixture of mismatch endonucleases comprises 0.38 pmol T7EI and 0.19 pmol T4E7.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0015] FIG. 1 is a diagram showing mismatch cleavage capabilities of the T7E1 and T4E7 endonucleases in isolation. The figure shows the cleavage of 1, 3 or 12 base pair insertions.

[0016] FIG. 2 shows the blend of T7E1 and T4E7 endonucleases and the capability of blend to detect mismatches of various sizes.

[0017] FIG. 3 shows a blend of T7E1 and T4E7 endonucleases and the capability to detect mismatches at CRISPR edited genomic sites. For each grouping of bars at each target site the left hand bar shows T7(1:10) vs the right hand bars that shows T7(1:10)+T4 (1:3).

DETAILED DESCRIPTION OF THE INVENTION

[0018] The methods and compositions of the invention described herein provide enzyme compositions and methods for detecting single and/or multiple nucleotide mismatches introduced by genomic editing events with programmable nucleases. These and other advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.

[0019] This invention pertains to the ability to detect single and/or multiple nucleotide mismatches on DNA fragments in vitro by enzymatic cleavage using a mixture of mismatch endonucleases. In some embodiments the mixture of mismatch endonucleases includes phage T7 endonuclease I and T4 endonuclease VII. This cleavage system discriminates DNA mismatches in both short (1 to 5) and long (6 or more) heteroduplexed double-stranded DNA (dsDNA) molecules. Though this invention is broadly applicable to the detection of mismatches present in any DNA heteroduplex sample, it offers improvement in the ability to detect successful genome editing events by targeted genomic editing nucleases, e.g., TALENS, RGENs, or ZFNs.

[0020] The invention is targeted to mismatch discrimination detection for CRISPR/Cas9 genome editing but is broadly applicable to any use of this enzyme blend to cleave any DNA source containing mismatched nucleotides, such as assays intended to detect naturally occurring mutations as well as experimentally induced mutations.

[0021] In one embodiment the invention is targeted to mismatch discrimination detection for programmable nuclease genome editing. In another embodiment the invention is targeted to mismatch discrimination for TALENs, ZFNs, or RGENs. In an additional embodiment the invention is targeted to mismatch discrimination detection for RGEN genome editing. In a further embodiment the invention is targeted to mismatch discrimination detection for Cas9 or Cpf1 genome editing. In a further embodiment the invention is applicable to the use of this enzyme blend to cleave any DNA source containing mismatched nucleotides, such as assays intended to detect naturally occurring mutations as well as experimentally induced mutations.

[0022] In one embodiment a double strand break (DSB) is introduced with a programmable nuclease system. The DSB is repaired through NHEJ or HDR events. Following NHEJ or HDR repair the repaired genome is optionally PCR amplified with primers spanning the predicted cut sites. The genomic editing events are then detected with a combination of mismatch endonucleases. In an additional embodiment the proposed method and compositions involve the use of a blend of at least two endonucleases. In one embodiment a mixture of T7EI and T4 Endonuclease VII (T4E7) is used to more efficiently cleave heteroduplexed DNA mismatches than can be resolved using a single enzyme. The mixture of T7E1 and T4E7 are able to cleave more types or repair fragments than either enzyme alone. The ability of the two enzymes to complement each other in function. Without being bound to any singular theory it is postulated that the T7EI in the mixture serves to cleave the majority of heteroduplexes containing 2 or more adjacent mismatches while the T4E7 serves to cleave single nucleotide mismatches. The basic procedure and premise is detailed in FIG. 1.

[0023] In FIG. 1, part a) shows a 1:1 mixture of 1 KB WT dsDNA and an otherwise identical 1 KB dsDNA strand that has a centered insertion/deletion of 12, 3, or 1 base(s) is prepared for hybridization. In part b) the samples are denatured at 95 degrees Celsius and heteroduplexes are formed by repeated heating and cooling to generate various mismatch dsDNA fragments. In isolation, T7E1 cleaves heteroduplexes with long and short mismatch bubbles (2-12) bases efficiently, but does not cleave single base mismatch bubbles. T4E7 cleaves large mismatch bubbles efficiently (12 bases), does not cleave short mismatch bubbles (2-3 bases), and is capable of cleaving single base mismatch bubbles with low efficiency.

[0024] Both enzymes are compatible in a simple nuclease cleavage reaction buffer (heteroduplex or HD Buffer) containing 10 mM Tris-HCl (pH 7.9), 50 mM NaCl, 10 mM MgCl2, and 1 mM DTT, and are highly active at 37 degrees Celsius.

[0025] In another embodiment the enzyme combination performs well on unpurified PCR heteroduplex products and works in PCR reaction buffer.

Example 1

[0026] Utilization of the T4/T7 Nuclease Blend to Cleave Heteroduplexes Containing 1, 2, 3, and 12 Nucleotide Mismatches.

[0027] The following example demonstrates the ability of the method of the invention to efficiently cleave mismatched heteroduplexes PCR products more efficiently than traditional approaches using single enzyme protocols. PCR assays were designed to amplify a 1 kb fragment of the Human HPRT1 gene. HPRT gBlocks Gene Fragments were designed to contain 0(WT), 1, 2, 3, or 12 base deletions positioned at around base 300 of these amplicons, such that mismatch heteroduplex cleavage assays would yield fragments of 300 and 700 bp, sizes that are readily distinguished. The HPRT gBlocks gene fragments were amplified using the HPRT Forward primer and HPRT Rev Primer. Primers and DNA fragments are shown in Table 1 (SEQ IDs #1-7). These synthetic gBlocks gene fragments represent hypothetical repair fragments following genomic editing events using programmable nucleases.

TABLE-US-00001 TABLE1 SequenceofoligonucleotidesandgBlocksusedtomakeheteroduplexes. SEQID Name Sequence NO: HPRT GGTTCCAGGTGATCAACCAA SEQID Forward NO:1 primer HPRTRev GTTCCAGTTCTAAGGACGTCTG SEQID primer NO:2 HPRTWT GAATGTTGTGATAAAAGGTGATGCTCACCTCTCCCACACCCTTTTATAGTTTAGGGA SEQID gBlock TTGTATTTCCAAGGTTTCTAGACTGAGAGCCCTTTTCATCTTTGCTCATTGACACTC NO:3 TGTACCCATTAATCCTCCTTATTAGCTCCCCTTCAATGGACACATGGGTAGTCAGGG TGCAGGTCTCAGAACTGTCCTTCAGGTTCCAGGTGATCAACCAAGTGCCTTGTCTGT AGTGTCAACTCATTGCTGCCCCTTCCTAGTAATCCCCATAATTTAGCTCTCCATTTC ATAGTCTTTCCTTGGGTGTGTTAAAAGTGACCATGGTACACTCAGCACGGATGAAAT GAAACAGTGTTTAGAAACGTCAGTCTTCTCTTTTGTAATGCCCTGTAGTCTCTCTGT ATGTTATATGTCACATTTTGTAATTAACAGCTTGCTGGTGAAAAGGACCCCACGAAG TGTTGGATATAAGCCAGACTGTAAGTGAATTACTTTTTTTGTCAATCATTTAACCAT CTTTAACCTAAAAGAGTTTTATGTGAAATGGCTTATAATTGCTTAGAGAATATTTGT AGAGAGGCACATTTGCCAGTATTAGATTTAAAAGTGATGTTTTCTTTATCTAAATGA TGAATTATGATTCTTTTTAGTTGTTGGATTTGAAATTCCAGACAAGTTTGTTGTAGG ATATGCCCTTGACTATAATGAATACTTCAGGGATTTGAATGTAAGTAATTGCTTCTT TTTCTCACTCATTTTTCAAAACACGCATAAAAATTTAGGAAAGAGAATTGTTTTCTC CTTCCAGCACCTCATAATTTGAACAGACTGATGGTTCCCATTAGTCACATAAAGCTG TAGTCTAGTACAGACGTCCTTAGAACTGGAACCTGGCCAGGCTAGGGTGACACTTCT TGTTGGCTGAAATAGTTGAACAGCTT HPRT GAATGTTGTGATAAAAGGTGATGCTCACCTCTCCCACACCCTTTTATAGTTTAGGGA SEQID 1DEL TTGTATTTCCAAGGTTTCTAGACTGAGAGCCCTTTTCATCTTTGCTCATTGACACTC NO:4 gBlock TGTACCCATTAATCCTCCTTATTAGCTCCCCTTCAATGGACACATGGGTAGTCAGGG TGCAGGTCTCAGAACTGTCCTTCAGGTTCCAGGTGATCAACCAAGTGCCTTGTCTGT AGTGTCAACTCATTGCTGCCCCTTCCTAGTAATCCCCATAATTTAGCTCTCCATTTC ATAGTCTTTCCTTGGGTGTGTTAAAAGTGACCATGGTACACTCAGCACGGATGAAAT GAAACAGTGTTTAGAAACGTCAGTCTTCTCTTTTGTAATGCCCTGTAGTCTCTCTGT ATGTTATATGTCACATTTTGTAATTAACAGCTTGCTGGTGAAAAGGACCCCAGAAGT TGTTGGATAAAGCCAGACTGTAAGTGAATTACTTTTTTTGTCAATCATTTAACCATC TTTAACCTAAAAGAGTTTTATGTGAAATGGCTTATAATTGCTTAGAGAATATTTGTA GAGAGGCACATTTGCCAGTATTAGATTTAAAAGTGATGTTTTCTTTATCTAAATGAT GAATTATGATTCTTTTTAGTTGTTGGATTTGAAATTCCAGACAAGTTTGTTGTAGGA TATGCCCTTGACTATAATGAATACTTCAGGGATTTGAATGTAAGTAATTGCTTCTTT TTCTCACTCATTTTTCAAAACACGCATAAAAATTTAGGAAAGAGAATTGTTTTCTCC TTCCAGCACCTCATAATTTGAACAGACTGATGGTTCCCATTAGTCACATAAAGCTGT AGTCTAGTACAGACGTCCTTAGAACTGGAACCTGGCCAGGCTAGGGTGACACTTCTT GTTGGCTGAAATAGTTGAACAGCTT HPRT GAATGTTGTGATAAAAGGTGATGCTCACCTCTCCCACACCCTTTTATAGTTTAGGGAT SEQTD 2DEL TGTATTTCCAAGGTTTCTAGACTGAGAGCCCTTTTCATCTTTGCTCATTGACACTCTG NO:5 gBlock TACCCATTAATCCTCCTTATTAGCTCCCCTTCAATGGACACATGGGTAGTCAGGGTGC AGGTCTCAGAACTGTCCTTCAGGTTCCAGGTGATCAACCAAGTGCCTTGTCTGTAGTG TCAACTCATTGCTGCCCCTTCCTAGTAATCCCCATAATTTAGCTCTCCATTTCATAGT CTTTCCTTGGGTGTGTTAAAAGTGACCATGGTACACTCAGCACGGATGAAATGAAACA GTGTTTAGAAACGTCAGTCTTCTCTTTTGTAATGCCCTGTAGTCTCTCTGTATGTTAT ATGTCACATTTTGTAATTAACAGCTTGCTGGTGAAAAGGACCCCGAAGTGTTGGATAT AAGCCAGACTGTAAGTGAATTACTTTTTTTGTCAATCATTTAACCATCTTTAACCTAA AAGAGTTTTATGTGAAATGGCTTATAATTGCTTAGAGAATATTTGTAGAGAGGCACAT TTGCCAGTATTAGATTTAAAAGTGATGTTTTCTTTATCTAAATGATGAATTATGATTC TTTTTAGTTGTTGGATTTGAAATTCCAGACAAGTTTGTTGTAGGATATGCCCTTGACT ATAATGAATACTTCAGGGATTTGAATGTAAGTAATTGCTTCTTTTTCTCACTCATTTT TCAAAACACGCATAAAAATTTAGGAAAGAGAATTGTTTTCTCCTTCCAGCACCTCATA ATTTGAACAGACTGATGGTTCCCATTAGTCACATAAAGCTGTAGTCTAGTACAGACGT CCTTAGAACTGGAACCTGGCCAGGCTAGGGTGACACTTCTTGTTGGCTGAAATAGTTG AACAGCTT HPRT GAATGTTGTGATAAAAGGTGATGCTCACCTCTCCCACACCCTTTTATAGTTTAGGGAT SEQID 3DEL TGTATTTCCAAGGTTTCTAGACTGAGAGCCCTTTTCATCTTTGCTCATTGACACTCTG NO:6 gBlock TACCCATTAATCCTCCTTATTAGCTCCCCTTCAATGGACACATGGGTAGTCAGGGTGC AGGTCTCAGAACTGTCCTTCAGGTTCCAGGTGATCAACCAAGTGCCTTGTCTGTAGTG TCAACTCATTGCTGCCCCTTCCTAGTAATCCCCATAATTTAGCTCTCCATTTCATAGT CTTTCCTTGGGTGTGTTAAAAGTGACCATGGTACACTCAGCACGGATGAAATGAAACA GTGTTTAGAAACGTCAGTCTTCTCTTTTGTAATGCCCTGTAGTCTCTCTGTATGTTAT ATGTCACATTTTGTAATTAACAGCTTGCTGGTGAAAAGGACCCGAAGTGTTGGATATA AGCCAGACTGTAAGTGAATTACTTTTTTTGTCAATCATTTAACCATCTTTAACCTAAA AGAGTTTTATGTGAAATGGCTTATAATTGCTTAGAGAATATTTGTAGAGAGGCACATT TGCCAGTATTAGATTTAAAAGTGATGTTTTCTTTATCTAAATGATGAATTATGATTCT TTTTAGTTGTTGGATTTGAAATTCCAGACAAGTTTGTTGTAGGATATGCCCTTGACTA TAATGAATACTTCAGGGATTTGAATGTAAGTAATTGCTTCTTTTTCTCACTCATTTTT CAAAACACGCATAAAAATTTAGGAAAGAGAATTGTTTTCTCCTTCCAGCACCTCATAA TTTGAACAGACTGATGGTTCCCATTAGTCACATAAAGCTGTAGTCTAGTACAGACGTC CTTAGAACTGGAACCTGGCCAGGCTAGGGTGACACTTCTTGTTGGCTGAAATAGTTGA ACAGCTT HPRT GAATGTTGTGATAAAAGGTGATGCTCACCTCTCCCACACCCTTTTATAGTTTAGGGAT SEQID 12DEL TGTATTTCCAAGGTTTCTAGACTGAGAGCCCTTTTCATCTTTGCTCATTGACACTCTG NO:7 gBlock TACCCATTAATCCTCCTTATTAGCTCCCCTTCAATGGACACATGGGTAGTCAGGGTGC AGGTCTCAGAACTGTCCTTCAGGTTCCAGGTGATCAACCAAGTGCCTTGTCTGTAGTG TCAACTCATTGCTGCCCCTTCCTAGTAATCCCCATAATTTAGCTCTCCATTTCATAGT CTTTCCTTGGGTGTGTTAAAAGTGACCATGGTACACTCAGCACGGATGAAATGAAACA GTGTTTAGAAACGTCAGTCTTCTCTTTTGTAATGCCCTGTAGTCTCTCTGTATGTTAT ATGTCACATTTTGTAATTAACAGCTTGCTGGTGAAAAGGACCCCAxxxxxxxxxxxTA TAAGCCAGACTGTAAGTGAATTACTTTTTTTGTCAATCATTTAACCATCTTTAACCTA AAAGAGTTTTATGTGAAATGGCTTATAATTGCTTAGAGAATATTTGTAGAGAGGCACA TTTGCCAGTATTAGATTTAAAAGTGATGTTTTCTTTATCTAAATGATGAATTATGATT CTTTTTAGTTGTTGGATTTGAAATTCCAGACAAGTTTGTTGTAGGATATGCCCTTGAC TATAATGAATACTTCAGGGATTTGAATGTAAGTAATTGCTTCTTTTTCTCACTCATTT TTCAAAACACGCATAAAAATTTAGGAAAGAGAATTGTTTTCTCCTTCCAGCACCTCAT AATTTGAACAGACTGATGGTTCCCATTAGTCACATAAAGCTGTAGTCTAGTACAGACG TCCTTAGAACTGGAACCTGGCCAGGCTAGGGTGACACTTCTTGTTGGCTGAAATAGTT GAACAGCTT

[0028] Following PCR equimolar mixtures (3 pmole each) of each deletion mutant and WT sequence were made in 1HD buffer and the DNA was denatured and hybridized by heating followed by a step down cooling protocol in a thermocycler to generate heteroduplex fragments. The WT sequence and each deletion mutant were denatured and slow cooled to generate various heteroduplex fragments with the following cycling parameters: 95 C..sup.10:00 cooled to 85 C. over 1 min, 85 C..sup.1:00 cooled to 75 C. over 1 min, 75 C..sup.1:00 cooled to 65 C. over 1 min, 65 C..sup.1:00 cooled to 55 C. over 1 min, 55 C..sup.1:00 cooled to 45 C. over 1 min, 45 C..sup.1:00 cooled to 35 C. over 1 min, 35 C..sup.1:00 cooled to 2 C. 5 over 1 min, 25 C..sup.1:00. These 50/50 mixtures of homoduplex/heteroduplex DNAs were then cleaved using either 0.38 pmol T7EI or 0.19 pmol T4E7 alone for 1 hr at 37 C. or a combination of both enzymes (0.38 pmol T7EI and 0.19 pmol T4E7 mix together) for 1 hr at 37 C. The reactions (20 l) were then diluted with 0.15 ml 0.1TE (10 mM Tris-HCl pH 7.5, 0.1 mM EDTA) and cleaved products were analyzed with a CE instrument, the Fragment Analyzer (Advanced Analytical). Experiments were performed in triplicate and results are shown in FIG. 2.

[0029] FIG. 2 a) shows the results following treatment with the single enzymes or the T4/T7 endonuclease mix on a Fragment Analyzer electropherogram. The Fragment Analyzer electropherogram in FIG. 2 a) shows the cleavage profiles with T7E1 alone, T4E7 alone, or mixture of both enzymes. Cut substrates include heteroduplexes with the indicated number of mismatched nucleotides (1, 2, 3, or 12). FIG. 2 b) shows quantitative data from FIG. 2a (top) as a function of cleavage percentage. Error bars represent the standard errors of the means.

[0030] Results show that as individual enzymes, T7EI alone recognized 2, 3, and 12 base mismatches better than the T4E7 enzyme alone. The T7EI enzyme alone cleaved all heteroduplexes except those having a single indel, which remained uncleaved. T4E7 enzyme alone weakly cleaved the single indel species, and also cleaved fragments having 2 or 3 base indels at a lower rate than the T7EI enzyme alone. Both enzymes alone cleaved the heteroduplex with a large 12 base indel. However, the T7EI+T4E7 cocktail mixture cleaved all of the heteroduplex fragment species. It was also noted that the combination of enzymes was able to cleave all fragment species more efficiently than either enzyme alone.

Example 2

[0031] The following example demonstrates the ability the combination of T7EI and T4E7 to more efficiently cleave mismatched heteroduplex PCR amplified genomic edited products as compared to T7EI alone.

[0032] Following genomic editing with a CRISPR genomic editing system target sites were PCR amplified. Following PCR amplification, the target sites were enzymatically treated with either T7E1 enzyme alone or a mixture of T7E1 enzyme and T4E7 enzyme. Following enzymatic the target sample were run on a capillary electrophoresis and the cleavage percentage was determined.

[0033] FIG. 3 shows the cleavage percentage of multiple loci following enzymatic treatment with T7E1 alone or a mixture of T7E1 enzyme and T4E7 enzyme. As the figure shows the mixture of T7E1 and T4E7 is able to more accurately determine the true cleavage percentage.

[0034] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0035] The use of the terms a and an and the and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms comprising, having, including and containing are to be construed as open-ended terms (i.e., meaning including, but no limited to) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., such as) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0036] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

EMBODIMENTS

[0037] A1. A method for detecting genomic editing events comprising: [0038] a) obtaining an edited genomic sequences; [0039] b) PCR amplifying the edited genomic sequences around the expected edited region to generate a plurality of PCR amplicons; [0040] c) reacting the plurality of PCR with a mixture of mismatch endonucleases to generate a plurality of cleaved fragments; [0041] d) detecting the cleaved fragments indicating a genomic editing event.

[0042] A2. The method of claim A1 wherein the edited genomic sequence edited with a programmable nuclease.

[0043] A3. The method of claim A2 wherein the programmable nuclease is a TALEN, a RGEN, or a ZFN.

[0044] A4. The method of claim A3 wherein the RGEN is a Cas9 enzyme or a Cpf1 enzyme.

[0045] A5. The method of claim A1 wherein the mixture of mismatch endonucleases comprises T7EI and T4E7.

[0046] A6. An enzyme composition for detecting edited genomic DNA comprising a mixture of mismatch endonucleases.

[0047] A7. The composition of claim A6 wherein the mixture of mismatch endonucleases consists of T7E1 and T4E7.

[0048] A8. The composition of claim A7 wherein the mixture of mismatch endonucleases consists of 0.38 pmol T7EI and 0.19 pmol T4E7.

REFERENCES

[0049] U.S. Pat. No. 5,698,400A DETECTION OF MUTATION BY RESOLVASE CLEAVAGE [0050] Mashal R. D., Koontz J., and Sklar J. Detection of mutations by cleavage of DNA heteroduplexes with bacteriophage resolvases. Nat Genet., 1995 9:177-83. [0051] Babon J. J., McKenzie M., and Cotton R. G. The use of resolvases T4 endonuclease VII and T7 endonuclease I in mutation detection. Methods Mol Biol., 2000, 152:187-99. [0052] Mean, R. J., Pierides, A., Deltas, C. C., and Koptides, M. Modification of the enzyme mismatch cleavage method using T7 endonuclease 1 and silver staining. BioTechniques, 2004, 36:758-760. [0053] Vouillot, L., Thelie, A., and Pollet, N. Comparison of T7E1 and Surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases. Genes, Genomes, Genetics, 2015, 5:407-415.