Skip to content
naulon

No retraction, correction or concern found when last checked on Nov 2, 2026.

Comparison of T7E1 and Surveyor Mismatch Cleavage Assays to Detect Mutations Triggered by Engineered Nucleases

Léna Vouillot, Aurore Thélie, Nicolas Pollet

G3 Genes|Genomes|Genetics · 2015

doi:10.1534/g3.114.015834

Abstract

Genome editing using engineered nucleases is used for targeted mutagenesis. But because genome editing does not target all loci with similar efficiencies, the mutation hit-rate at a given locus needs to be evaluated. The analysis of mutants obtained using engineered nucleases requires specific methods for mutation detection, and the enzyme mismatch cleavage method is used commonly for this purpose. This method uses enzymes that cleave heteroduplex DNA at mismatches and extrahelical loops formed by single or multiple nucleotides. Bacteriophage resolvases and single-stranded nucleases are used commonly in the assay but have not been compared side-by-side on mutations obtained by engineered nucleases. We present the first comparison of the sensitivity of T7E1 and Surveyor EMC assays on deletions and point mutations obtained by zinc finger nuclease targeting in frog embryos. We report the mutation detection limits and efficiencies of T7E1 and Surveyor. In addition, we find that T7E1 outperforms the Surveyor nuclease in terms of sensitivity with deletion substrates, whereas Surveyor is better for detecting single nucleotide changes. We conclude that T7E1 is the preferred enzyme to scan mutations triggered by engineered nucleases.

Shown for reading. Its publisher allows quoting it, not republishing it.

References 56

  1. 1.

    Babon J. J. Youil R. Cotton R. G. H., 1995 Improved strategy for mutation detection—a modification to the enzyme mismatch cleavage method. Nucleic Acids Res. 23: 5082–5084.

    Not in our library10.1093/nar/23.24.5082

  2. 2.

    Behrensdorf H. A. Pignot M. Windhab N. Kappel A., 2002 Rapid parallel mutation scanning of gene fragments using a microelectronic protein-DNA chip format. Nucleic Acids Res. 30: e64.

    Not in our library10.1093/nar/gnf063

  3. 3.

    Bentley A. MacLennan B. Calvo J. Dearolf C. R., 2000 Targeted recovery of mutations in Drosophila. Genetics 156: 1169–1173.

    Not in our library10.1093/genetics/156.3.1169

  4. 4.

    Beumer K. J. Trautman J. K. Bozas A. Liu J.-L. Rutter J., 2008 Efficient gene targeting in Drosophila by direct embryo injection with zinc-finger nucleases. Proc. Natl. Acad. Sci. USA 105: 19821–19826.

    Not in our library10.1073/pnas.0810475105

  5. 5.

    Bhattacharyya A. Lilley D. M., 1989 Single base mismatches in DNA. Long- and short-range structure probed by analysis of axis trajectory and local chemical reactivity. J. Mol. Biol. 209: 583–597.

    Not in our library10.1016/0022-2836(89)90596-2

  6. 6.

    Coghill E. L. Hugill A. Parkinson N. Davison C. Glenister P., 2002 A gene-driven approach to the identification of ENU mutants in the mouse. Nat. Genet. 30: 255–256.

    Not in our library10.1038/ng847

  7. 7.

    Colbert T. Till B. J. Tompa R. Reynolds S. Steine M. N., 2001 High-throughput screening for induced point mutations. Plant Physiol. 126: 480–484.

    Not in our library10.1104/pp.126.2.480

  8. 8.

    Comai L. Young K. Till B. J. Reynolds S. H. Greene E. A., 2004 Efficient discovery of DNA polymorphisms in natural populations by Ecotilling. Plant J. 37: 778–786.

    Not in our library10.1111/j.0960-7412.2003.01999.x

  9. 9.
  10. 10.

    Déclais A.-C. Lilley D. M., 2008 New insight into the recognition of branched DNA structure by junction-resolving enzymes. Curr. Opin. Struct. Biol. 18: 86–95.

    Not in our library10.1016/j.sbi.2007.11.001

  11. 11.

    Déclais A.-C. Liu J. Freeman A. D. J. Lilley D. M. J., 2006 Structural recognition between a four-way DNA junction and a resolving enzyme. J. Mol. Biol. 359: 1261–1276.

    Not in our library10.1016/j.jmb.2006.04.037

  12. 12.

    Dodgson J. B. Wells R. D., 1977 Action of single-strand specific nucleases on model DNA heteroduplexes of defined size and sequence. Biochemistry (Mosc.) 16: 2374–2379.

    Not in our library10.1021/bi00630a010

  13. 13.

    Freeman A. D. J. Déclais A.-C. Lilley D. M. J., 2013 The importance of the N-terminus of T7 endonuclease I in the interaction with DNA junctions. J. Mol. Biol. 425: 395–410.

    Not in our library10.1016/j.jmb.2012.11.029

  14. 14.

    Gaj T. Gersbach C. A. Barbas C. F. 3rd, 2013 ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol. 31: 397–405.

    Not in our library10.1016/j.tibtech.2013.04.004

  15. 15.

    Geurts A. M. Cost G. J. Freyvert Y. Zeitler B. Miller J. C., 2009 Knockout rats via embryo microinjection of zinc-finger nucleases. Science 325: 433.

    Not in our library10.1126/science.1172447

  16. 16.

    Gohlke C. Murchie A. I. Lilley D. M. Clegg R. M., 1994 Kinking of DNA and RNA helices by bulged nucleotides observed by fluorescence resonance energy transfer. Proc. Natl. Acad. Sci. USA 91: 11660–11664.

    Not in our library10.1073/pnas.91.24.11660

  17. 17.

    Guo X. Zhang T. Hu Z. Zhang Y. Shi Z., 2014 Efficient RNA/Cas9-mediated genome editing in Xenopus tropicalis. Development 141: 707–714.

    Not in our library10.1242/dev.099853

  18. 18.

    Guschin D. Y. Waite A. J. Katibah G. E. Miller J. C. Holmes M. C., 2010 A rapid and general assay for monitoring endogenous gene modification. Methods Mol. Biol. 649: 247–256.

    Not in our library10.1007/978-1-60761-753-2_15

  19. 19.

    Hadden J. M. Déclais A.-C. Carr S. B. Lilley D. M. J. Phillips S. E. V., 2007 The structural basis of Holliday junction resolution by T7 endonuclease I. Nature 449: 621–624.

    Not in our library10.1038/nature06158

  20. 20.

    Ho S. N. Hunt H. D. Horton R. M. Pullen J. K. Pease L. R., 1989 Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77: 51–59.

    Not in our library10.1016/0378-1119(89)90358-2

  21. 21.

    Holkers M. Maggio I. Liu J. Janssen J. M. Miselli F., 2013 Differential integrity of TALE nuclease genes following adenoviral and lentiviral vector gene transfer into human cells. Nucleic Acids Res. 41: e63.

    Not in our library10.1093/nar/gks1446

  22. 22.

    Howard J. T. Ward J. Watson J. N. Roux K. H., 1999 Heteroduplex cleavage analysis using S1 nuclease. Biotechniques 27: 18–19.

    Not in our library10.2144/99271bm01

  23. 23.

    Huang M. C. Cheong W. C. Lim L. S. Li M.-H., 2012 A simple, high sensitivity mutation screening using Ampligase mediated T7 endonuclease I and Surveyor nuclease with microfluidic capillary electrophoresis. Electrophoresis 33: 788–796.

    Not in our library10.1002/elps.201100460

  24. 24.

    Isalan M., 2012 Zinc-finger nucleases: how to play two good hands. Nat. Methods 9: 32–34.

    Not in our library10.1038/nmeth.1805

  25. 25.

    Ishibashi S. Cliffe R. Amaya E., 2012 Highly efficient bi-allelic mutation rates using TALENs in Xenopus tropicalis. Biol. Open 1: 1273–1276.

    Not in our library10.1242/bio.20123228

  26. 26.

    Judo M. S. B. Wedel A. B. Wilson C., 1998 Stimulation and suppression of PCR-mediated recombination. Nucleic Acids Res. 26: 1819–1825.

    Not in our library10.1093/nar/26.7.1819

  27. 27.

    Kim Y. Kweon J. Kim J.-S., 2013 TALENs and ZFNs are associated with different mutation signatures. Nat. Methods 10: 185.

    Not in our library10.1038/nmeth.2364

  28. 28.

    Lahr D. J. G. Katz L. A., 2009 Reducing the impact of PCR-mediated recombination in molecular evolution and environmental studies using a new-generation high-fidelity DNA polymerase. Biotechniques 47: 857–866.

    Not in our library10.2144/000113219

  29. 29.

    Lei Y. Guo X. Liu Y. Cao Y. Deng Y., 2012 Efficient targeted gene disruption in Xenopus embryos using engineered transcription activator-like effector nucleases (TALENs). Proc. Natl. Acad. Sci. USA 109: 17484–17489.

    Not in our library10.1073/pnas.1215421109

  30. 30.

    Maier D. A. Brennan A. L. Jiang S. Binder-Scholl G. K. Lee G., 2013 Efficient clinical scale gene modification via zinc finger nuclease-targeted disruption of the HIV co-receptor CCR5. Hum. Gene Ther. 24: 245–258.

    Not in our library10.1089/hum.2012.172

  31. 31.

    Mashal R. D. Koontz J. Sklar J., 1995 Detection of mutations by cleavage of DNA heteroduplexes with bacteriophage resolvases. Nat. Genet. 9: 177–183.

    Not in our library10.1038/ng0295-177

  32. 32.

    Miller J. C. Tan S. Qiao G. Barlow K. A. Wang J., 2011 A TALE nuclease architecture for efficient genome editing. Nat. Biotechnol. 29: 143–148.

    Not in our library10.1038/nbt.1755

  33. 33.
  34. 34.

    Oleykowski C. A. Bronson Mullins C. R. Godwin A. K. Yeung A. T., 1998 Mutation detection using a novel plant endonuclease. Nucleic Acids Res. 26: 4597–4602.

    Not in our library10.1093/nar/26.20.4597

  35. 35.

    Perry J. A. Wang T. L. Welham T. J. Gardner S. Pike J. M., 2003 A TILLING reverse genetics tool and a web-accessible collection of mutants of the legume Lotus japonicus. Plant Physiol. 131: 866–871.

    Not in our library10.1104/pp.102.017384

  36. 36.

    Qiu P. Shandilya H. Alessio J. M. D. Connor K. O. Durocher J., 2004 Mutation detection using Surveyor nuclease. Biotechniques 36: 702–707.

    Not in our library10.2144/04364pf01

  37. 37.

    Van Rensburg R. Beyer I. Yao X.-Y. Wang H. Denisenko O., 2013 Chromatin structure of two genomic sites for targeted transgene integration in induced pluripotent stem cells and hematopoietic stem cells. Gene Ther. 20: 201–214.

    Not in our library10.1038/gt.2012.25

  38. 38.

    Sakurai T. Watanabe S. Kamiyoshi A. Sato M. Shindo T., 2014 A single blastocyst assay optimized for detecting CRISPR/Cas9 system-induced indel mutations in mice. BMC Biotechnol. 14: 69.

    Not in our library10.1186/1472-6750-14-69

  39. 39.

    Sanjana N. E. Cong L. Zhou Y. Cunniff M. M. Feng G., 2012 A transcription activator-like effector toolbox for genome engineering. Nat. Protoc. 7: 171–192.

    Not in our library10.1038/nprot.2011.431

  40. 40.

    Slade A. J. Fuerstenberg S. I. Loeffler D. Steine M. N. Facciotti D., 2005 A reverse genetic, nontransgenic approach to wheat crop improvement by TILLING. Nat. Biotechnol. 23: 75–81.

    Not in our library10.1038/nbt1043

  41. 41.
  42. 42.

    Taylor G. R. Deeble J., 1999 Enzymatic methods for mutation scanning. Genet. Anal. Biomol. Eng. 14: 181–186.

    Not in our library10.1016/s1050-3862(98)00029-1

  43. 43.

    Terns R. M. Terns M. P., 2014 CRISPR-based technologies: prokaryotic defense weapons repurposed. Trends Genet. TIG 30: 111–118.

    Not in our library10.1016/j.tig.2014.01.003

  44. 44.

    Tesson L. Usal C. Ménoret S. Leung E. Niles B. J., 2011 Knockout rats generated by embryo microinjection of TALENs. Nat. Biotechnol. 29: 695–696.

    Not in our library10.1038/nbt.1940

  45. 45.

    Till B. J. Burtner C. Comai L. Henikoff S., 2004 Mismatch cleavage by single-strand specific nucleases. Nucleic Acids Res. 32: 2632–2641.

    Not in our library10.1093/nar/gkh599

  46. 46.

    Triques K. Sturbois B. Gallais S. Dalmais M. Chauvin S., 2007 Characterization of Arabidopsis thaliana mismatch specific endonucleases: application to mutation discovery by TILLING in pea. Plant J. 51: 1116–1125.

    Not in our library10.1111/j.1365-313x.2007.03201.x

  47. 47.

    Triques K. Piednoir E. Dalmais M. Schmidt J. Le Signor C., 2008 Mutation detection using ENDO1: application to disease diagnostics in humans and TILLING and Eco-TILLING in plants. BMC Mol. Biol. 9: 42.

    Not in our library10.1186/1471-2199-9-42

  48. 48.

    Tsuji T. Niida Y., 2008 Development of a simple and highly sensitive mutation screening system by enzyme mismatch cleavage with optimized conditions for standard laboratories. Electrophoresis 29: 1473–1483.

    Not in our library10.1002/elps.200700729

  49. 49.

    Voskarides K. Deltas C., 2009 Screening for mutations in kidney-related genes using SURVEYOR nuclease for cleavage at heteroduplex mismatches. J. Mol. Diagn. 11: 311–318.

    Not in our library10.2353/jmoldx.2009.080144

  50. 50.

    Wagner R. Debbie P. Radman M., 1995 Mutation detection using immobilized mismatch binding protein (MutS). Nucleic Acids Res. 23: 3944–3948.

    Not in our library10.1093/nar/23.19.3944

  51. 51.

    Wienholds E. van Eeden F. Kosters M. Mudde J. Plasterk R. H. A., 2003 Efficient target-selected mutagenesis in zebrafish. Genome Res. 13: 2700–2707.

    Not in our library10.1101/gr.1725103

  52. 52.

    Yang B. Wen X. Kodali N. S. Oleykowski C. A. Miller C. G., 2000 Purification, cloning, and characterization of the CEL I nuclease. Biochemistry (Mosc.) 39: 3533–3541.

    Not in our library10.1021/bi992376z

  53. 53.

    Yeung A. T. Hattangadi D. Blakesley L. Nicolas E., 2005 Enzymatic mutation detection technologies. Biotechniques 38: 749–758.

    Not in our library10.2144/05385rv01

  54. 54.

    Ymlahi-Ouazzani Q. Bronchain O. J. Paillard E. Ballagny C. Chesneau A., 2010 Reduced levels of survival motor neuron protein leads to aberrant motoneuron growth in a Xenopus model of muscular atrophy. Neurogenetics 11: 27–40.

    Not in our library10.1007/s10048-009-0200-6

  55. 55.

    Youil R. Kemper B. W. Cotton R. G., 1995 Screening for mutations by enzyme mismatch cleavage with T4 endonuclease VII. Proc. Natl. Acad. Sci. USA 92: 87.

    Not in our library10.1073/pnas.92.1.87

  56. 56.

    Young J. J. Cherone J. M. Doyon Y. Ankoudinova I. Faraji F. M., 2011 Efficient targeted gene disruption in the soma and germ line of the frog Xenopus tropicalis using engineered zinc-finger nucleases. Proc. Natl. Acad. Sci. USA 108: 7052–7057.

    Not in our library10.1073/pnas.1102030108

Cited by papers we hold 8

Reads

Read by people on this page

None yet.

Checked or read by AI through naulon

None yet.

A person counts once a day per paper. We keep no cookie and no address: a reader is a hash that changes every day. Any month under five reads shows no number.

Are you an author?

Is this your paper?

Claim it to add your note and be paid when agents cite it. Free.