Resection and repair of a Cas9 double-strand break at CTG trinucleotide repeats induces local and extensive chromosomal deletions


Autoři: Valentine Mosbach aff001;  David Viterbo aff001;  Stéphane Descorps-Declère aff001;  Lucie Poggi aff001;  Wilhelm Vaysse-Zinkhöfer aff001;  Guy-Franck Richard aff001
Působiště autorů: Institut Pasteur, CNRS, UMR3525, Paris, France aff001;  Institut Pasteur, Center of Bioinformatics, Biostatistics and Integrative Biology (C3BI), Paris, France aff002;  Sorbonne Universités, Collège doctoral, Paris, France aff003
Vyšlo v časopise: Resection and repair of a Cas9 double-strand break at CTG trinucleotide repeats induces local and extensive chromosomal deletions. PLoS Genet 16(7): e32767. doi:10.1371/journal.pgen.1008924
Kategorie: Research Article
doi: 10.1371/journal.pgen.1008924

Souhrn

Microsatellites are short tandem repeats, ubiquitous in all eukaryotes and represent ~2% of the human genome. Among them, trinucleotide repeats are responsible for more than two dozen neurological and developmental disorders. Targeting microsatellites with dedicated DNA endonucleases could become a viable option for patients affected with dramatic neurodegenerative disorders. Here, we used the Streptococcus pyogenes Cas9 to induce a double-strand break within the expanded CTG repeat involved in myotonic dystrophy type 1, integrated in a yeast chromosome. Repair of this double-strand break generated unexpected large chromosomal deletions around the repeat tract. These deletions depended on RAD50, RAD52, DNL4 and SAE2, and both non-homologous end-joining and single-strand annealing pathways were involved. Resection and repair of the double-strand break (DSB) were totally abolished in a rad50Δ strain, whereas they were impaired in a sae2Δ mutant, only on the DSB end containing most of the repeat tract. This observation demonstrates that Sae2 plays significant different roles in resecting a DSB end containing a repeated and structured sequence as compared to a non-repeated DSB end. In addition, we also discovered that gene conversion was less efficient when the DSB could be repaired using a homologous template, suggesting that the trinucleotide repeat may interfere with gene conversion too. Altogether, these data show that SpCas9 may not be the best choice when inducing a double-strand break at or near a microsatellite, especially in mammalian genomes that contain many more dispersed repeated elements than the yeast genome.

Klíčová slova:

Genetic loci – Guide RNA – Chromosomal deletions – Repeated sequences – Saccharomyces cerevisiae – Southern blot – Surgical resection – Trinucleotide repeats – Cloning – DNA repair – Yeast


Zdroje

1. Richard G-F, Kerrest A, Dujon B. Comparative genomics and molecular dynamics of DNA repeats in eukaryotes. Microbiol Mol Biol Rev. 2008;72: 686–727. doi: 10.1128/MMBR.00011-08 19052325

2. International Human Genome Sequencing Consortium. Finishing the euchromatic sequence of the human genome. Nature. 2004;431: 931–945. doi: 10.1038/nature03001 15496913

3. Orr HT, Zoghbi HY. Trinucleotide repeat disorders. Annu Rev Neurosci. 2007;30: 575–621. doi: 10.1146/annurev.neuro.29.051605.113042 17417937

4. Gacy AM, Goellner G, Juranic N, Macura S, McMurray CT. Trinucleotide repeats that expand in human disease form hairpin structures in vitro. Cell. 1995;81: 533–540. doi: 10.1016/0092-8674(95)90074-8 7758107

5. Liu G, Chen X, Bissler JJ, Sinden RR, Leffak M. Replication-dependent instability at (CTG) x (CAG) repeat hairpins in human cells. Nat Chem Biol. 2010;6: 652–9. doi: 10.1038/nchembio.416 20676085

6. Axford MM, Wang YH, Nakamori M, Zannis-Hadjopoulos M, Thornton CA, Pearson CE. Detection of Slipped-DNAs at the Trinucleotide Repeats of the Myotonic Dystrophy Type I Disease Locus in Patient Tissues. PLoS Genet. 2013;9: 1–13. doi: 10.1371/journal.pgen.1003866 24367268

7. Anand RP, Shah KA, Niu H, Sung P, Mirkin SM, Freudenreich CH. Overcoming natural replication barriers: differential helicase requirements. Nucleic Acids Res. 2012;40: 1091–105. doi: 10.1093/nar/gkr836 21984413

8. Nguyen JHG, Viterbo D, Anand RP, Verra L, Sloan L, Richard G-F, et al. Differential requirement of Srs2 helicase and Rad51 displacement activities in replication of hairpin-forming CAG/CTG repeats. Nucleic Acids Res. 45: 4519–4531. doi: 10.1093/nar/gkx088 28175398

9. Pelletier R, Krasilnikova MM, Samadashwily GM, Lahue R, Mirkin SM. Replication and expansion of trinucleotide repeats in yeast. Mol Cell Biol. 2003;23: 1349–57. doi: 10.1128/mcb.23.4.1349-1357.2003 12556494

10. Samadashwily G, Raca G, Mirkin SM. Trinucleotide repeats affect DNA replication in vivo. Nat Genet. 1997;17: 298–304. doi: 10.1038/ng1197-298 9354793

11. Viterbo D, Michoud G, Mosbach V, Dujon B, Richard G-F. Replication stalling and heteroduplex formation within CAG/CTG trinucleotide repeats by mismatch repair. DNA Repair. 2016;42: 94–106. doi: 10.1016/j.dnarep.2016.03.002 27045900

12. Sutherland GR, Baker E, Richards RI. Fragile sites still breaking. Trends Genet. 1998;14: 501–506. doi: 10.1016/s0168-9525(98)01628-x 9865156

13. Balakumaran BS, Freudenreich CH, Zakian VA. CGG/CCG repeats exhibit orientation-dependent instability and orientation-independent fragility in Saccharomyces cerevisiae. Hum Mol Genet. 2000;9: 93–100. doi: 10.1093/hmg/9.1.93 10587583

14. Freudenreich CH, Kantrow SM, Zakian VA. Expansion and length-dependent fragility of CTG repeats in yeast. Science. 1998;279: 853–856. doi: 10.1126/science.279.5352.853 9452383

15. Haber J E. Genome stability. Summer Scholl. New York: Garland Science; 2014.

16. Fairhead C, Dujon B. Consequences of unique double-stranded breaks in yeast chromosomes: death or homozygosis. Mol Gen Genet. 1993;240: 170–180. doi: 10.1007/BF00277054 8355651

17. Haber JE. In vivo biochemistry: physical monitoring of recombination induced by site-specific endonucleases. BioEssays. 1995;17: 609–620. doi: 10.1002/bies.950170707 7646483

18. Plessis A, Perrin A, Haber JE, Dujon B. Site-specific recombination determined by I-Sce I, a mitochondrial group I intron-encoded endonuclease expressed in the yeast nucleus. Genetics. 1992;130: 451–460. 1551570

19. Nelms BE, Maser RS, MacKay JF, Lagally MG, Petrini JHJ. In Situ Visualization of DNA Double-Strand Break Repair in Human Fibroblasts. Science. 1998;280: 590–592. doi: 10.1126/science.280.5363.590 9554850

20. Mosbach V, Poggi L, Viterbo D, Charpentier M, Richard G-F. TALEN-induced double-strand break repair of CTG trinucleotide repeats. Cell Rep. 2018;22: 2146–2159. doi: 10.1016/j.celrep.2018.01.083 29466740

21. Richard G-F, Viterbo D, Khanna V, Mosbach V, Castelain L, Dujon B. Highly specific contractions of a single CAG/CTG trinucleotide repeat by TALEN in yeast. PLoS ONE. 2014;9: e95611. doi: 10.1371/journal.pone.0095611 24748175

22. DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res. 2013;41: 4336–43. doi: 10.1093/nar/gkt135 23460208

23. Chen H, Lisby M, Symington LS. RPA coordinates DNA end resection and prevents formation of DNA hairpins. Mol Cell. 2013;50: 589–600. doi: 10.1016/j.molcel.2013.04.032 23706822

24. Richard G-F, Goellner GM, McMurray CT, Haber JE. Recombination-induced CAG trinucleotide repeat expansions in yeast involve the MRE11/RAD50/XRS2 complex. EMBO J. 2000;19: 2381–2390. doi: 10.1093/emboj/19.10.2381 10811629

25. Kleinstiver BP, Pattanayak V, Prew MS, Tsai SQ, Nguyen NT, Zheng Z, et al. High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects. Nature. 2016;529: 490–495. doi: 10.1038/nature16526 26735016

26. Slaymaker IM, Gao L, Zetsche B, Scott DA, Yan WX, Zhang F. Rationally engineered Cas9 nucleases with improved specificity. Science. 2016;351: 84–88. doi: 10.1126/science.aad5227 26628643

27. Chen JS, Dagdas YS, Kleinstiver BP, Welch MM, Sousa AA, Harrington LB, et al. Enhanced proofreading governs CRISPR–Cas9 targeting accuracy. Nature. 2017;550: 407–410. doi: 10.1038/nature24268 28931002

28. Lee JK, Jeong E, Lee J, Jung M, Shin E, Kim Y, et al. Directed evolution of CRISPR-Cas9 to increase its specificity. Nat Commun. 2018;9: 3048. doi: 10.1038/s41467-018-05477-x 30082838

29. Casini A, Olivieri M, Petris G, Montagna C, Reginato G, Maule G, et al. A highly specific SpCas9 variant is identified by in vivo screening in yeast. Nat Biotechnol. 2018;36: 265–271. doi: 10.1038/nbt.4066 29431739

30. Richard G-F, Dujon B, Haber JE. Double-strand break repair can lead to high frequencies of deletions within short CAG/CTG trinucleotide repeats. Mol Gen Genet. 1999;261: 871–882. doi: 10.1007/s004380050031 10394925

31. Richard G-F, Cyncynatus C, Dujon B. Contractions and expansions of CAG/CTG trinucleotide repeats occur during ectopic gene conversion in yeast, by a MUS81-independent mechanism. J Mol Biol. 2003;326: 769–782. doi: 10.1016/s0022-2836(02)01405-5 12581639

32. Field D, Wills C. Abundant microsatellite polymorphism in Saccharomyces cerevisiae, and the different distributions of microsatellites in eight prokaryotes and S. cerevisiae, result from strong mutation pressures and a variety of selective forces. Proc Natl Acad Sci USA. 1998;95: 1647–1652. doi: 10.1073/pnas.95.4.1647 9465070

33. Malpertuy A, Dujon B, Richard G-F. Analysis of microsatellites in 13 hemiascomycetous yeast species: mechanisms involved in genome dynamics. J Mol Evol. 2003;56: 730–741. doi: 10.1007/s00239-002-2447-5 12911036

34. Richard G-F, Dujon B. Distribution and variability of trinucleotide repeats in the genome of the yeast Saccharomyces cerevisiae. Gene. 1996;174: 165–174. doi: 10.1016/0378-1119(96)00514-8 8863744

35. Richard G-F, Hennequin C, Thierry A, Dujon B. Trinucleotide repeats and other microsatellites in yeasts. Res Microbiol. 1999;150: 589–602. doi: 10.1016/s0923-2508(99)00131-x 10672999

36. Haeussler M, Schönig K, Eckert H, Eschstruth A, Mianné J, Renaud J-B, et al. Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol. 2016;17. doi: 10.1186/s13059-016-1012-2 27380939

37. Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF, et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol. 2016;34: 184–191. doi: 10.1038/nbt.3437 26780180

38. Rothstein R, Helms C, Rosenberg N. Concerted deletions and inversions are caused by mitotic recombination between delta sequences in Saccharomyces cerevisiae. Mol Cell Biol. 1987;7: 1198–1207. doi: 10.1128/mcb.7.3.1198 3550432

39. Welcker AJ, de Montigny J, Potier S, Souciet JL. Involvement of very short DNA tandem repeats and the influence of the RAD52 gene on the occurrence of deletions in Saccharomyces cerevisiae. Genetics. 2000;156: 549–557. 11014805

40. Cinesi C, Aeschbach L, Yang B, Dion V. Contracting CAG/CTG repeats using the CRISPR-Cas9 nickase. Nat Commun. 2016;7: 13272. doi: 10.1038/ncomms13272 27827362

41. Dabrowska M, Juzwa W, Krzyzosiak WJ, Olejniczak M. Precise Excision of the CAG Tract from the Huntingtin Gene by Cas9 Nickases. Front Neurosci. 2018;12. doi: 10.3389/fnins.2018.00075 29535594

42. Lemos BR, Kaplan AC, Bae JE, Ferrazzoli AE, Kuo J, Anand RP, et al. CRISPR/Cas9 cleavages in budding yeast reveal templated insertions and strand-specific insertion/deletion profiles. Proc Natl Acad Sci. 2018;115: E2040–E2047. doi: 10.1073/pnas.1716855115 29440496

43. Wilson TE, Grawunder U, Lieber MR. Yeast DNA ligase IV mediates non-homologous DNA end joining. Nature. 1997;388: 495–498. doi: 10.1038/41365 9242411

44. Frank-Vaillant M, Marcand S. NHEJ regulation by mating type is exercised through a novel protein, Lif2p, essential to the Ligase IV pathway. Genes&Development. 2001;15: 3005–3012.

45. Charpentier M, Khedher AHY, Menoret S, Brion A, Lamribet K, Dardillac E, et al. CtIP fusion to Cas9 enhances transgene integration by homology-dependent repair. Nat Commun. 2018;9: 1133. doi: 10.1038/s41467-018-03475-7 29556040

46. van Overbeek M, Capurso D, Carter MM, Thompson MS, Frias E, Russ C, et al. DNA Repair Profiling Reveals Nonrandom Outcomes at Cas9-Mediated Breaks. Mol Cell. 2016;63: 633–646. doi: 10.1016/j.molcel.2016.06.037 27499295

47. Haber JE. The many interfaces of Mre11. Cell. 1998;95: 583–586. doi: 10.1016/s0092-8674(00)81626-8 9845359

48. Lengsfeld BM, Rattray AJ, Bhaskara V, Ghirlando R, Paull TT. Sae2 Is an Endonuclease that Processes Hairpin DNA Cooperatively with the Mre11/Rad50/Xrs2 Complex. Mol Cell. 2007;28: 638–651. doi: 10.1016/j.molcel.2007.11.001 18042458

49. Mimitou EP, Symington LS. Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing. Nature. 2008;455: 770–4. doi: 10.1038/nature07312 18806779

50. So CC, Martin A. DSB structure impacts DNA recombination leading to class switching and chromosomal translocations in human B cells. PLOS Genet. 2019;15: e1008101. doi: 10.1371/journal.pgen.1008101 30946744

51. Storici F, Bebenek K, Kunkel TA, Gordenin DA, Resnick MA. RNA-templated DNA repair. Nature. 2007;447: 338–341. doi: 10.1038/nature05720 17429354

52. Zhu Z, Chung WH, Shim EY, Lee SE, Ira G. Sgs1 helicase and two nucleases Dna2 and Exo1 resect DNA double-strand break ends. Cell. 2008;134: 981–94. doi: 10.1016/j.cell.2008.08.037 18805091

53. Orr-Weaver TL, Szostak JW, Rothstein RJ. Yeast transformation: a model system for the study of recombination. Proc Natl Acad Sci U S A. 1981;78: 6354–6358. doi: 10.1073/pnas.78.10.6354 6273866

54. Holmes AM, Haber JE. Double-strand break repair in yeast requires both leading and lagging strand DNA polymerases. Cell. 1999;96: 415–424. doi: 10.1016/s0092-8674(00)80554-1 10025407

55. Muller H, Annaluru N, Schwerzmann JW, Richardson SM, Dymond JS, Cooper EM, et al. Assembling large DNA segments in yeast. Methods Mol Biol Clifton NJ. 2012;852: 133–150. doi: 10.1007/978-1-61779-564-0_11 22328431

56. Sikorski RS, Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989;122: 19–27. 2659436

57. Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinforma Oxf Engl. 2009;25: 1754–1760. doi: 10.1093/bioinformatics/btp324 19451168

58. Viterbo D, Marchal A, Mosbach V, Poggi L, Vaysse-Zinkhöfer W, Richard G-F. A fast, sensitive and cost-effective method for nucleic acid detection using non-radioactive probes. Biol Methods Protoc. 2018;3. doi: 10.1093/biomethods/bpy006 32161800

59. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009;25: 2078–9. doi: 10.1093/bioinformatics/btp352 19505943

60. DePristo MA, Banks E, Poplin R, Garimella KV, Maguire JR, Hartl C, et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet. 2011;43: 491–8. doi: 10.1038/ng.806 21478889

61. Koboldt DC, Zhang Q, Larson DE, Shen D, McLellan MD, Lin L, et al. VarScan 2: somatic mutation and copy number alteration discovery in cancer by exome sequencing. Genome Res. 2012;22: 568–76. doi: 10.1101/gr.129684.111 22300766

62. Fungtammasan A, Ananda G, Hile SE, Su MS-W, Sun C, Harris R, et al. Accurate typing of short tandem repeats from genome-wide sequencing data and its applications. Genome Res. 2015 [cited 13 Oct 2016]. doi: 10.1101/gr.185892.114 25823460

63. Millot G. Comprendre et réaliser les tests statistiques à l’aide de R. 2nd ed. Brussels: de boeck; 2011.


Článek vyšel v časopise

PLOS Genetics


2020 Číslo 7

Nejčtenější v tomto čísle

Tomuto tématu se dále věnují…


Kurzy Doporučená témata Časopisy
Přihlášení
Zapomenuté heslo

Nemáte účet?  Registrujte se

Zapomenuté heslo

Zadejte e-mailovou adresu se kterou jste vytvářel(a) účet, budou Vám na ni zaslány informace k nastavení nového hesla.

Přihlášení

Nemáte účet?  Registrujte se

VIRTUÁLNÍ ČEKÁRNA ČR Jste praktický lékař nebo pediatr? Zapojte se! Jste praktik nebo pediatr? Zapojte se!

×