A fly model establishes distinct mechanisms for synthetic CRISPR/Cas9 sex distorters


Autoři: Barbara Fasulo aff001;  Angela Meccariello aff001;  Maya Morgan aff001;  Carl Borufka aff001;  Philippos Aris Papathanos aff002;  Nikolai Windbichler aff001
Působiště autorů: Department of Life Sciences, Imperial College London, Sir Alexander Fleming Building, South Kensington Campus, London, United Kingdom aff001;  Department of Entomology, Robert H. Smith Faculty of Agriculture, Food and Environment, Hebrew University of Jerusalem, Rehovot, Israel aff002
Vyšlo v časopise: A fly model establishes distinct mechanisms for synthetic CRISPR/Cas9 sex distorters. PLoS Genet 16(3): e32767. doi:10.1371/journal.pgen.1008647
Kategorie: Research Article
doi: 10.1371/journal.pgen.1008647

Souhrn

Synthetic sex distorters have recently been developed in the malaria mosquito, relying on endonucleases that target the X-chromosome during spermatogenesis. Although inspired by naturally-occurring traits, it has remained unclear how they function and, given their potential for genetic control, how portable this strategy is across species. We established Drosophila models for two distinct mechanisms for CRISPR/Cas9 sex-ratio distortion—“X-shredding” and “X-poisoning”—and dissected their target-site requirements and repair dynamics. X-shredding resulted in sex distortion when Cas9 endonuclease activity occurred during the meiotic stages of spermatogenesis but not when Cas9 was expressed from the stem cell stages onwards. Our results suggest that X-shredding is counteracted by the NHEJ DNA repair pathway and can operate on a single repeat cluster of non-essential sequences, although the targeting of a number of such repeats had no effect on the sex ratio. X-poisoning by contrast, i.e. targeting putative haplolethal genes on the X chromosome, induced a high bias towards males (>92%) when we directed Cas9 cleavage to the X-linked ribosomal target gene RpS6. In the case of X-poisoning sex distortion was coupled to a loss in reproductive output, although a dominant-negative effect appeared to drive the mechanism of female lethality. These model systems will guide the study and the application of sex distorters to medically or agriculturally important insect target species.

Klíčová slova:

DNA repair – Drosophila melanogaster – Embryos – Guide RNA – Invertebrate genomics – Repeated sequences – X chromosomes – X-linked traits


Zdroje

1. Hamilton WD. Extraordinary sex ratios. A sex-ratio theory for sex linkage and inbreeding has new implications in cytogenetics and entomology. Science. 1967;156(3774):477–88. Epub 1967/04/28. doi: 10.1126/science.156.3774.477 6021675.

2. Sweeny TL, Barr AR. Sex Ratio Distortion Caused by Meiotic Drive in a Mosquito, Culex pipiens L. Genetics. 1978;88(3):427–46. 17248804; PubMed Central PMCID: PMC1224591.

3. Newton M.E. WRLaSDI. A cytogenetic analysis of meiotic drive in the mosquito Aedes aegypti. Genetica. 1976;46:297–318.

4. Windbichler N, Papathanos PA, Crisanti A. Targeting the X chromosome during spermatogenesis induces Y chromosome transmission ratio distortion and early dominant embryo lethality in Anopheles gambiae. PLoS Genet. 2008;4(12):e1000291. Epub 2008/12/06. doi: 10.1371/journal.pgen.1000291 19057670; PubMed Central PMCID: PMC2585807.

5. Galizi R, Doyle LA, Menichelli M, Bernardini F, Deredec A, Burt A, et al. A synthetic sex ratio distortion system for the control of the human malaria mosquito. Nat Commun. 2014;5:3977. Epub 2014/06/10. doi: 10.1038/ncomms4977 24915045; PubMed Central PMCID: PMC4057611.

6. Galizi R, Hammond A, Kyrou K, Taxiarchi C, Bernardini F, O'Loughlin SM, et al. A CRISPR-Cas9 sex-ratio distortion system for genetic control. Sci Rep. 2016;6:31139. Epub 2016/08/03. doi: 10.1038/srep31139 27484623; PubMed Central PMCID: PMC4971495.

7. Hall AB, Basu S, Jiang X, Qi Y, Timoshevskiy VA, Biedler JK, et al. SEX DETERMINATION. A male-determining factor in the mosquito Aedes aegypti. Science. 2015;348(6240):1268–70. Epub 2015/05/23. doi: 10.1126/science.aaa2850 25999371; PubMed Central PMCID: PMC5026532.

8. Burt A, Deredec A. Self-limiting population genetic control with sex-linked genome editors. Proc Biol Sci. 2018;285(1883). Epub 2018/07/25. doi: 10.1098/rspb.2018.0776 30051868; PubMed Central PMCID: PMC6083257.

9. Bernardini F, Galizi R, Menichelli M, Papathanos P-A, Dritsou V, Marois E, et al. Site-specific genetic engineering of the Anopheles gambiae Y chromosome. Proceedings of the National Academy of Sciences. 2014;111(21):7600–5. doi: 10.1073/pnas.1404996111 24821795; PubMed Central PMCID: PMC4040617.

10. White-Cooper H. Tissue, cell type and stage-specific ectopic gene expression and RNAi induction in the Drosophila testis. Spermatogenesis. 2012;2(1):11–22. doi: 10.4161/spmg.19088 22553486

11. Kondo S, Ueda R. Highly improved gene targeting by germline-specific Cas9 expression in Drosophila. Genetics. 2013;195(3):715–21. Epub 2013/09/03. doi: 10.1534/genetics.113.156737 24002648; PubMed Central PMCID: PMC3813859.

12. Papathanos PA, Windbichler N. Redkmer: An Assembly-Free Pipeline for the Identification of Abundant and Specific X-Chromosome Target Sequences for X-Shredding by CRISPR Endonucleases. CRISPR J. 2018;1(1):88–98. doi: 10.1089/crispr.2017.0012 30627701; PubMed Central PMCID: PMC6319322.

13. Chang CH, Larracuente AM. Heterochromatin-Enriched Assemblies Reveal the Sequence and Organization of the. Genetics. 2019;211(1):333–48. Epub 2018/11/12. doi: 10.1534/genetics.118.301765 30420487; PubMed Central PMCID: PMC6325706.

14. Marygold SJ, Roote J, Reuter G, Lambertsson A, Ashburner M, Millburn GH, et al. The ribosomal protein genes and Minute loci of Drosophila melanogaster. Genome Biol. 2007;8(10):R216. doi: 10.1186/gb-2007-8-10-r216 17927810; PubMed Central PMCID: PMC2246290.

15. McKim KS, Dahmus JB, Hawley RS. Cloning of the Drosophila melanogaster meiotic recombination gene mei-218: a genetic and molecular analysis of interval 15E. Genetics. 1996;144(1):215–28. 8878687; PubMed Central PMCID: PMC1207495.

16. Stewart MJ, Denell R. The Drosophila ribosomal protein S6 gene includes a 3' triplication that arose by unequal crossing-over. Mol Biol Evol. 1993;10(5):1041–7. doi: 10.1093/oxfordjournals.molbev.a040053 8412647.

17. Port F, Chen HM, Lee T, Bullock SL. Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila. Proc Natl Acad Sci U S A. 2014;111(29):E2967–76. Epub 2014/07/09. doi: 10.1073/pnas.1405500111 25002478; PubMed Central PMCID: PMC4115528.

18. Port F, Bullock SL. Augmenting CRISPR applications in Drosophila with tRNA-flanked sgRNAs. Nat Methods. 2016;13(10):852–4. Epub 2016/09/07. doi: 10.1038/nmeth.3972 27595403; PubMed Central PMCID: PMC5215823.

19. Romeijn RJ, Gorski MM, van Schie MA, Noordermeer JN, Mullenders LH, Ferro W, et al. Lig4 and rad54 are required for repair of DNA double-strand breaks induced by P-element excision in Drosophila. Genetics. 2005;169(2):795–806. Epub 2004/11/15. doi: 10.1534/genetics.104.033464 15545651; PubMed Central PMCID: PMC1449100.

20. Staeva-Vieira E, Yoo S, Lehmann R. An essential role of DmRad51/SpnA in DNA repair and meiotic checkpoint control. EMBO J. 2003;22(21):5863–74. doi: 10.1093/emboj/cdg564 14592983; PubMed Central PMCID: PMC275421.

21. Lu KL, Yamashita YM. Germ cell connectivity enhances cell death in response to DNA damage in the. Elife. 2017;6. Epub 2017/08/15. doi: 10.7554/eLife.27960 28809158; PubMed Central PMCID: PMC5577909.

22. Kaufman TC. A Short History and Description of. Genetics. 2017;206(2):665–89. doi: 10.1534/genetics.117.199950 28592503; PubMed Central PMCID: PMC5499179.

23. Smits AH, Ziebell F, Joberty G, Zinn N, Mueller WF, Clauder-Münster S, et al. Biological plasticity rescues target activity in CRISPR knock outs. Nat Methods. 2019;16(11):1087–93. Epub 2019/10/28. doi: 10.1038/s41592-019-0614-5 31659326.

24. Dilthey AT, Jain C, Koren S, Phillippy AM. Strain-level metagenomic assignment and compositional estimation for long reads with MetaMaps. Nat Commun. 2019;10(1):3066. Epub 2019/07/11. doi: 10.1038/s41467-019-10934-2 31296857; PubMed Central PMCID: PMC6624308.

25. Menon DU, Coarfa C, Xiao W, Gunaratne PH, Meller VH. siRNAs from an X-linked satellite repeat promote X-chromosome recognition in Drosophila melanogaster. Proc Natl Acad Sci U S A. 2014;111(46):16460–5. Epub 2014/11/03. doi: 10.1073/pnas.1410534111 25368194; PubMed Central PMCID: PMC4246271.

26. Kim M, Ekhteraei-Tousi S, Lewerentz J, Larsson J. The X-linked 1.688 Satellite in. Genetics. 2018;208(2):623–32. Epub 2017/12/13. doi: 10.1534/genetics.117.300581 29242291; PubMed Central PMCID: PMC5788526.

27. Kuhn GC, Küttler H, Moreira-Filho O, Heslop-Harrison JS. The 1.688 repetitive DNA of Drosophila: concerted evolution at different genomic scales and association with genes. Mol Biol Evol. 2012;29(1):7–11. Epub 2011/06/28. doi: 10.1093/molbev/msr173 21712468.

28. Champer SE, Oh SY, Liu C, Wen Z, Clark AG, Messer PW, et al. Computational and experimental performance of CRISPR homing gene drive strategies with multiplexed gRNAs. bioRxiv. 2019:679902. doi: 10.1101/679902

29. Chan YS, Naujoks DA, Huen DS, Russell S. Insect population control by homing endonuclease-based gene drive: an evaluation in Drosophila melanogaster. Genetics. 2011;188(1):33–44. doi: 10.1534/genetics.111.127506 21368273; PubMed Central PMCID: PMC3120159.

30. Lindsley DL, Grell EH. Spermiogenesis without chromosomes in Drosophila melanogaster. Genetics. 1969;61(1):Suppl:69–78. Epub 1969/01/01. 5345403.

31. Devlin EE, Dacosta L, Mohandas N, Elliott G, Bodine DM. A transgenic mouse model demonstrates a dominant negative effect of a point mutation in the RPS19 gene associated with Diamond-Blackfan anemia. Blood. 2010;116(15):2826–35. Epub 2010/07/06. doi: 10.1182/blood-2010-03-275776 20606162; PubMed Central PMCID: PMC2974590.

32. Buchman A, Akbari OS. Site-specific transgenesis of the Drosophila melanogaster Y-chromosome using CRISPR/Cas9. Insect Mol Biol. 2019;28(1):65–73. Epub 2018/10/08. doi: 10.1111/imb.12528 30079589.

33. Vibranovski MD, Zhang YE, Kemkemer C, Lopes HF, Karr TL, Long M. Re-analysis of the larval testis data on meiotic sex chromosome inactivation revealed evidence for tissue-specific gene expression related to the drosophila X chromosome. BMC Biol. 2012;10:49; author reply 50. Epub 2012/06/12. doi: 10.1186/1741-7007-10-49 22691264; PubMed Central PMCID: PMC3391172.

34. Mikhaylova LM, Nurminsky DI. Lack of global meiotic sex chromosome inactivation, and paucity of tissue-specific gene expression on the Drosophila X chromosome. BMC Biol. 2011;9:29. Epub 2011/05/04. doi: 10.1186/1741-7007-9-29 21542906; PubMed Central PMCID: PMC3104377.

35. Meccariello A, Salvemini M, Primo P, Hall B, Koskinioti P, Dalikova M, et al. Maleness-on-the-Y (MoY) orchestrates male sex determination in major agricultural fruit fly pests. Science. 2019;365(6460):1457–60. Epub 2019/08/31. doi: 10.1126/science.aax1318 31467189.

36. Krzywinska E, Dennison NJ, Lycett GJ, Krzywinski J. A maleness gene in the malaria mosquito Anopheles gambiae. Science. 2016;353(6294):67–9. doi: 10.1126/science.aaf5605 27365445.

37. Hall AB, Papathanos PA, Sharma A, Cheng C, Akbari OS, Assour L, et al. Radical remodeling of the Y chromosome in a recent radiation of malaria mosquitoes. Proc Natl Acad Sci U S A. 2016;113(15):E2114–23. Epub 2016/03/29. doi: 10.1073/pnas.1525164113 27035980; PubMed Central PMCID: PMC4839409.

38. McKenna A, Shendure J. FlashFry: a fast and flexible tool for large-scale CRISPR target design. BMC Biol. 2018;16(1):74. Epub 2018/07/05. doi: 10.1186/s12915-018-0545-0 29976198; PubMed Central PMCID: PMC6033233.

39. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al. RNA-guided human genome engineering via Cas9. Science. 2013;339(6121):823–6. Epub 2013/01/05. doi: 10.1126/science.1232033 23287722; PubMed Central PMCID: PMC3712628.

40. Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell. 2015;163(3):759–71. Epub 2015/10/01. doi: 10.1016/j.cell.2015.09.038 26422227; PubMed Central PMCID: PMC4638220.

41. Pinello L, Canver MC, Hoban MD, Orkin SH, Kohn DB, Bauer DE, et al. Analyzing CRISPR genome-editing experiments with CRISPResso. Nat Biotechnol. 2016;34(7):695–7. Epub 2016/07/13. doi: 10.1038/nbt.3583 27404874; PubMed Central PMCID: PMC5242601.


Článek vyšel v časopise

PLOS Genetics


2020 Číslo 3

Nejčtenější v tomto čísle

Tomuto tématu se dále věnují…


Kurzy

Zvyšte si kvalifikaci online z pohodlí domova

Nová éra v léčbě migrény
nový kurz
Autoři: MUDr. Eva Medová, MUDr. Tomáš Nežádal, Ph.D.

Imunitní trombocytopenie (ITP) u dospělých pacientů
Autoři: prof. MUDr. Tomáš Kozák, Ph.D., MBA

Pěnová skleroterapie
Autoři: MUDr. Marek Šlais

White paper - jak vidíme optimální péči o zubní náhrady
Autoři: MUDr. Jindřich Charvát, CSc.

Hemofilie - série kurzů

Všechny kurzy
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!

×