1887

Abstract

Control of biological populations remains a critical goal to address the challenges facing ecosystems and agriculture and those posed by human disease, including pests, parasites, pathogens and invasive species. A particular architecture of the CRISPR/Cas biotechnology – a gene drive – has the potential to modify or eliminate populations on a massive scale. Super-Mendelian inheritance has now been demonstrated in both fungi and metazoans, including disease vectors such as mosquitoes. Studies in yeast and fly model systems have developed a number of molecular safeguards to increase biosafety and control over drive systems , including titration of nuclease activity, anti-CRISPR-dependent inhibition and use of non-native DNA target sites. We have developed a CRISPR/Cas9 gene drive in that allows for the safe and rapid examination of alternative drive designs and control mechanisms. In this study, we tested whether non-homologous end-joining (NHEJ) had occurred within diploid cells displaying a loss of the target allele following drive activation and did not detect any instances of NHEJ within multiple sampled populations. We also demonstrated successful multiplexing using two additional non-native target sequences. Furthermore, we extended our analysis of ‘resistant’ clones that still harboured both the drive and target selection markers following expression of Cas9; mutation or NHEJ-based repair could not explain the majority of these heterozygous clones. Finally, we developed a second-generation gene drive in yeast with a guide RNA cassette integrated within the drive locus with a near 100 % success rate; resistant clones in this system could also be reactivated during a second round of Cas9 induction.

Loading

Article metrics loading...

/content/journal/acmi/10.1099/acmi.0.000059
2019-09-11
2019-09-22
Loading full text...

Full text loading...

/deliver/fulltext/acmi/10.1099/acmi.0.000059/acmi000059.html?itemId=/content/journal/acmi/10.1099/acmi.0.000059&mimeType=html&fmt=ahah

References

  1. Sternberg SH, Doudna JA. Expanding the biologist's toolkit with CRISPR-Cas9. Mol Cell 2015;58:568–574 [CrossRef]
    [Google Scholar]
  2. Doudna JA, Charpentier E. Genome editing. the new frontier of genome engineering with CRISPR-Cas9. Science 2014;346:1258096 [CrossRef]
    [Google Scholar]
  3. Wright AV, Nuñez JK, Doudna JA. Biology and applications of CRISPR systems: harnessing nature's toolbox for genome engineering. Cell 2016;164:29–44 [CrossRef]
    [Google Scholar]
  4. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 2012;337:816–821 [CrossRef]
    [Google Scholar]
  5. Jinek M, East A, Cheng A, Lin S, Ma E et al. RNA-programmed genome editing in human cells. Elife 2013;2:e00471 [CrossRef]
    [Google Scholar]
  6. DiCarlo JE, Chavez A, Dietz SL, Esvelt KM, Church GM. Safeguarding CRISPR-Cas9 gene drives in yeast. Nat Biotechnol 2015;33:1250–1255 [CrossRef]
    [Google Scholar]
  7. Gantz VM, Jasinskiene N, Tatarenkova O, Fazekas A, Macias VM et al. Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi. Proc Natl Acad Sci USA 2015;112:E6736–E6743 [CrossRef]
    [Google Scholar]
  8. Hammond A, Galizi R, Kyrou K, Simoni A, Siniscalchi C et al. A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae. Nat Biotechnol 2016;34:78–83 [CrossRef]
    [Google Scholar]
  9. Esvelt KM, Gemmell NJ. Conservation demands safe gene drive. PLoS Biol 2017;15:e2003850 [CrossRef]
    [Google Scholar]
  10. Esvelt KM, Smidler AL, Catteruccia F, Church GM. Concerning RNA-guided gene drives for the alteration of wild populations. Elife 2014;3:e03401 [CrossRef]
    [Google Scholar]
  11. Akbari OS, Bellen HJ, Bier E, Bullock SL, Burt A et al. Biosafety. Safeguarding gene drive experiments in the laboratory. Science 2015;349:927–929 [CrossRef]
    [Google Scholar]
  12. Basgall EM, Goetting SC, Goeckel ME, Giersch RM, Roggenkamp E et al. Gene drive inhibition by the anti-CRISPR proteins AcrIIA2 and AcrIIA4 in Saccharomyces cerevisiae. Microbiology 2018;164:464–474 [CrossRef]
    [Google Scholar]
  13. Roggenkamp E, Giersch RM, Schrock MN, Turnquist E, Halloran M et al. Tuning CRISPR-Cas9 Gene Drives in Saccharomyces cerevisiae. G3 2018;8:999–1018 [CrossRef]
    [Google Scholar]
  14. Del Amo VL et al. Split-gene drive system provides flexible application for safe laboratory investigation and potential field deployment. bioRxiv 2019;684597
    [Google Scholar]
  15. Amo LD V et al. Small-Molecule control of super-Mendelian inheritance in gene drives. bioRxiv 2019;665620
    [Google Scholar]
  16. Vella MR, Gunning CE, Lloyd AL, Gould F. Evaluating strategies for reversing CRISPR-Cas9 gene drives. Sci Rep 2017;7:11038 [CrossRef]
    [Google Scholar]
  17. Roggenkamp E, Giersch RM, Wedeman E, Eaton M, Turnquist E et al. CRISPR-UnLOCK: multipurpose Cas9-based strategies for conversion of yeast libraries and strains. Front Microbiol 2017;8:1773 [CrossRef]
    [Google Scholar]
  18. Halder V, Porter CBM, Chavez A, Shapiro RS. Design, execution, and analysis of CRISPR-Cas9-based deletions and genetic interaction networks in the fungal pathogen candida albicans. Nature protocols 2019
    [Google Scholar]
  19. Shapiro RS, Chavez A, Porter CBM, Hamblin M, Kaas CS et al. A CRISPR-Cas9-based gene drive platform for genetic interaction analysis in candida albicans. Nat Microbiol 2018;3:73–82 [CrossRef]
    [Google Scholar]
  20. Champer J, Liu J, Oh SY, Reeves R, Luthra A et al. Reducing resistance allele formation in CRISPR gene drive. Proc Natl Acad Sci U S A 2018;115:5522–5527 [CrossRef]
    [Google Scholar]
  21. Kyrou K, Hammond AM, Galizi R, Kranjc N, Burt A et al. A CRISPR-Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes. Nat Biotechnol 2018;36:1062–1066 [CrossRef]
    [Google Scholar]
  22. Grunwald HA, Gantz VM, Poplawski G, Xu X-RS, Bier E et al. Super-Mendelian inheritance mediated by CRISPR-Cas9 in the female mouse germline. Nature 2019;566:105109 [CrossRef]
    [Google Scholar]
  23. Goeckel ME, Basgall EM, Lewis IC, Goetting SC, Yan Y et al. Modulating CRISPR gene drive activity through nucleocytoplasmic localization of Cas9 in S. cerevisiae. Fungal Biol Biotechnol 2019;6:2 [CrossRef]
    [Google Scholar]
  24. Yan Y, Finnigan GC. Development of a multi-locus CRISPR gene drive system in budding yeast. Sci Rep 2018;8:17277 [CrossRef]
    [Google Scholar]
  25. Finnigan GC, Thorner J. mCAL: a new approach for versatile multiplex action of Cas9 using one sgRNA and loci flanked by a programmed target sequence. G3 2016;6:2147–2156 [CrossRef]
    [Google Scholar]
  26. Champer J, Chung J, Lee YL, Liu C, Yang E et al. Molecular Safeguarding of CRISPR gene drive experiments. Elife 2019;8:e41439 [CrossRef]
    [Google Scholar]
  27. Sambrook J, Russell DW. Molecular Cloning: A Laboratory Manual , 3rd edn. Cold Spring Harbor Laboratory Press; 2001
    [Google Scholar]
  28. Finnigan GC, Thorner J. Complex in vivo ligation using homologous recombination and high-efficiency plasmid rescue from Saccharomyces cerevisiae. Bio Protoc 2015;5:e1521 [CrossRef]
    [Google Scholar]
  29. DiCarlo JE, Norville JE, Mali P, Rios X, Aach J et al. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res 2013;41:4336–4343 [CrossRef]
    [Google Scholar]
  30. Świat MA, Dashko S, den Ridder M, Wijsman M, van der Oost J et al. FnCpf1: a novel and efficient genome editing tool for Saccharomyces cerevisiae. Nucleic Acids Res 2017;45:12585–12598 [CrossRef]
    [Google Scholar]
  31. Lian J, HamediRad M, Hu S, Zhao H. Combinatorial metabolic engineering using an orthogonal tri-functional CRISPR system. Nat Commun 2017;8:1688 [CrossRef]
    [Google Scholar]
  32. Unckless RL, Clark AG, Messer PW. Evolution of resistance against CRISPR/Cas9 gene drive. Genetics 2017;205:827–841 [CrossRef]
    [Google Scholar]
  33. Hammond AM, Kyrou K, Bruttini M, North A, Galizi R et al. The creation and selection of mutations resistant to a gene drive over multiple generations in the malaria mosquito. PLoS Genet 2017;13:e1007039 [CrossRef]
    [Google Scholar]
  34. Chen B, Gilbert LA, Cimini BA, Schnitzbauer J, Zhang W et al. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 2013;155:1479–1491 [CrossRef]
    [Google Scholar]
  35. Buchman A, Marshall JM, Ostrovski D, Yang T, Akbari OS. Synthetically engineered Medea gene drive system in the worldwide crop pest Drosophila suzukii. Proc Natl Acad Sci USA 2018;115:4725–4730 [CrossRef]
    [Google Scholar]
  36. Noble C, Adlam B, Church GM, Esvelt KM, Nowak MA. Current CRISPR gene drive systems are likely to be highly invasive in wild populations. eLife 2018;7: [CrossRef]
    [Google Scholar]
  37. Noble C, Min J, Olejarz J, Buchthal J, Chavez A et al. Daisy-chain gene drives for the alteration of local populations. Proc Natl Acad Sci USA 2019;116:8275–8282 [CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/acmi/10.1099/acmi.0.000059
Loading
/content/journal/acmi/10.1099/acmi.0.000059
Loading

Data & Media loading...

Supplementary material 1

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error