#PAGE_PARAMS# #ADS_HEAD_SCRIPTS# #MICRODATA#

Generation of deletions and precise point mutations in Dictyostelium discoideum using the CRISPR nickase


Autoři: Hoshie Iriki aff001;  Takefumi Kawata aff001;  Tetsuya Muramoto aff001
Působiště autorů: Department of Biology, Faculty of Science, Toho University, Funabashi, Chiba, Japan aff001
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pone.0224128

Souhrn

The CRISPR/Cas9 system enables targeted genome modifications across a range of eukaryotes. Although we have reported that transient introduction of all-in-one vectors that express both Cas9 and sgRNAs can efficiently induce multiple gene knockouts in Dictyostelium discoideum, concerns remain about off-target effects and false-positive amplification during mutation detection via PCR. To minimise these effects, we modified the system to permit gene deletions of greater than 1 kb via use of paired sgRNAs and Cas9 nickase. An all-in-one vector expressing the Cas9 nickase and sgRNAs was transiently introduced into D. discoideum, and the resulting mutants showed long deletions with a relatively high efficiency of 10–30%. By further improving the vector, a new dual sgRNA expression vector was also constructed to allow simultaneous insertion of two sgRNAs via one-step cloning. By applying this system, precise point mutations and genomic deletions were generated in the target locus via simultaneous introduction of the vector and a single-stranded oligonucleotide template without integrating a drug resistance cassette. These systems enable simple and straightforward genome editing that requires high specificity, and they can serve as an alternative to the conventional homologous recombination-based gene disruption method in D. discoideum.

Klíčová slova:

CRISPR – Deletion mutation – Dictyostelium discoideum – Point mutation – Polymerase chain reaction – Vector cloning – Oligonucleotides


Zdroje

1. Kessin RH. Dictyostelium—Evolution, cell biology, and the development of multicellularity. Cambridge, UK: Cambridge Univ. Press; 2001. 318 p.

2. Eichinger L, Pachebat JA, Glockner G, Rajandream MA, Sucgang R, Berriman M, et al. The genome of the social amoeba Dictyostelium discoideum. Nature. 2005;435(7038):43–57. Epub 2005/05/06. doi: 10.1038/nature03481 15875012.

3. Gaudet P, Pilcher KE, Fey P, Chisholm RL. Transformation of Dictyostelium discoideum with plasmid DNA. Nat Protoc. 2007;2(6):1317–24. Epub 2007/06/05. doi: 10.1038/nprot.2007.179 17545968.

4. Linkner J, Nordholz B, Junemann A, Winterhoff M, Faix J. Highly effective removal of floxed Blasticidin S resistance cassettes from Dictyostelium discoideum mutants by extrachromosomal expression of Cre. European journal of cell biology. 2012;91(2):156–60. doi: 10.1016/j.ejcb.2011.11.001 22154549.

5. Muramoto T, Iriki H, Watanabe J, Kawata T. Recent Advances in CRISPR/Cas9-Mediated Genome Editing in Dictyostelium. Cells. 2019;8(1). Epub 2019/01/16. doi: 10.3390/cells8010046 30642074.

6. Sekine R, Kawata T, Muramoto T. CRISPR/Cas9 mediated targeting of multiple genes in Dictyostelium. Sci Rep. 2018;8(1):8471. Epub 2018/06/02. doi: 10.1038/s41598-018-26756-z 29855514.

7. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337(6096):816–21. Epub 2012/06/30. doi: 10.1126/science.1225829 22745249.

8. Chu VT, Weber T, Wefers B, Wurst W, Sander S, Rajewsky K, et al. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat Biotechnol. 2015;33(5):543–8. Epub 2015/03/25. doi: 10.1038/nbt.3198 25803306.

9. Lin S, Staahl BT, Alla RK, Doudna JA. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. Elife. 2014;3:e04766. Epub 2014/12/17. doi: 10.7554/eLife.04766 25497837.

10. Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol. 2013;31(9):827–32. Epub 2013/07/23. doi: 10.1038/nbt.2647 23873081.

11. Anderson KR, Haeussler M, Watanabe C, Janakiraman V, Lund J, Modrusan Z, et al. CRISPR off-target analysis in genetically engineered rats and mice. Nat Methods. 2018;15(7):512–4. Epub 2018/05/23. doi: 10.1038/s41592-018-0011-5 29786090.

12. Pattanayak V, Lin S, Guilinger JP, Ma E, Doudna JA, Liu DR. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat Biotechnol. 2013;31(9):839–43. Epub 2013/08/13. doi: 10.1038/nbt.2673 23934178.

13. Tsai SQ, Zheng Z, Nguyen NT, Liebers M, Topkar VV, Thapar V, et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol. 2015;33(2):187–97. Epub 2014/12/17. doi: 10.1038/nbt.3117 25513782.

14. Fu Y, Sander JD, Reyon D, Cascio VM, Joung JK. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol. 2014;32(3):279–84. Epub 2014/01/28. doi: 10.1038/nbt.2808 24463574.

15. Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, Trevino AE, et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell. 2013;154(6):1380–9. Epub 2013/09/03. doi: 10.1016/j.cell.2013.08.021 23992846.

16. Shen B, Zhang W, Zhang J, Zhou J, Wang J, Chen L, et al. Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nat Methods. 2014;11(4):399–402. Epub 2014/03/04. doi: 10.1038/nmeth.2857 24584192.

17. Krokan HE, Bjoras M. Base excision repair. Cold Spring Harb Perspect Biol. 2013;5(4):a012583. Epub 2013/04/03. doi: 10.1101/cshperspect.a012583 23545420.

18. Ghezraoui H, Piganeau M, Renouf B, Renaud JB, Sallmyr A, Ruis B, et al. Chromosomal translocations in human cells are generated by canonical nonhomologous end-joining. Mol Cell. 2014;55(6):829–42. Epub 2014/09/10. doi: 10.1016/j.molcel.2014.08.002 25201414.

19. Vriend LE, Prakash R, Chen CC, Vanoli F, Cavallo F, Zhang Y, et al. Distinct genetic control of homologous recombination repair of Cas9-induced double-strand breaks, nicks and paired nicks. Nucleic acids research. 2016;44(11):5204–17. Epub 2016/03/24. doi: 10.1093/nar/gkw179 27001513.

20. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339(6121):819–23. Epub 2013/01/05. doi: 10.1126/science.1231143 23287718.

21. Mali P, Aach J, Stranges PB, Esvelt KM, Moosburner M, Kosuri S, et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol. 2013;31(9):833–8. Epub 2013/08/03. doi: 10.1038/nbt.2675 23907171.

22. Cho SW, Kim S, Kim Y, Kweon J, Kim HS, Bae S, et al. Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res. 2014;24(1):132–41. Epub 2013/11/21. doi: 10.1101/gr.162339.113 24253446.

23. Paschke P, Knecht DA, Silale A, Traynor D, Williams TD, Thomason PA, et al. Rapid and efficient genetic engineering of both wild type and axenic strains of Dictyostelium discoideum. PLoS One. 2018;13(5):e0196809. Epub 2018/05/31. doi: 10.1371/journal.pone.0196809 29847546.

24. Veltman DM, Akar G, Bosgraaf L, Van Haastert PJ. A new set of small, extrachromosomal expression vectors for Dictyostelium discoideum. Plasmid. 2009;61(2):110–8. Epub 2008/12/10. doi: 10.1016/j.plasmid.2008.11.003 19063918.

25. Park J, Bae S, Kim JS. Cas-Designer: a web-based tool for choice of CRISPR-Cas9 target sites. Bioinformatics. 2015;31(24):4014–6. doi: 10.1093/bioinformatics/btv537 26358729.

26. Mann SK, Yonemoto WM, Taylor SS, Firtel RA. DdPK3, which plays essential roles during Dictyostelium development, encodes the catalytic subunit of cAMP-dependent protein kinase. Proc Natl Acad Sci U S A. 1992;89(22):10701–5. Epub 1992/11/15. doi: 10.1073/pnas.89.22.10701 1332055.

27. Anjard C, Etchebehere L, Pinaud S, Veron M, Reymond CD. An unusual catalytic subunit for the cAMP-dependent protein kinase of Dictyostelium discoideum. Biochemistry. 1993;32(37):9532–8. Epub 1993/09/21. doi: 10.1021/bi00088a003 8373760.

28. Adikusuma F, Pfitzner C, Thomas PQ. Versatile single-step-assembly CRISPR/Cas9 vectors for dual gRNA expression. PLoS One. 2017;12(12):e0187236. Epub 2017/12/07. doi: 10.1371/journal.pone.0187236 29211736.

29. Gopalappa R, Suresh B, Ramakrishna S, Kim HH. Paired D10A Cas9 nickases are sometimes more efficient than individual nucleases for gene disruption. Nucleic acids research. 2018;46(12):e71. Epub 2018/03/28. doi: 10.1093/nar/gky222 29584876.

30. Brinkman EK, Chen T, de Haas M, Holland HA, Akhtar W, van Steensel B. Kinetics and Fidelity of the Repair of Cas9-Induced Double-Strand DNA Breaks. Mol Cell. 2018;70(5):801–13 e6. Epub 2018/05/29. doi: 10.1016/j.molcel.2018.04.016 29804829.

31. Leatherbarrow EL, Harper JV, Cucinotta FA, O’Neill P. Induction and quantification of gamma-H2AX foci following low and high LET-irradiation. Int J Radiat Biol. 2006;82(2):111–8. Epub 2006/03/21. doi: 10.1080/09553000600599783 16546909.

32. Sharma PM, Ponnaiya B, Taveras M, Shuryak I, Turner H, Brenner DJ. High throughput measurement of gammaH2AX DSB repair kinetics in a healthy human population. PLoS One. 2015;10(3):e0121083. Epub 2015/03/21. doi: 10.1371/journal.pone.0121083 25794041.

33. Shibata A, Conrad S, Birraux J, Geuting V, Barton O, Ismail A, et al. Factors determining DNA double-strand break repair pathway choice in G2 phase. EMBO J. 2011;30(6):1079–92. Epub 2011/02/15. doi: 10.1038/emboj.2011.27 21317870.

34. Muramoto T, Chubb JR. Live imaging of the Dictyostelium cell cycle reveals widespread S phase during development, a G2 bias in spore differentiation and a premitotic checkpoint. Development. 2008;135(9):1647–57. Epub 2008/03/28. doi: 10.1242/dev.020115 18367554.

35. Hudson JJ, Hsu DW, Guo K, Zhukovskaya N, Liu PH, Williams JG, et al. DNA-PKcs-dependent signaling of DNA damage in Dictyostelium discoideum. Curr Biol. 2005;15(20):1880–5. Epub 2005/10/26. doi: 10.1016/j.cub.2005.09.039 16243037.

36. Jasin M, Haber JE. The democratization of gene editing: Insights from site-specific cleavage and double-strand break repair. DNA Repair (Amst). 2016;44:6–16. Epub 2016/06/05. doi: 10.1016/j.dnarep.2016.05.001 27261202.

37. Kalousi A, Soutoglou E. Nuclear compartmentalization of DNA repair. Curr Opin Genet Dev. 2016;37:148–57. Epub 2016/06/09. doi: 10.1016/j.gde.2016.05.013 27266837.

38. Hustedt N, Durocher D. The control of DNA repair by the cell cycle. Nat Cell Biol. 2016;19(1):1–9. Epub 2016/12/23. doi: 10.1038/ncb3452 28008184.

39. Weijer CJ, Duschl G, David CN. A revision of the Dictyostelium discoideum cell cycle. J Cell Sci. 1984;70:111–31. Epub 1984/08/01. 6389576.

40. McVey M, Lee SE. MMEJ repair of double-strand breaks (director’s cut): deleted sequences and alternative endings. Trends Genet. 2008;24(11):529–38. Epub 2008/09/24. doi: 10.1016/j.tig.2008.08.007 18809224.

41. Owens DDG, Caulder A, Frontera V, Harman JR, Allan AJ, Bucakci A, et al. Microhomologies are prevalent at Cas9-induced larger deletions. Nucleic acids research. 2019;47(14):7402–17. Epub 2019/05/28. doi: 10.1093/nar/gkz459 31127293.

42. Muramoto T, Muller I, Thomas G, Melvin A, Chubb JR. Methylation of H3K4 Is required for inheritance of active transcriptional states. Curr Biol. 2010;20(5):397–406. Epub 2010/03/02. doi: 10.1016/j.cub.2010.01.017 20188556.

43. Raj A, van Oudenaarden A. Nature, nurture, or chance: stochastic gene expression and its consequences. Cell. 2008;135(2):216–26. Epub 2008/10/30. doi: 10.1016/j.cell.2008.09.050 18957198.

44. Stevense M, Muramoto T, Muller I, Chubb JR. Digital nature of the immediate-early transcriptional response. Development. 2010;137(4):579–84. Epub 2010/01/30. doi: 10.1242/dev.043836 20110323.

45. Veltman DM, Keizer-Gunnink I, Haastert PJ. An extrachromosomal, inducible expression system for Dictyostelium discoideum. Plasmid. 2009;61(2):119–25. Epub 2008/12/03. doi: 10.1016/j.plasmid.2008.11.002 19046986.

46. Yang S, Sleight SC, Sauro HM. Rationally designed bidirectional promoter improves the evolutionary stability of synthetic genetic circuits. Nucleic acids research. 2013;41(1):e33. Epub 2012/10/25. doi: 10.1093/nar/gks972 23093602.

47. Komor AC, Kim YB, Packer MS, Zuris JA, Liu DR. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature. 2016;533(7603):420–4. Epub 2016/04/21. doi: 10.1038/nature17946 27096365.

48. Komor AC, Zhao KT, Packer MS, Gaudelli NM, Waterbury AL, Koblan LW, et al. Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity. Sci Adv. 2017;3(8):eaao4774. Epub 2017/09/07. doi: 10.1126/sciadv.aao4774 28875174.

49. Nishida K, Arazoe T, Yachie N, Banno S, Kakimoto M, Tabata M, et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science. 2016;353(6305). Epub 2016/08/06. doi: 10.1126/science.aaf8729 27492474.

50. Hsu DW, Chubb JR, Muramoto T, Pears CJ, Mahadevan LC. Dynamic acetylation of lysine-4-trimethylated histone H3 and H3 variant biology in a simple multicellular eukaryote. Nucleic acids research. 2012;40(15):7247–56. Epub 2012/05/19. doi: 10.1093/nar/gks367 22600736.

51. Devreotes P, Horwitz AR. Signaling networks that regulate cell migration. Cold Spring Harb Perspect Biol. 2015;7(8):a005959. Epub 2015/08/05. doi: 10.1101/cshperspect.a005959 26238352.

52. Insall R. The interaction between pseudopods and extracellular signalling during chemotaxis and directed migration. Curr Opin Cell Biol. 2013;25(5):526–31. Epub 2013/06/12. doi: 10.1016/j.ceb.2013.04.009 23747069.


Článek vyšel v časopise

PLOS One


2019 Číslo 10
Nejčtenější tento týden
Nejčtenější v tomto čísle
Kurzy

Zvyšte si kvalifikaci online z pohodlí domova

plice
INSIGHTS from European Respiratory Congress
nový kurz

Současné pohledy na riziko v parodontologii
Autoři: MUDr. Ladislav Korábek, CSc., MBA

Svět praktické medicíny 3/2024 (znalostní test z časopisu)

Kardiologické projevy hypereozinofilií
Autoři: prof. MUDr. Petr Němec, Ph.D.

Střevní příprava před kolonoskopií
Autoři: MUDr. Klára Kmochová, Ph.D.

Všechny kurzy
Kurzy Podcasty Doporučená témata Časopisy
Přihlášení
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

#ADS_BOTTOM_SCRIPTS#