Advertisement

CRISPR pp 335-348 | Cite as

Precise Genome Editing of Drosophila with CRISPR RNA-Guided Cas9

  • Scott J. Gratz
  • Melissa M. Harrison
  • Jill Wildonger
  • Kate M. O’Connor-Giles
Part of the Methods in Molecular Biology book series (MIMB, volume 1311)

Abstract

The readily programmable CRISPR-Cas9 system is transforming genome engineering. We and others have adapted the S. pyogenes CRISPR-Cas9 system to precisely engineer the Drosophila genome and demonstrated that these modifications are efficiently transmitted through the germline. Here we provide a detailed protocol for engineering small indels, defined deletions, and targeted insertion of exogenous DNA sequences within one month using a rapid DNA injection-based approach.

Key words

CRISPR Cas9 Genome engineering Nonhomologous end joining Homology-directed repair Drosophila 

Abbreviations

CRISPR

Clustered regularly interspaced short palindromic repeats

crRNA

CRISPR RNA

DSB

Double-strand break

dsDNA

Double-stranded DNA

gRNA

Guide RNA

HDR

Homology-directed repair

Indel

Insertion-deletion

NHEJ

Nonhomologous end joining

PAM

Protospacer adjacent motif

ssDNA

Single-stranded DNA

tracrRNA

Trans-activating CRISPR RNA

Notes

Acknowledgements

We are grateful to members of the Harrison, O’Connor-Giles, and Wildonger labs for their help in establishing CRISPR-Cas9 protocols in Drosophila, and to Dustin Rubinstein for comments on this chapter. Our work has been funded by start-up funds from the University of Wisconsin to M.M.H., J.W., and K.O.C.G. and grants from the National Institutes of Health to J.W. (R00 NS072252) and K.O.C.G. (R00 NS060985 and R01 NS078179). Plasmids and transgenic fly lines described here are available through the nonprofit distributor Addgene and the Bloomington Drosophila Stock Center, respectively. Detailed reagent information is available at http://flycrispr.molbio.wisc.edu.

References

  1. 1.
    Bassett AR, Tibbit C, Ponting CP, Liu JL (2013) Highly efficient targeted mutagenesis of drosophila with the CRISPR/Cas9 system. Cell Rep 4(1):220–228. doi: 10.1016/j.celrep.2013.06.020, S2211-1247(13)00312-4 [pii]CrossRefPubMedCentralPubMedGoogle Scholar
  2. 2.
    Gratz SJ, Cummings AM, Nguyen JN, Hamm DC, Donohue LK, Harrison MM, Wildonger J, O'Connor-Giles KM (2013) Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease. Genetics 194(4):1029–1035. doi: 10.1534/genetics.113.152710 CrossRefPubMedCentralPubMedGoogle Scholar
  3. 3.
    Gratz SJ, Ukken FP, Rubinstein CD, Thiede G, Donohue LK, Cummings AM, O’Connor-Giles KM (2014) Highly specific and efficient CRISPR/Cas9-catalyzed homology-directed repair in Drosophila. Genetics 196(4):961–971. doi: 10.1534/genetics.113.160713 CrossRefPubMedCentralPubMedGoogle Scholar
  4. 4.
    Kondo S, Ueda R (2013) Highly improved gene targeting by germline-specific Cas9 expression in Drosophila. Genetics 195(3):715–721. doi: 10.1534/genetics.113.156737 CrossRefPubMedCentralPubMedGoogle Scholar
  5. 5.
    Ren X, Sun J, Housden BE, Hu Y, Roesel C, Lin S, Liu LP, Yang Z, Mao D, Sun L, Wu Q, Ji JY, Xi J, Mohr SE, Xu J, Perrimon N, Ni JQ (2013) Optimized gene editing technology for Drosophila melanogaster using germ line-specific Cas9. Proc Natl Acad Sci U S A 110(47):19012–19017. doi: 10.1073/pnas.1318481110 CrossRefPubMedCentralPubMedGoogle Scholar
  6. 6.
    Sebo ZL, Lee HB, Peng Y, Guo Y (2013) A simplified and efficient germline-specific CRISPR/Cas9 system for Drosophila genomic engineering. Fly (Austin) 8(1)Google Scholar
  7. 7.
    Yu Z, Ren M, Wang Z, Zhang B, Rong YS, Jiao R, Gao G (2013) Highly efficient genome modifications mediated by CRISPR/Cas9 in Drosophila. Genetics 195(1):289–291. doi: 10.1534/genetics.113.153825 CrossRefPubMedCentralPubMedGoogle Scholar
  8. 8.
    Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096):816–821. doi: 10.1126/science.1225829, science.1225829 [pii]CrossRefPubMedGoogle Scholar
  9. 9.
    Cho SW, Kim S, Kim Y, Kweon J, Kim HS, Bae S, Kim JS (2014) Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res 24(1):132–141. doi: 10.1101/gr.162339.113 CrossRefPubMedCentralPubMedGoogle Scholar
  10. 10.
    Cradick TJ, Fine EJ, Antico CJ, Bao G (2013) CRISPR/Cas9 systems targeting beta-globin and CCR5 genes have substantial off-target activity. Nucleic Acids Res 41(20):9584–9592. doi: 10.1093/nar/gkt714 CrossRefPubMedCentralPubMedGoogle Scholar
  11. 11.
    Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK, Sander JD (2013) High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol 31(9):822–826. doi: 10.1038/nbt.2623 CrossRefPubMedCentralPubMedGoogle Scholar
  12. 12.
    Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, Cradick TJ, Marraffini LA, Bao G, Zhang F (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31(9):827–832. doi: 10.1038/nbt.2647 CrossRefPubMedCentralPubMedGoogle Scholar
  13. 13.
    Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (2013) RNA-guided human genome engineering via Cas9. Science 339(6121):823–826. doi: 10.1126/science.1232033 CrossRefPubMedCentralPubMedGoogle Scholar
  14. 14.
    Pattanayak V, Lin S, Guilinger JP, Ma E, Doudna JA, Liu DR (2013) High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat Biotechnol 31(9):839–843. doi: 10.1038/nbt.2673 CrossRefPubMedCentralPubMedGoogle Scholar
  15. 15.
    Semenova E, Jore MM, Datsenko KA, Semenova A, Westra ER, Wanner B, van der Oost J, Brouns SJ, Severinov K (2011) Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence. Proc Natl Acad Sci U S A 108(25):10098–10103. doi: 10.1073/pnas.1104144108 CrossRefPubMedCentralPubMedGoogle Scholar
  16. 16.
    Wiedenheft B, van Duijn E, Bultema JB, Waghmare SP, Zhou K, Barendregt A, Westphal W, Heck AJ, Boekema EJ, Dickman MJ, Doudna JA (2011) RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions. Proc Natl Acad Sci U S A 108(25):10092–10097. doi: 10.1073/pnas.1102716108 CrossRefPubMedCentralPubMedGoogle Scholar
  17. 17.
    Yang H, Wang H, Shivalila CS, Cheng AW, Shi L, Jaenisch R (2013) One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell 154(6):1370–1379. doi: 10.1016/j.cell.2013.08.022 CrossRefPubMedCentralPubMedGoogle Scholar
  18. 18.
    Banga SS, Boyd JB (1992) Oligonucleotide-directed site-specific mutagenesis in Drosophila melanogaster. Proc Natl Acad Sci U S A 89(5):1735–1739CrossRefPubMedCentralPubMedGoogle Scholar
  19. 19.
    Beumer KJ, Trautman JK, Mukherjee K, Carroll D (2013) Donor DNA utilization during gene targeting with zinc-finger nucleases. G3 (Bethesda). doi: 10.1534/g3.112.005439
  20. 20.
    Hwang WY, Fu Y, Reyon D, Maeder ML, Kaini P, Sander JD, Joung JK, Peterson RT, Yeh JR (2013) Heritable and precise zebrafish genome editing using a CRISPR-Cas system. PLoS One 8(7), e68708. doi: 10.1371/journal.pone.0068708, PONE-D-13-13968 [pii]CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Scott J. Gratz
    • 1
  • Melissa M. Harrison
    • 2
  • Jill Wildonger
    • 3
  • Kate M. O’Connor-Giles
    • 1
    • 4
    • 5
  1. 1.Genetics Training ProgramUniversity of Wisconsin-MadisonMadisonUSA
  2. 2.Department of Biomolecular ChemistryUniversity of Wisconsin School of Medicine and Public HealthMadisonUSA
  3. 3.Department of BiochemistryUniversity of Wisconsin-MadisonMadisonUSA
  4. 4.Laboratory of GeneticsUniversity of Wisconsin-MadisonMadisonUSA
  5. 5.Laboratory of Cell and Molecular BiologyUniversity of Wisconsin-MadisonMadisonUSA

Personalised recommendations