Advertisement

Next-Generation Sequencing of Genome-Wide CRISPR Screens

  • Edwin H. Yau
  • Tariq M. RanaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1712)

Abstract

Genome-wide functional genomic screens utilizing the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system have proven to be a powerful tool for systematic genomic perturbation in mammalian cells and provide an alternative to previous screens utilizing RNA interference technology. The wide availability of these libraries through public plasmid repositories as well as the decreasing cost and speed in quantifying these screens using high-throughput next-generation sequencing (NGS) allows for the adoption of the technology in a variety of laboratories interested in diverse biologic questions. Here, we describe the protocol to generate next-generation sequencing libraries from genome-wide CRISPR genomic screens.

Key words

CRISPR Cas9 Genome-wide screen Genome engineering GeCKO 

Notes

Acknowledgments

We thank the Rana lab members, John Shimishata, and Steven Head at The Scripps Research Institute Genomic Core. This work was supported in part by grants from the National Institutes of Health to T.M.R., E.Y, is supported by the National Cancer Institute of the National Institutes of Health under award number T32CA121938. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

References

  1. 1.
    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 337:816–821CrossRefPubMedGoogle Scholar
  2. 2.
    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 339:823–826CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome-engineering using CRISPR/Cas systems. Science 339:819–823CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Doudna JA, Charpentier E (2014) Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346:1258096CrossRefPubMedGoogle Scholar
  5. 5.
    Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157:1262–1278CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Sander JD, Joung JK (2014) CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol 4:347–355CrossRefGoogle Scholar
  7. 7.
    Sternberg SH, Doudna JA (2015) Expanding the biologist’s toolkit with CRISPR-Cas0. Mol Cell 58:568–574CrossRefPubMedGoogle Scholar
  8. 8.
    Wang T, Birsoy K, Hughes NW, Krupczak KM, Post Y, Wei JJ, Lander ES, Sabatini DM (2015) Identification and characterization of essential genes in the human genome. Science 350:1096–1101CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Hart T, Chandrashekhar M, Aregger M, Steinhart Z, Brown KR, MacLeod G, Mis M, Zimmermann M, Fradet-Turcotte A, Sun S, Mero P, Dirks P, Sidhu S, Roth FP, Rissland OS, Durocher D, Angers S, Moffat J (2015) High-resolution CRISPR screen reveal fitness genes and genotype-specific cancer liabilities. Cell 163:1515–1526CrossRefPubMedGoogle Scholar
  10. 10.
    Morgens DW, Deans RM, Li A, Bassik MC (2016) Systematic comparison of CRISPR/Cas9 and RNAi screens for essential genes. Nat Biotechnol 34:634–636CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelsen TS, Heckl D, Ebert BL, Root DE, Doench JG, Zhang F (2014) Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343:84–87CrossRefPubMedGoogle Scholar
  12. 12.
    Wang T, Wei JJ, Sabatini DM, Lander ES (2014) Genetic screens in human cells using the CRISPR-Cas9 system. Science 343:80–84CrossRefPubMedGoogle Scholar
  13. 13.
    Gilbert LA, Horlbeck MA, Adamson B, Villalta JE, Chen Y, Whitehead EH, Gulmaraes C, Panning B, Pioegh HL, Bassik MC, Qi LS, Kampmann M, Weissman JS (2014) Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159:647–661CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Virreira Winter S, Zychlinsky A, Bardoel BW (2016) Genome-wide CRISPR screen reveals novel host factors required for Staphylococcus aureus α-hemolysin-mediated toxicity. Sci Rep 6:24242CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Kampmann M, Bassik MC, Weissman JS (2014) Functional genomics platform for pooled screening and mammalian genetic interaction maps. Nat Protoc 9:1825–1847CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF, Smith I, Tothova Z, Wilen C, Orchard R, Virgin HW, Listgarten J, Root DE (2016) Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol 34:184–191CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Chen S, Sanjana NE, Zheng K, Shalem O, Lee K, Shi X, Scott DA, Song J, Pan JQ, Weissleder R, Lee H, Zhang F, Sharp PA (2015) Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis. Cell 160:1246–1260CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  1. 1.Division of Hematology-Oncology, Department of Internal MedicineUniversity of California, San DiegoLa JollaUSA
  2. 2.Solid Tumor Therapeutics Program, Moores Cancer CenterUniversity of California, San DiegoLa JollaUSA
  3. 3.Department of PediatricsUniversity of California, San DiegoLa JollaUSA
  4. 4.Institute for Genomic MedicineUniversity of California, San DiegoLa JollaUSA

Personalised recommendations