Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening

An Author Correction to this article was published on 22 October 2018

This article has been updated

Abstract

Forward genetic screens are powerful tools for the unbiased discovery and functional characterization of specific genetic elements associated with a phenotype of interest. Recently, the RNA-guided endonuclease Cas9 from the microbial CRISPR (clustered regularly interspaced short palindromic repeats) immune system has been adapted for genome-scale screening by combining Cas9 with pooled guide RNA libraries. Here we describe a protocol for genome-scale knockout and transcriptional activation screening using the CRISPR-Cas9 system. Custom- or ready-made guide RNA libraries are constructed and packaged into lentiviral vectors for delivery into cells for screening. As each screen is unique, we provide guidelines for determining screening parameters and maintaining sufficient coverage. To validate candidate genes identified by the screen, we further describe strategies for confirming the screening phenotype, as well as genetic perturbation, through analysis of indel rate and transcriptional activation. Beginning with library design, a genome-scale screen can be completed in 9–15 weeks, followed by 4–5 weeks of validation.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Approaches to genetic perturbation: shRNA knockdown, Cas9 knockout, and dCas9 transcriptional activation.
Figure 2: Timeline and overview of experiments.
Figure 3: GeCKO and SAM libraries for genome-scale knockout and activation screens.
Figure 4: Anticipated results for genome-scale knockout and activation screens.

Similar content being viewed by others

Change history

  • 13 April 2017

    In the version of this article initially published, the wrong version of Supplementary Data 4 was provided, and links to the GitHub page hosting the same files (which will provide ongoing access to updated future versions) were omitted. This error and omission have been corrected for the PDF and HTML versions of this article.

  • 22 October 2018

    In the published version of this paper, Step 64 of the Procedure reads, "Refer to Steps 37-39 for NGS analysis of the sgRNA distribution." This step should refer the reader to Steps 35-39. This text has not been corrected in the original paper.

References

  1. Boutros, M. & Ahringer, J. The art and design of genetic screens: RNA interference. Nat. Rev. Genet. 9, 554–566 (2008).

    Article  CAS  PubMed  Google Scholar 

  2. Echeverri, C.J. & Perrimon, N. High-throughput RNAi screening in cultured cells: a user's guide. Nat. Rev. Genet. 7, 373–384 (2006).

    Article  CAS  PubMed  Google Scholar 

  3. Shalem, O., Sanjana, N.E. & Zhang, F. High-throughput functional genomics using CRISPR-Cas9. Nat. Rev. Genet. 16, 299–311 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Elbashir, S.M. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498 (2001).

    Article  CAS  PubMed  Google Scholar 

  5. Fire, A. et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811 (1998).

    Article  CAS  PubMed  Google Scholar 

  6. McManus, M.T. & Sharp, P.A. Gene silencing in mammals by small interfering RNAs. Nat. Rev. Genet. 3, 737–747 (2002).

    Article  CAS  PubMed  Google Scholar 

  7. Meister, G. & Tuschl, T. Mechanisms of gene silencing by double-stranded RNA. Nature 431, 343–349 (2004).

    Article  CAS  PubMed  Google Scholar 

  8. Berns, K. et al. A large-scale RNAi screen in human cells identifies new components of the p53 pathway. Nature 428, 431–437 (2004).

    Article  CAS  PubMed  Google Scholar 

  9. Boutros, M. et al. Genome-wide RNAi analysis of growth and viability in Drosophila cells. Science 303, 832–835 (2004).

    Article  CAS  PubMed  Google Scholar 

  10. Moffat, J. & Sabatini, D.M. Building mammalian signalling pathways with RNAi screens. Nat. Rev. Mol. Cell Biol. 7, 177–187 (2006).

    Article  CAS  PubMed  Google Scholar 

  11. Paddison, P.J. et al. A resource for large-scale RNA-interference-based screens in mammals. Nature 428, 427–431 (2004).

    Article  CAS  PubMed  Google Scholar 

  12. Root, D.E., Hacohen, N., Hahn, W.C., Lander, E.S. & Sabatini, D.M. Genome-scale loss-of-function screening with a lentiviral RNAi library. Nat. Methods 3, 715–719 (2006).

    Article  CAS  PubMed  Google Scholar 

  13. Silva, J.M. et al. Second-generation shRNA libraries covering the mouse and human genomes. Nat. Genet. 37, 1281–1288 (2005).

    Article  CAS  PubMed  Google Scholar 

  14. Birmingham, A. et al. 3′ UTR seed matches, but not overall identity, are associated with RNAi off-targets. Nat. Methods 3, 199–204 (2006).

    Article  CAS  PubMed  Google Scholar 

  15. Jackson, A.L. et al. Expression profiling reveals off-target gene regulation by RNAi. Nat. Biotechnol. 21, 635–637 (2003).

    Article  CAS  PubMed  Google Scholar 

  16. Jackson, A.L. & Linsley, P.S. Recognizing and avoiding siRNA off-target effects for target identification and therapeutic application. Nat. Rev. Drug Discov. 9, 57–67 (2010).

    Article  CAS  PubMed  Google Scholar 

  17. Wright, A.V., Nunez, J.K. & Doudna, J.A. Biology and applications of CRISPR systems: harnessing nature's toolbox for genome engineering. Cell 164, 29–44 (2016).

    Article  CAS  PubMed  Google Scholar 

  18. Marraffini, L.A. CRISPR-Cas immunity in prokaryotes. Nature 526, 55–61 (2015).

    Article  CAS  PubMed  Google Scholar 

  19. Hsu, P.D., Lander, E.S. & Zhang, F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262–1278 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Garneau, J.E. et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468, 67–71 (2010).

    Article  CAS  PubMed  Google Scholar 

  21. Deltcheva, E. et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471, 602–607 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Gasiunas, G., Barrangou, R., Horvath, P. & Siksnys, V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl. Acad. Sci. USA 109, E2579–E2586 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Rouet, P., Smih, F. & Jasin, M. Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease. Mol. Cell Biol. 14, 8096–8106 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Chavez, A. et al. Highly efficient Cas9-mediated transcriptional programming. Nat. Methods 12, 326–328 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Gilbert, L.A. et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159, 647–661 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Gilbert, L.A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–451 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Konermann, S. et al. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517, 583–588 (2015).

    Article  CAS  PubMed  Google Scholar 

  31. Maeder, M.L. et al. CRISPR RNA-guided activation of endogenous human genes. Nat. Methods 10, 977–979 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Mali, P. et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat. Biotechnol. 31, 833–838 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Perez-Pinera, P. et al. RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Nat. Methods 10, 973–976 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Qi, L.S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Konermann, S. et al. Optical control of mammalian endogenous transcription and epigenetic states. Nature 500, 472–476 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Chavez, A. et al. Comparison of Cas9 activators in multiple species. Nat. Methods 13, 563–567 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Shalem, O. et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343, 84–87 (2014).

    Article  CAS  PubMed  Google Scholar 

  38. Wang, T., Wei, J.J., Sabatini, D.M. & Lander, E.S. Genetic screens in human cells using the CRISPR-Cas9 system. Science 343, 80–84 (2014).

    Article  CAS  PubMed  Google Scholar 

  39. Koike-Yusa, H., Li, Y., Tan, E.P., Velasco-Herrera Mdel, C. & Yusa, K. Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library. Nat. Biotechnol. 32, 267–273 (2014).

    Article  CAS  PubMed  Google Scholar 

  40. Zhou, Y. et al. High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells. Nature 509, 487–491 (2014).

    Article  CAS  PubMed  Google Scholar 

  41. Doench, J.G. et al. Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nat. Biotechnol. 32, 1262–1267 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Chen, S. et al. Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis. Cell 160, 1246–1260 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Shi, J. et al. Discovery of cancer drug targets by CRISPR-Cas9 screening of protein domains. Nat. Biotechnol. 33, 661–667 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Parnas, O. et al. A genome-wide CRISPR screen in primary immune cells to dissect regulatory networks. Cell 162, 675–686 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Wang, T. et al. Identification and characterization of essential genes in the human genome. Science 350, 1096–1101 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Wong, A.S. et al. Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM. Proc. Natl. Acad. Sci. USA 113, 2544–2549 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Doench, J.G. et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat. Biotechnol. 34, 184–191 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Jain, I.H. et al. Hypoxia as a therapy for mitochondrial disease. Science 352, 54–61 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Marceau, C.D. et al. Genetic dissection of Flaviviridae host factors through genome-scale CRISPR screens. Nature 535, 159–163 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Zhang, R. et al. A CRISPR screen defines a signal peptide processing pathway required by flaviviruses. Nature 535, 164–168 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Blondel, C.J. et al. CRISPR/Cas9 screens reveal requirements for host cell sulfation and fucosylation in bacterial type III secretion system-mediated cytotoxicity. Cell Host Microbe 20, 226–237 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Orchard, R.C. et al. Discovery of a proteinaceous cellular receptor for anorovirus. Science 353, 933–936 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Canver, M.C. et al. BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis. Nature 527, 192–197 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Korkmaz, G. et al. Functional genetic screens for enhancer elements in the human genome using CRISPR-Cas9. Nat. Biotechnol. 34, 192–198 (2016).

    Article  CAS  PubMed  Google Scholar 

  55. Diao, Y. et al. A new class of temporarily phenotypic enhancers identified by CRISPR/Cas9-mediated genetic screening. Genome Res. 26, 397–405 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Sanjana, N.E. et al. High-resolution interrogation of functional elements in the noncoding genome. Science 353, 1545–1549 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Fulco, C.P. et al. Systematic mapping of functional enhancer-promoter connections with CRISPR interference. Science 354, 769–773 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Fu, Y. et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol. 31, 822–826 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Hsu, P.D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Pattanayak, V. et al. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat. Biotechnol. 31, 839–843 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Evers, B. et al. CRISPR knockout screening outperforms shRNA and CRISPRi in identifying essential genes. Nat. Biotechnol. 34, 631–633 (2016).

    Article  CAS  PubMed  Google Scholar 

  62. Munoz, D.M. et al. CRISPR screens provide a comprehensive assessment of cancer vulnerabilities but generate false-positive hits for highly amplified genomic regions. Cancer Discov. 6, 900–913 (2016).

    Article  CAS  PubMed  Google Scholar 

  63. Aguirre, A.J. et al. Genomic copy number dictates a gene-independent cell response to CRISPR/Cas9 targeting. Cancer Discov. 6, 914–929 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Morgens, D.W., Deans, R.M., Li, A. & Bassik, M.C. Systematic comparison of CRISPR/Cas9 and RNAi screens for essential genes. Nat. Biotechnol. 34, 634–636 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Hasson, S.A. et al. High-content genome-wide RNAi screens identify regulators of parkin upstream of mitophagy. Nature 504, 291–295 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Moffat, J. et al. A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell 124, 1283–1298 (2006).

    Article  CAS  PubMed  Google Scholar 

  67. Neumann, B. et al. High-throughput RNAi screening by time-lapse imaging of live human cells. Nat. Methods 3, 385–390 (2006).

    Article  CAS  PubMed  Google Scholar 

  68. Sanjana, N.E., Shalem, O. & Zhang, F. Improved vectors and genome-wide libraries for CRISPR screening. Nat. Methods 11, 783–784 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Konig, R. et al. A probability-based approach for the analysis of large-scale RNAi screens. Nat. Methods 4, 847–849 (2007).

    Article  PubMed  CAS  Google Scholar 

  70. Luo, B. et al. Highly parallel identification of essential genes in cancer cells. Proc. Natl. Acad. Sci. USA 105, 20380–20385 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Li, W. et al. MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens. Genome Biol. 15, 554 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  72. Ran, F.A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Ran, F.A. et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154, 1380–1389 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Zetsche, B. et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163, 759–771 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Dahlman, J.E. et al. Orthogonal gene knockout and activation with a catalytically active Cas9 nuclease. Nat. Biotechnol. 33, 1159–1161 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Kiani, S. et al. Cas9 gRNA engineering for genome editing, activation and repression. Nat. Methods 12, 1051–1054 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank O. Shalem, D.A. Scott and P.D. Hsu for helpful discussions and insights; R. Belliveau for overall research support; R. Macrae for critical reading of the manuscript; and the entire Zhang laboratory for support and advice. O.O.A. was supported by a Paul and Daisy Soros Fellowship, a Friends of the McGovern Institute Fellowship, and the Poitras Center for Affective Disorders. J.S.G. was supported by a DOE Computational Science Graduate Fellowship. F.Z. was supported by the NIH through the National Institute of Mental Health (NIMH; grants 5DP1-MH100706 and 1R01-MH110049), the National Science Foundation (NSF), the Howard Hughes Medical Institute (HHMI), the New York Stem Cell Foundation, the Simons Foundation, the Paul G. Allen Family Foundation, and the Vallee Foundation, and James and Patricia Poitras, Robert Metcalfe, and David Cheng. F.Z. is a New York Stem Cell Foundation-Robertson Investigator. Reagents are available through Addgene; support forums and computational tools are available via the Zhang laboratory website (http://www.genome-engineering.org).

Author information

Authors and Affiliations

Authors

Contributions

J.J., S.K., J.S.G., O.O.A., R.J.P., M.D.B., N.E.S. and F.Z. designed and performed the experiments. J.J., S.K. and F.Z. wrote the manuscript with help from all authors.

Corresponding author

Correspondence to Feng Zhang.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Data 1-4

Design _library.py, Design_targeted_library.py, Count_spacers.py, and Calculate_indel.py. See https://github.com/fengzhanglab/Screening_Protocols_manuscript for latest version. (Note: In the version of this article initially published, the wrong version of Supplementary Data 4 was provided, and the GitHub link was omitted. This error and omission are now corrected as of 13 April 2017.) (ZIP 414 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Joung, J., Konermann, S., Gootenberg, J. et al. Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening. Nat Protoc 12, 828–863 (2017). https://doi.org/10.1038/nprot.2017.016

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2017.016

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing