Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening

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.

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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.

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.

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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).

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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.

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Correspondence to Feng Zhang.

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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)

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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

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