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CRISPR interference (CRISPRi) for sequence-specific control of gene expression

Nature Protocols volume 8, pages 2180–2196 (2013)Cite this article

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Abstract

Sequence-specific control of gene expression on a genome-wide scale is an important approach for understanding gene functions and for engineering genetic regulatory systems. We have recently described an RNA-based method, CRISPR interference (CRISPRi), for targeted silencing of transcription in bacteria and human cells. The CRISPRi system is derived from the Streptococcus pyogenes CRISPR (clustered regularly interspaced palindromic repeats) pathway, requiring only the coexpression of a catalytically inactive Cas9 protein and a customizable single guide RNA (sgRNA). The Cas9-sgRNA complex binds to DNA elements complementary to the sgRNA and causes a steric block that halts transcript elongation by RNA polymerase, resulting in the repression of the target gene. Here we provide a protocol for the design, construction and expression of customized sgRNAs for transcriptional repression of any gene of interest. We also provide details for testing the repression activity of CRISPRi using quantitative fluorescence assays and native elongating transcript sequencing. CRISPRi provides a simplified approach for rapid gene repression within 1–2 weeks. The method can also be adapted for high-throughput interrogation of genome-wide gene functions and genetic interactions, thus providing a complementary approach to RNA interference, which can be used in a wider variety of organisms.

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Figure 1: The CRISPRi system for transcription repression in bacteria and human cells.
Figure 2: General workflow for the design, cloning and expression of sgRNAs.
Figure 3: Design of the sgRNAs.
Figure 4: Extensions to the base-pairing region with mismatched nucleotides decrease repression activity.
Figure 5: Cloning strategy for concatenating multiple sgRNA expression cassettes onto the same plasmid.

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Acknowledgements

We thank the Jonathan Weissman lab and Wendell Lim lab for their support. L.S.Q. acknowledges support from the UCSF Center for Systems and Synthetic Biology. This work was supported by US National Institutes of Health (NIH) grant no. P50 GM081879 (L.S.Q., W.A.L.), the Howard Hughes Medical Institute (M.H.L., L.A.G., J.S.W., W.A.L.), a Howard Hughes Collaborative Initiative Award (J.S.W.) and a Ruth L. Kirschstein National Research Service Award (M.H.L.). X.W. is supported by A Foundation for the Author of National Excellent Doctoral Dissertation grant (grant number 201158).

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Authors and Affiliations

  1. Department of Cellular and Molecular Pharmacology, University of California, San Francisco (UCSF), San Francisco, California, USA

    Matthew H Larson, Luke A Gilbert, Wendell A Lim, Jonathan S Weissman & Lei S Qi

  2. Howard Hughes Medical Institute, UCSF, San Francisco, California, USA

    Matthew H Larson, Luke A Gilbert, Wendell A Lim & Jonathan S Weissman

  3. California Institute for Quantitative Biomedical Research, San Francisco, California, USA

    Matthew H Larson, Luke A Gilbert, Wendell A Lim, Jonathan S Weissman & Lei S Qi

  4. Bioinformatics Division, Tsinghua National Laboratory for Information Science and Technology Department of Automation, Center for Synthetic and Systems Biology, Tsinghua University, Beijing, China

    Xiaowo Wang

  5. UCSF Center for Systems and Synthetic Biology, UCSF, San Francisco, California, USA

    Wendell A Lim & Lei S Qi

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  1. Matthew H Larson
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L.S.Q., M.H.L., L.A.G. and X.W. wrote the manuscript. J.S.W., W.A.L. and L.S.Q. supervised the research.

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Correspondence to Lei S Qi.

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Larson, M., Gilbert, L., Wang, X. et al. CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nat Protoc 8, 2180–2196 (2013). https://doi.org/10.1038/nprot.2013.132

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