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Nat Protoc. 2013 Nov;8(11):2180-96. doi: 10.1038/nprot.2013.132. Epub 2013 Oct 17.

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

Author information

1
1] Department of Cellular and Molecular Pharmacology, University of California, San Francisco (UCSF), San Francisco, California, USA. [2] Howard Hughes Medical Institute, UCSF, San Francisco, California, USA. [3] California Institute for Quantitative Biomedical Research, San Francisco, California, USA.

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.

PMID:
24136345
PMCID:
PMC3922765
DOI:
10.1038/nprot.2013.132
[Indexed for MEDLINE]
Free PMC Article

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