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

By Matthew H. Larson, Luke A. Gilbert, Xiaowo Wang, Wendell A. Lim, Jonathan S. Weissman, Lei S. Qi. Nature Protocols, 2013

1,150 citations recorded by the source corpus.

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.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/53633cc15e299d5ad3c726f677cbd9f7746a553a

How researchers see this paper

The researchers whose trusted networks rank it highest, strongest advocate first. Every researcher gets a different order.

In Molecular Biology

Citations in the corpus

CRISPR/Cas9 in Genome Editing and Beyond. (2016, methodology): “, gene overexpression, TALE- or ZF-mediated regulations), CRISPRi/a combine the advantages of design simplicity (37, 174, 188) with high specificity (37, 175, 180, 189), directly control endogenous gene expression at the transcriptional level, and can act on both coding and noncoding sequences (40, ”
Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System (2013, neutral): “sgRNAs were cloned into a lentiviral U6based expression vector derived from pSico, which coexpresses mCherry and a puromycin resistance cassette from a CMV promoter (Larson et al., 2013).”
sgRNAcas9: A Software Package for Designing CRISPR sgRNA and Evaluating Potential Off-Target Cleavage Sites (2014, background): “The clustered, regularly interspaced short palindromic repeats (CRISPR) in combination with a CRISPR-associated nuclease 9 (Cas9) were recently demonstrated to be versatile tools for genome engineering [1–6].”
Target specificity of the CRISPR-Cas9 system (2014, neutral): “Fusing dCas9 with transcription repressor domains such as the Krueppel-associated box (KRAB) leads to stronger silencing of mammalian genes, a technology termed CRISPRi [24].”