title: Targeted Transcriptional Repression in Bacteria Using CRISPR Interference (CRISPRi).
abstract: Clustered regularly interspersed short palindromic repeats (CRISPR) interference (CRISPRi) is a powerful technology for sequence-specifically repressing gene expression in bacterial cells. CRISPRi requires only a single protein and a custom-designed guide RNA for specific gene targeting. In Escherichia coli, CRISPRi repression efficiency is high (~300-fold), and there are no observable off-target effects. The method can be scaled up as a general strategy for the repression of many genes simultaneously using multiple designed guide RNAs. Here we provide a protocol for efficient guide RNA design, cloning, and assay of the CRISPRi system in E. coli. In principle, this protocol can be used to construct CRISPRi systems for gene repression in other species of bacteria.
authors.affiliation: Biophysics Graduate Program, San Francisco, CA, USA.
authors.forename: John S
authors.initials: JS
authors.lastname: Hawkins
authors.forename: Spencer
authors.initials: S
authors.lastname: Wong
authors.forename: Jason M
authors.initials: JM
authors.lastname: Peters
authors.forename: Ricardo
authors.initials: R
authors.lastname: Almeida
authors.forename: Lei S
authors.initials: LS
authors.lastname: Qi
mesh_terms: D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics*; D003001:Cloning, Molecular; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D020868:Gene Silencing; D005818:Genetic Engineering / Q000379:methods*; D016133:Polymerase Chain Reaction; D012313:RNA / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics*
chemical_list: D012313:RNA