CRISPRi-seq for genome-wide fitness quantification in bacteria.

Details

Serval ID
serval:BIB_18CADA206BE7
Type
Article: article from journal or magazin.
Publication sub-type
Review (review): journal as complete as possible of one specific subject, written based on exhaustive analyses from published work.
Collection
Publications
Institution
Title
CRISPRi-seq for genome-wide fitness quantification in bacteria.
Journal
Nature protocols
Author(s)
de Bakker V., Liu X., Bravo A.M., Veening J.W.
ISSN
1750-2799 (Electronic)
ISSN-L
1750-2799
Publication state
Published
Issued date
02/2022
Peer-reviewed
Oui
Volume
17
Number
2
Pages
252-281
Language
english
Notes
Publication types: Journal Article ; Research Support, Non-U.S. Gov't ; Review
Publication Status: ppublish
Abstract
CRISPR interference (CRISPRi) is a powerful tool to link essential and nonessential genes to specific phenotypes and to explore their functions. Here we describe a protocol for CRISPRi screenings to assess genome-wide gene fitness in a single sequencing step (CRISPRi-seq). We demonstrate the use of the protocol in Streptococcus pneumoniae, an important human pathogen; however, the protocol can easily be adapted for use in other organisms. The protocol includes a pipeline for single-guide RNA library design, workflows for pooled CRISPRi library construction, growth assays and sequencing steps, a read analysis tool (2FAST2Q) and instructions for fitness quantification. We describe how to make an IPTG-inducible system with small libraries that are easy to handle and cost-effective and overcome bottleneck issues, which can be a problem when using similar, transposon mutagenesis-based methods. Ultimately, the procedure yields a fitness score per single-guide RNA target for any given growth condition. A genome-wide screening can be finished in 1 week with a constructed library. Data analysis and follow-up confirmation experiments can be completed in another 2-3 weeks.
Keywords
Clustered Regularly Interspaced Short Palindromic Repeats
Pubmed
Web of science
Create date
14/01/2022 18:26
Last modification date
12/03/2022 7:29
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