Transcriptional Knockdown in Pneumococci Using CRISPR Interference

Morten Kjos

doi:10.1007/978-1-4939-9199-0_8

Transcriptional Knockdown in Pneumococci Using CRISPR Interference

Methods Mol Biol. 2019:1968:89-98. doi: 10.1007/978-1-4939-9199-0_8.

Author

Morten Kjos¹

Affiliation

¹ Faculty of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Ås, Norway. morten.kjos@nmbu.no.

PMID: 30929208
DOI: 10.1007/978-1-4939-9199-0_8

Abstract

Sequence-specific knockdown of gene expression using CRISPR interference (CRISPRi) has recently been developed for Streptococcus pneumoniae. By coexpression of a catalytically inactive Cas9-protein (dCas9) and a single guide RNA (sgRNA), CRISPRi can be used to knock down transcription of any gene of interest. Gene specificity is mediated by a 20 bp sequence on the sgRNA, and new genes can be targeted by replacing this 20 bp sequence. Here, a protocol is provided for design of sgRNAs and construction of CRIPSRi strains in S. pneumoniae, based on the vectors published by Liu et al. (Mol Syst Biol 13:931, 2017).

Keywords: CRISPRi; Inverse PCR; Knockdown; dCas9; sgRNA.

MeSH terms

Bacterial Proteins / genetics
CRISPR-Cas Systems / genetics
Clustered Regularly Interspaced Short Palindromic Repeats / genetics*
Gene Expression Regulation, Bacterial / genetics
Polymerase Chain Reaction
RNA, Guide, CRISPR-Cas Systems / genetics
Streptococcus pneumoniae / genetics*

Substances

Bacterial Proteins
RNA, Guide, CRISPR-Cas Systems