Application of the CRISPRi system to repress sepF expression in Mycobacterium smegmatis

Jing Xiao; Hongyan Jia; Liping Pan; Zihui Li; Lingna Lv; Boping Du; Lanyue Zhang; Fengjiao Du; Yinxia Huang; Tingming Cao; Qi Sun; Rongrong Wei; Aiying Xing; Zongde Zhang

doi:10.1016/j.meegid.2018.06.033

Application of the CRISPRi system to repress sepF expression in Mycobacterium smegmatis

Infect Genet Evol. 2019 Aug:72:183-190. doi: 10.1016/j.meegid.2018.06.033. Epub 2018 Jul 6.

Authors

Jing Xiao¹, Hongyan Jia¹, Liping Pan¹, Zihui Li¹, Lingna Lv¹, Boping Du¹, Lanyue Zhang¹, Fengjiao Du¹, Yinxia Huang¹, Tingming Cao¹, Qi Sun¹, Rongrong Wei¹, Aiying Xing¹, Zongde Zhang²

Affiliations

¹ Beijing Key Laboratory for Drug Resistance Tuberculosis Research, Laboratory of Molecular Biology, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing 101149, China.
² Beijing Key Laboratory for Drug Resistance Tuberculosis Research, Laboratory of Molecular Biology, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing 101149, China. Electronic address: zzd417@ccmu.edu.cn.

PMID: 31242975
DOI: 10.1016/j.meegid.2018.06.033

Abstract

Despite technical advances in introducing genomic deletions and modulating gene expression, direct inactivation of essential genes in mycobacteria remains difficult. In this study, we described clustered regularly interspaced short palindromic repeat interference (CRISPRi) technology to repress the expression of sepF (MSMEG_4219) based on nuclease-deficient CRISPR-associated protein 9 (Cas9) and small guide RNA (sgRNA) specific to the target sequence in Mycobacterium smegmatis. Using this CRISPRi approach, we achieved the repression of sepF by up to 98% in M. smegmatis without off-target effects. The depleted Msm_sepF strains resulted in growth and morphology changes including elongated, filamentous and branched bacterial cells, but the levels of the interacting partners ftsZ and murG were not modified in M. smegmatis. The sepF gene was proven to be an essential gene in M. smegmatis. This study provided an improved and detailed technical procedure for the application of CRISPRi technology in mycobacteria, and this approach was demonstrated to be a simple and efficient tool for regulating the expression of essential genes in M. smegmatis.

Keywords: CRISPR interference; Gene expression; Mycobacterium smegmatis; dcas9; sepF.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Bacterial Proteins / genetics
CRISPR-Associated Protein 9
CRISPR-Cas Systems* / drug effects
CRISPR-Cas Systems* / genetics
Gene Editing* / methods
Gene Knockdown Techniques*
Genes, Essential
Mycobacterium smegmatis / drug effects
Mycobacterium smegmatis / genetics*
Mycobacterium smegmatis / growth & development

Substances

Bacterial Proteins
CRISPR-Associated Protein 9