Application of combined CRISPR screening for genetic and chemical-genetic interaction profiling in Mycobacterium tuberculosis

Abstract

CRISPR screening, including CRISPR interference (CRISPRi) and CRISPR-knockout (CRISPR-KO) screening, has become a powerful technology in the genetic screening of eukaryotes. In contrast with eukaryotes, CRISPR-KO screening has not yet been applied to functional genomics studies in bacteria. Here, we constructed genome-scale CRISPR-KO and also CRISPRi libraries in Mycobacterium tuberculosis (Mtb). We first examined these libraries to identify genes essential for Mtb viability. Subsequent screening identified dozens of genes associated with resistance/susceptibility to the antitubercular drug bedaquiline (BDQ). Genetic and chemical validation of the screening results suggested that it provided a valuable resource to investigate mechanisms of action underlying the effects of BDQ and to identify chemical-genetic synergies that can be used to optimize tuberculosis therapy. In summary, our results demonstrate the potential for efficient genome-wide CRISPR-KO screening in bacteria and establish a combined CRISPR screening approach for high-throughput investigation of genetic and chemical-genetic interactions in Mtb.

A CRISPR-Cas12a-based platform for ultrasensitive rapid highly specific detection of Mycobacterium tuberculosis in clinical application

“…Clustered regularly interspaced short palindromic repeat-associated proteins (CRISPR-Cas) is immune system of archaea and bacteria, which could resist the invasion of foreign nucleic acid, such as phage and plasmids ( Liu et al., 2022 ). They have been a popular tool for gene editing, function determining and transcription regulation ( Yan et al., 2022 ). Beyond above, the CRISPR-Cas system has also been employed in biosensing applications for pathogen detection based on the collateral cleavage activity of Cas effectors (such as Cas9, Cas12, and Cas13) ( Koonin et al., 2017 ).…”

Blunted blades: new CRISPR-derived technologies to dissect microbial multi-drug resistance and biofilm formation

“…Given the global prevalence and severity of Mycobacterium tuberculosis (Mtb), it is not surprising that the CRISPRi technology has been quickly added to the arsenal that aids in understanding the mechanisms of mycobacterial MDR ( 81 ). In the Mtb field, CRISPRi is a powerful tool for examining essential genes and has also been effective in conjunction with KO libraries, including CRISPR-KO and Tn-seq libraries ( 52 ). When used in conjunction with KO libraries, essential genes identified by CRISPRi can be further explored to determine whether a polar effect contributes to their essentiality ( 52 ).…”

“…In the Mtb field, CRISPRi is a powerful tool for examining essential genes and has also been effective in conjunction with KO libraries, including CRISPR-KO and Tn-seq libraries ( 52 ). When used in conjunction with KO libraries, essential genes identified by CRISPRi can be further explored to determine whether a polar effect contributes to their essentiality ( 52 ). Using this strategy, the Sun lab found 29 essential genes that impacted resistance or susceptibility to the antimycobacterial bedaquiline (BDQ), which inhibits the mycobacterial ATP synthase ( 52 ).…”

“…When used in conjunction with KO libraries, essential genes identified by CRISPRi can be further explored to determine whether a polar effect contributes to their essentiality ( 52 ). Using this strategy, the Sun lab found 29 essential genes that impacted resistance or susceptibility to the antimycobacterial bedaquiline (BDQ), which inhibits the mycobacterial ATP synthase ( 52 ). This was achieved by growing both CRISPR-KO and CRISPRi libraries to late-log phase, mimicking in vivo mycobacterial growth conditions before adding BDQ ( 52 ).…”

“…Using this strategy, the Sun lab found 29 essential genes that impacted resistance or susceptibility to the antimycobacterial bedaquiline (BDQ), which inhibits the mycobacterial ATP synthase ( 52 ). This was achieved by growing both CRISPR-KO and CRISPRi libraries to late-log phase, mimicking in vivo mycobacterial growth conditions before adding BDQ ( 52 ). While both libraries detected genes resistant and susceptible to BDQ treatment, 29 essential genes were only identifiable by CRISPRi.…”