Reprogramming Mycobacterium Tuberculosis CRISPR System for Gene Editing and Genome-Wide RNA Interference Screening

Abstract

Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), which is still the leading cause of mortality from a single infectious disease worldwide. The development of novel anti-TB drugs and vaccines is severely hampered by the complicated and time-consuming genetic manipulation techniques for M. tuberculosis. Here, we harnessed an endogenous type III-A CRISPR/Cas10 system of M. tuberculosis for efficient gene editing and RNA interference (RNAi). This simple and easy method only needs to transform a single mini-CRISPR array plasmid, thus avoiding the introduction of exogenous protein and minimizing proteotoxicity. We demonstrated that M. tuberculosis genes can be efficiently and specifically knocked in/out by this system as confirmed by DNA high-throughput sequencing. This system was further applied to single- and multiple-gene RNAi. Moreover, we successfully performed genome-wide RNAi screening to identify M. tuberculosis genes regulating in vitro and intracellular growth. This system can be extensively used for exploring the functional genomics of M. tuberculosis and facilitate the development of novel anti-TB drugs and vaccines.

The future of CRISPR in Mycobacterium tuberculosis infection

“…Collectively, these lines of evidence indicate that, in contrast to previous assumptions, MTBC CRISPR-Cas system retains at least some functionality: crRNA processing, crRNP assembly, target nucleotide recognition and target degradation by ancillary RNAse activity are detected under certain conditions in M. tuberculosis . Some gene silencing strategies take advantage of the endogenous activity of Cas proteins in M. tuberculosis as will be discussed later [ 40 , 41 ].…”

“…The endogenous type III-A CRISPR system of M. tuberculosis was leveraged for gene knock-in/knockout (KO) and for single/multiple gene RNAi to precisely dissect the functions of specific genes. This approach relies on the use of 40-bp gRNAs targeting the coding strands coupled with an HDR template to insert or replace specific genes of M. tuberculosis and also for massive mutagenesis and screening of genes related to bacterial growth [ 41 ].…”

CRISPR-Based Approaches for Gene Regulation in Non-Model Bacteria

“…These assays can use small, targeted libraries, such as essential genes or genes in a metabolic pathway ( Shields et al, 2020 ; Göttl et al, 2021 ), or large genome-wide libraries targeting nearly all genes in the bacterial genome ( Lee et al, 2019 ; Jiang et al, 2020 ). Additionally, CRISPRi libraries can be constructed in two major forms—pooled libraries, where cells containing different gRNA are mixed during library construction ( Bosch et al, 2021 ; Rahman et al, 2021 ), a strategy known as multiplexing, or arrayed libraries where different gRNA designs are constructed individually in different clonal populations, typically arrayed in microtiter plates ( Liu et al, 2017 ; Göttl et al, 2021 ). Pooled competitive selections are more common due to the ease of DNA construction and analysis of large, genome-scale gRNA libraries with >10,000 designs by next-generation sequencing ( Lee et al, 2019 ; Bosch et al, 2021 ).…”

“…Researchers developed CRISPRi technology by deactivating the nuclease activity of select Cas enzymes to create mutant dCas proteins that bind, but do not cleave, the DNA target ( Qi et al, 2013 ). Most CRISPRi systems repress a gene’s expression through steric inhibition of RNA polymerase binding or extension ( Qi et al, 2013 ), although some repress gene expression through RNA cleavage ( Zhang K. et al, 2020 ; Rahman et al, 2021 ). Gene repression over 100-fold has been reported for several diverse CRISPRi tools and can approach near knockout levels of gene expression ( Qi et al, 2013 ; Miao et al, 2019 ).…”

“…Several Type I CRISPRi/a tools have been designed, but due to the large number of genes in these systems, most tools are implemented by reprogramming the host species’ endogenous CRISPR system for gene repression ( Luo et al, 2015 ; Xu et al, 2021 ; Villegas Kcam et al, 2022 ). Only a handful of CRISPRi tools from other systems have been reported for transcriptional regulation in bacteria, likely due to the novelty of the system ( Rahman et al, 2021 ) or high cellular toxicity observed upon expression ( Zhang K. et al, 2020 ).…”