CRISPR Tools To Control Gene Expression in Bacteria

Abstract

SUMMARY CRISPR-Cas systems have been engineered as powerful tools to control gene expression in bacteria. The most common strategy relies on the use of Cas effectors modified to bind target DNA without introducing DNA breaks. These effectors can either block the RNA polymerase or recruit it through activation domains. Here, we discuss the mechanistic details of how Cas effectors can modulate gene expression by blocking transcription initiation or acting as transcription roadblocks. CRISPR-Cas tools can be further engineered to obtain fine-tuned control of gene expression or target multiple genes simultaneously. Several caveats in using these tools have also been revealed, including off-target effects and toxicity, making it important to understand the design rules of engineered CRISPR-Cas effectors in bacteria. Alternatively, some types of CRISPR-Cas systems target RNA and could be used to block gene expression at the posttranscriptional level. Finally, we review applications of these tools in high-throughput screens and the progress and challenges in introducing CRISPR knockdown to other species, including nonmodel bacteria with industrial or clinical relevance. A deep understanding of how CRISPR-Cas systems can be harnessed to control gene expression in bacteria and build powerful tools will certainly open novel research directions.

Rapid and highly efficient genomic engineering with a novel iEditing device for programming versatile extracellular electron transfer of electroactive bacteria

“…Among the various genome engineering tools, CRISPR has attracted the most attention because of its high efficiency and easily programmable ability (Xu et al ., 2020). However, the off‐target effect is a major concern in analysing the generated phenotypes and the selection of engineered biosystems (Broeders et al ., 2019; Vigouroux and Bikard, 2020). Other defects include bacterial cellular toxicity and big size (Li and Peng, 2019; Pausch et al ., 2020), limiting system construction and cellular delivery.…”

Programmable Gene Knockdown in Diverse Bacteria Using Mobile‐CRISPRi

“…The efficiency of Mobile-CRISPRi transfer and integration varies by strain, such that two strains of the same species can produce vastly different numbers of transconjugants (e.g., P. aeruginosa PAO1 and PA14 differ by >100-fold; Peters et al, 2019). Cell surface features (e.g., capsules) as well as defense systems that destroy horizontally transferred DNA (e.g., restriction enzymes and other CRISPR systems) can reduce transfer and integration efficiency (Thomas & Nielsen, 2005). Extending the mating time to 24 hr may improve recovery of transconjugants for low-efficiency recipients.…”

A multiplex CRISPR interference tool for virulence gene interrogation in Legionella pneumophila

“…These self-targeting crRNAs form a surveillance complex together with the tracrRNA and with dCas9 which, in the absence of cleavage, imposes transcriptional gene repression by sterically precluding RNA polymerase activity at the gene to which the complex was directed, a technology referred to as CRISPR i nterference (CRISPR i ) 14 , 16 . Gene silencing is advantageous to the study of essential genes that are otherwise intolerable to deletion, as well as for interrogating genes of interest without laborious null strain construction 17 . The recent development of mobile-CRISPRi has allowed for gene silencing to be performed in a number of gamma proteobacteria and Bacillales Firmicutes 18 , yet this and earlier studies, which implemented CRISPRi 19 , nearly always silenced only one gene or pairs of genes, with few exceptions 20 – 23 .…”