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

Abstract

CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) have become ubiquitous approaches to control gene expression in bacteria due to their simple design and effectiveness. By regulating transcription of a target gene(s), CRISPRi/a can dynamically engineer cellular metabolism, implement transcriptional regulation circuitry, or elucidate genotype-phenotype relationships from smaller targeted libraries up to whole genome-wide libraries. While CRISPRi/a has been primarily established in the model bacteria Escherichia coli and Bacillus subtilis, a growing numbering of studies have demonstrated the extension of these tools to other species of bacteria (here broadly referred to as non-model bacteria). In this mini-review, we discuss the challenges that contribute to the slower creation of CRISPRi/a tools in diverse, non-model bacteria and summarize the current state of these approaches across bacterial phyla. We find that despite the potential difficulties in establishing novel CRISPRi/a in non-model microbes, over 190 recent examples across eight bacterial phyla have been reported in the literature. Most studies have focused on tool development or used these CRISPRi/a approaches to interrogate gene function, with fewer examples applying CRISPRi/a gene regulation for metabolic engineering or high-throughput screens and selections. To date, most CRISPRi/a reports have been developed for common strains of non-model bacterial species, suggesting barriers remain to establish these genetic tools in undomesticated bacteria. More efficient and generalizable methods will help realize the immense potential of programmable CRISPR-based transcriptional control in diverse bacteria.

CRISPR-Cas tools for simultaneous transcription & translation control in bacteria

“…Our results here suggest that dCas13 mediated activation (tlCRISPRa) could offer advantages over DNA-targeting dCas9 (txCRISPRa) by enabling control of individual genes in multi-gene operons. txCRISPRa is also subject to relatively stringent target requirements that have limited its generalizability to arbitrary gene targets (19, 58, 59). If tlCRISPRa with dCas13 is subject to less stringent or even simply different target requirements, it will expand the range of accessible gene targets in bacteria.…”

Cas9-mediated endogenous plasmid loss in Borrelia burgdorferi

“…The study of plasmid-encoded functions in bacteria other than the Lyme disease spirochetes can therefore be facilitated by implementation of a Cas9-mediated plasmid curation protocol. Translation of this approach across bacterial phyla is likely feasible, as demonstrated by the successful broad implementation of CRISPR-based methods of gene regulation [ 76 ].…”

Cas9-mediated endogenous plasmid loss in Borrelia burgdorferi

“…The study of plasmid-encoded functions in bacteria other than the Lyme disease spirochetes can therefore be facilitated by implementation of a Cas9-mediated plasmid curation protocol. Translation of this approach across bacterial phyla is likely feasible, as demonstrated by the successful broad implementation of CRISPR-based methods of gene regulation [66].…”