Strategies for Developing CRISPR‐Based Gene Editing Methods in Bacteria

Abstract

Gene editing is a fundamental technique for the identification of the linkages from genetic determinants to significant biological phenotypes, and the engineering of industrial strains to produce fine chemicals. Recently, the primitive bacterial immunity systems, clustered regularly interspaced short palindromic repeat (CRISPR)‐Cas systems, have been engineered as programmable nucleases to deliver double‐strand breaks (DSBs) at user‐defined loci. The DSB is repaired via either homology‐direct repair or the non‐homologous end joining pathway, generating a mutant in various organisms, and leading a revolution in the field of gene editing. This paper reviews the structural and molecular details of CRISPR‐Cas systems, describing their applications and challenges of genome targeting in bacterial cells. Moreover, the DSB repair pathways in bacteria are summarized and a guideline for selecting repair machines and maximizing the recombination efficiency is presented. In addition, the plasmid curing approaches in bacteria are outlined for iterative gene editing or downstream biological applications. Furthermore, CRISPR‐based transcriptional regulation, base editing, and transposition are also introduced. Finally, this paper prospects the future directions for improving CRISPR‐based gene editing methods in bacteria.

Programmable adenine deamination in bacteria using a Cas9–adenine-deaminase fusion

“…1,2 The recently discovered clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins have been engineered for robust genetic manipulation in a variety of organisms, including eukaryotes and diverse bacterial species, owing to their programmable and strong DNA-cleavage capacity. [3][4][5][6][7][8] Given that most bacterial species lack the non-homologous end joining (NHEJ) double-strand DNA break repair mechanism, genome cleavage by CRISPR/Cas systems is lethal to most bacteria. Therefore, CRISPR/Cas systems have been explored as effective selection agents to eliminate unedited cells, dramatically simplifying the genetic manipulation processes in bacteria.…”

CRISPR-Assisted Multiplex Base Editing System in Pseudomonas putida KT2440

“…A unique characteristic of the CRISPR/Cas9 system is the feasibility of concurrent multiplex genome editing, yet genome editing of two loci or more in eukaryotes has been the primary focus ( Jakoèiunas et al, 2016 ) and few studies of multiplex genome editing have been reported for prokaryotes, which can be ascribed to the weak HDR and poor or lack of non-homologous end joining (NHEJ) repair ( Wu et al, 2019b ). Although multiplex genome editing of two loci by the CRISPR/Cas9 system had been tested in P. putida KT2440, the efficiency was extremely low and could not be applied readily in experiments ( Aparicio et al, 2018 ).…”

Efficient Genome Editing by a Miniature CRISPR-AsCas12f1 Nuclease in Bacillus anthracis

“…When a one-plasmid-based CRISPR-Cas system was used for two or more gene edits in same parent strain, the rate of editing plasmid curing was shown to be the major constraint and more attention must be paid to overcome this (Wu et al, 2019). According to published studies and our results, some recombinant plasmids, despite including a temperature sensitive origin of replication, are very difficult to cure at the non-permissive temperature in B. anthracis and some other bacillus species (Hartz et al, 2021).…”