Reversed paired-gRNA plasmid cloning strategy for efficient genome editing in Escherichia coli

Abstract

BackgroundThe CRISPR-Cas9 system is a powerful tool for genome editing in various organisms. Several of its applications, including the generation of large deletions, require co-expression of two distinct guide RNAs (gRNAs). However, the instability of paired-gRNA plasmids prevents these applications from being scalable in Escherichia coli. Coexpressing paired gRNAs under the driving of independent but identical promoters in the same direction triggers plasmid recombination, due to the presence of direct repeats (DRs). ResultsIn this study, plasmid deletion between DRs occurred with high frequencies during plasmid construction and subsequent duplication processes, when three DRs-involved paired-gRNA plasmids cloning strategies were tested. This recombination phenomenon was RecA-independent, in agreement with the replication slippage model. To completely eliminate the DRs-induced plasmid instability, a reversed paired-gRNA plasmids (RPGPs) cloning strategy was developed by converting DRs to the more stable invert repeats (IRs). ConclusionsUsing RPGPs, we achieved a rapid deletion of chromosome fragments up to 100 kb with high efficiency of 83.33% in Escherichia coli. This study provides general solutions to construct stable plasmids containing short DRs, which can improve the performances of CRISPR systems that rely on paired gRNAs, and also facilitate other applications involving repeated genetic parts.

Advances in CRISPR/Cas9

“…Using double gRNAs to target two sites on the same chromosome which are far away from each other may lead to large segment deletion [118]. Recently, researchers eliminated the direct repeats (DRs) through Reversed Paired-gRNA Plasmid Cloning Strategy to resolve the problem of instability of double gRNAs in the same direction, realizing the rapid deletion of 100 kb chromosome segment in E. coli [119]. Multiple CRISPR technology has extensive application prospects in cellular recorders, genetic circuits, biosensors, combinatorial genetic perturbations, large-scale genome engineering, and the rewiring of metabolic pathways [120].…”

Development and application of a rapid all-in-one plasmid CRISPR-Cas9 system for iterative genome editing in Bacillus subtilis

“…In order to ensure the orthogonality of the promoters, we chose the non-carbohydrate bacitracin inducible promoter P liaI to express the Cas9 nuclease [21]. To reduce the possibility of internal recombination of the two gRNA expression cassettes, we reversed the transcription direction of the expression cassette gRNA rep and gRNA target [22]. Together, the all-in-one plasmid allows sequential genome editing and plasmid curing.…”