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

A growing number of CRISPR-Cas9 associated applications require co-expression of two distinct gRNAs. However, coexpressing paired gRNAs under the driving of independent but identical promoters in the same direction triggers plasmid instability, due to the presence of direct repeats (DRs). In this study, deletion between DRs occurred with high frequencies during plasmid construction and 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). Using 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 relied on paired gRNAs, and also facilitate other applications involving repeated genetic parts.