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

ABSTRACT: BACKGROUND:Co-expression of two distinct guide RNAs (gRNAs) has been used to facilitate the application of CRISPR/Cas9 system in fields such as large genomic deletion. The paired gRNAs are often placed adjacently in the same direction and expressed individually by two identical promoters, constituting direct repeats (DRs) which are susceptible to self-homologous recombination. As a result, the paired-gRNA plasmids cannot remain stable, which greatly prevents extensible applications of CRISPR/Cas9 system. RESULTS:To address this limitation, different DRs-involved paired-gRNA plasmids were designed and the events of recombination were characterized. Deletion between DRs occurred with high frequencies during plasmid construction and subsequent plasmid propagation. This recombination event was

SUBMITTER: Ding T 

PROVIDER: S-EPMC7063769 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature