| Summary: | Single-stranded DNA-binding proteins (SSBs) are essential for all living organisms. Whether SSBs can repair DNA double-strand breaks (DSBs) and improve the efficiency of CRISPR/Cas9-mediated genome editing has not been determined. Here, based on a pCas/pTargetF system, we constructed pCas-SSB and pCas-T4L by replacing the λ-Red recombinases with <i>Escherichia coli</i> SSB and phage T4 DNA ligase in pCas, respectively. Inactivation of the <i>E. coli lacZ</i> gene with homologous donor dsDNA increased the gene editing efficiency of pCas-SSB/pTargetF by 21.4% compared to pCas/pTargetF. Inactivation of the <i>E. coli lacZ</i> gene via NHEJ increased the gene editing efficiency of pCas-SSB/pTargetF by 33.2% compared to pCas-T4L/pTargetF. Furthermore, the gene-editing efficiency of pCas-SSB/pTargetF in <i>E. coli</i> (Δ<i>recA</i>, Δ<i>recBCD</i>, Δ<i>SSB</i>) with or without donor dsDNA did not differ. Additionally, pCas-SSB/pTargetF with donor dsDNA successfully deleted the <i>wp116</i> gene in <i>Pseudomonas</i> sp. UW4. These results demonstrate that <i>E. coli</i> SSB repairs DSBs caused by CRISPR/Cas9 and effectively improves CRISPR/Cas9 genome editing in <i>E. coli</i> and <i>Pseudomonas</i>.
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