Single-Stranded DNA-Binding Proteins Mediate DSB Repair and Effectively Improve CRISPR/Cas9 Genome Editing in <i>Escherichia coli</i> and <i>Pseudomonas</i>

Abstract

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 Escherichia coli SSB and phage T4 DNA ligase in pCas, respectively. Inactivation of the E. coli lacZ gene with homologous donor dsDNA increased the gene editing efficiency of pCas-SSB/pTargetF by 21.4% compared to pCas/pTargetF. Inactivation of the E. coli lacZ 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 E. coli (ΔrecA, ΔrecBCD, ΔSSB) with or without donor dsDNA did not differ. Additionally, pCas-SSB/pTargetF with donor dsDNA successfully deleted the wp116 gene in Pseudomonas sp. UW4. These results demonstrate that E. coli SSB repairs DSBs caused by CRISPR/Cas9 and effectively improves CRISPR/Cas9 genome editing in E. coli and Pseudomonas.

Keywords

• single-stranded DNA-binding protein
• DNA double-strand breaks
• dsDNA repair
• CRISPR/Cas9 genome editing
• homologous recombination
• nonhomologous end joining