A CRISPR/Cas9-based single-stranded DNA recombineering system for genome editing of Rhodococcus opacus PD630

• DOI: 10.1016/j.synbio.2021.08.001
• Publication Date: 2021-08-10T23:54:18Z
• Authors: Youxiang Liang; Yuwen Wei; Song Jiao; Huimin Yu
• Keywords: Recombineering; Guide RNA; CRISPR interference; Gene targeting

Genome engineering of Rhodococcus opacus PD630, an important microorganism used for the bioconversion lignin, is currently dependent on inefficient homologous recombination. Although a CRISPR interference procedure gene repression has previously been developed R. CRISPR/Cas9 system knockout yet to be reported strain. In this study, we found that cytotoxicity Cas9 and deficiency in pathways repairing DNA double-strand breaks (DSBs) were major causes failure conventional technologies opacus, even when augmented with recombinases Che9c60 Che9c61. We successfully efficient single-stranded (ssDNA) recombineering coupled counter-selection, which facilitated rapid scarless editing genome. A two-plasmid system, comprising driven by weak promoter Pniami, designed prevent cytotoxicity, single-guide RNA (sgRNA) under control strong constitutive promoter, was proven appropriate respect cleavage function. novel recombinase, RrRecT derived from ruber prophage, identified first time, recombination short ssDNA donors (40–80 nt) targeted lagging strand enabled us obtain efficiency up 103-fold higher than endogenous pathways. Finally, incorporating into single plasmid then co-transforming cells sgRNA plasmids donors, efficiently achieved disruption base mutation efficiencies ranging 22 % 100 %. Simultaneous double genes also confirmed, although at lower efficiency. This effective genome tool will accelerate metabolism.