Fig. 1
Strategy for the construction of a CRISPR–Cas9 two-plasmid system in P. putida KT2440. a pSC101 replicon in pCASsac was replaced with RK2 replicon together with oriT fragment, creating pCAS-RK2K. In pCAS-RK2K, cas9 gene was linked with its native promoter, and gRNA cassettes were transcribed by P rhaB promoter so as to guide Cas9 protein targeting pRO1600 replicon in pSEVA-gRNAT. The λ-Red recombination system was under control of arabinose promoter to enhance the repairing efficiency in KT2440. SacB, a commonly used counterselectable marker function as a self-curing tool. b The P trc -laclq inducible system was eliminated from pSEVA644 and gRNA cassettes were inserted, generating pSEVA-gRNAF. The upstream homologous arm (UHA) and downstream homologous arm (DHA) were amplified from genome and connected by overlap-extension PCR. Next, the combinational homologous arm was assembled into pSEVA-gRNAF, giving rise to pSEVA-gRNAT