Developing a robust genome editing tool based on an endogenous Type I-B CRISPR-Cas system in Saccharopolyspora spinosa

Saccharopolyspora spinosa is an industrial rare actinomycete capable of producing important environmental-friendly biopesticides, spinosyns. However, exploitation of S. spinosa has been limited due to its genetic inaccessibility and lack of effective genome engineering tools. Here, we characterized the activity of an endogenous type I-B CRISPR-Cas system as well as its recognized protospacer adjacent motifs (PAMs) based on bioinformatics analysis combined with plasmid interference assay in S. spinosa. By reprogramming this endogenous CRISPR-Cas system, we achieved 100% editing efficiency for gene deletion. Using this tool, the genetic barrier composed of the Restriction-Modification (RM) systems was systematically disarmed. We showed that by disarming one type I RM system and two type II RM systems simultaneously, the transformation efficiency of foreign plasmids was significantly increased. Based on the engineered strain with simultaneous deletion of these three RM genes, we achieved the deletion of 75-kb spinosyns biosynthetic gene cluster as well as gene insertion at high efficiency. Collectively, we developed a reliable and high-efficiency genome editing tool based on the endogenous type I CRISPR-Cas system combined with the disarmament of the RM systems in S. spinosa. This is the first time to establish an endogenous CRISPR-Cas-based genome editing tool in the non-model industrial actinomycetes. Overall design: To investigate the transcription of the endogenous Type I-B CRISPR-Cas system, we then performed gene expression profiling analysis using data obtained from RNA-seq of cultured S. spinosa strain at three time points. Grant ID: 2019YFA0905400 Grant title: Network Reconstruction and System Optimization of Actinomycetes Drug Synthesis Biological Systems Funding source: National Key Research and Development Program Grant ID: 32270095 Funding source: National Natural Science Foundation of China