Characterization of Cas proteins for CRISPR-Cas editing in streptomycetes.

Application of the well-characterized Streptococcus pyogenes CRISPR-Cas9 system in actinomycetes streptomycetes has enabled high efficiency multiplex genome editing and CRISPRi-mediated transcriptional regulation in these prolific bioactive metabolite producers. Nonetheless， SpCas9 has its limitations and can be ineffective depending on the strains and target sites. Here， we built and tested alternative CRISPR-Cas constructs based on the standalone pCRISPomyces-2 editing plasmid. We showed that Streptococcus thermophilus CRISPR1 (sth1Cas9)， Staphylococcus aureus Cas9 (saCas9)， and Francisella tularensis subsp. Novicida U112 Cpf1 (fnCpf1) are functional in multiple streptomycetes， enabling efficient homology directed repair (HDR)-mediated knock-in and deletion. In strains where spCas9 was non-functional， these alternative Cas systems enabled precise genomic modifications within biosynthetic gene clusters for the discovery， production and diversification of natural products. These additional Cas proteins provide us with the versatility to overcome the limitations of individual CRISPR-Cas systems for genome editing and transcriptional regulation of these industrially important bacteria. This article is protected by copyright. All rights reserved.