CASCADE-Cas3 Enables Highly Efficient Genome Engineering inStreptomycesSpecies

Abstract Type I CRISPR systems are widespread in bacteria and archaea. The main differences compared to more widely applied type II systems are multi-effector CASCADE needed for crRNA processing and target recognition, as well as the processive nature of the hallmark nuclease Cas3. Given the widespread nature of type I systems, the processive nature of Cas3, as well as the recombinogenic overhangs created by Cas3, we hypothesized that Cas3 would be uniquely positioned to enable efficient genome engineering in streptomycetes. Here, we report a new type I based CRISPR genome engineering tool for streptomycetes. The plasmid system, called pCRISPR-Cas3, utilizes a compact type I-C CRISPR system and enables highly efficient genome engineering. pCRISPR-Cas3, outperforms pCRISPR-Cas9 and facilitates targeted and random sized deletions, as well as substitutions of large genomic regions such as biosynthetic gene clusters. Without additional modifications, pCRISPR-Cas3 enabled genome engineering in several Streptomyces species at high efficiencies.