Supplementary Information to publication "CASCADE-Cas3 Enables Highly Efficient Genome Engineering in Streptomyces Species"

Supplementary Information for manuscript "CASCADE-Cas3 Enables Highly Efficient Genome Engineering in Streptomyces Species". Includes additional data related to genome mining, bioinformatics, as well as plasmids and primers used in the study. Abstract: Type I CRISPR systems are widespread in bacteria and archaea. Compared to more widely applied type II systems, type I systems differ in the 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 and 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. Furthermore we demonstrate its ability to effectively perform 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.