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.
Funding
5-year funding extension (2021-2025) of the Novo Nordisk Foundation Center for Biosustainability
Novo Nordisk Foundation
Find out more...Integration of Informatics and Metabolic Engineering for the discovery of Novel Antibiotics (IIMENA)
Novo Nordisk Foundation
Find out more...