Harnessing the Streptomyces-originating type I-E CRISPR/Cas system for efficient genome editing in Streptomyces.

Since their discovery, CRISPR/Cas systems have significantly expanded the genetic toolbox, aiding in the exploration and enhanced production of natural products across various microbes. Among these, class 2 CRISPR/Cas systems are simpler and more broadly used, but they frequently fail to function effectively in many Streptomyces strains. In this study, we present an engineered class 1 type I CRISPR/Cas system derived from Streptomyces avermitilis, which enables efficient gene editing in phylogenetically distant Streptomyces strains. Through a plasmid interference assay, we identified the effective protospacer adjacent motif as 5'-AAN-3'. Utilizing this system, we achieved targeted chromosomal deletions ranging from 8 bp to 100 kb, with efficiencies exceeding 92%. We further utilized this system to insert DNA fragments into different Streptomyces genomes, facilitating the heterologous expression of exogenous genes and the activation of endogenous natural product biosynthetic gene clusters. Overall, we established a type I CRISPR/Cas-based gene-editing methodology that significantly advances the exploration of Streptomyces, known for their rich natural product resources. This is the first report of a gene editing tool developed based on the endogenous class 1 type I CRISPR/Cas system in Streptomyces spp. Our work enriches the Streptomyces gene manipulation toolbox and advances the discovery of valuable natural products within these organisms.