Inducible CRISPRi enables efficient and high-fidelity genome editing in Streptomyces

Streptomycetes are prolific producers of bioactive natural products, but many of the biosynthetic gene clusters (BGCs) are silent in the laboratory. Genetic manipulation is important to unlock their full potential. CRISPR-Cas-based genome editing has greatly advanced genetic engineering in Streptomyces. However, several challenges remain, including Cas nuclease toxicity, unintended genomic rearrangements, and elimination of the delivery plasmid. Here, we present a novel genome editing strategy that harnesses cumate-inducible CRISPR interference (CRISPRi) to transiently knockdown essential genes such as divIVA or dnaA as counterselectable marker. This enforces loss of the vector backbone, promotes homologous recombination, and yields markerless mutants by loss of the antibiotic resistance cassette during the final recombination step. We demonstrate the versatility of the ICE system (Inducible CRISPRi targeting an Essential gene) by (i) deleting four BGCs in Streptomyces coelicolor M145, (ii) inserting both a promoter and a large BGC, and (iii) introducing precise single-nucleotide substitutions. Furthermore, deletion of the prodigiosin BGC elicited expression of a poorly expressed BGC for prolinolexin lipopeptides in Streptomyces roseifaciens DSM 106196T. Considering that different essential genes may be targeted, we anticipate that inducible CRISPRi-based counterselection may be adaptable to genome editing strategies in a broad range of microbial systems.