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

TY - JOUR

T1 - Inducible CRISPRi enables efficient and high-fidelity genome editing in Streptomyces

AU - Bai, Chaoxian

AU - Zhu, Hua

AU - Bayona, Lina M.

AU - Du, Chao

AU - van Wezel, Gilles P.

N1 - Data archiving: no NIOO data

PY - 2026/6/24

Y1 - 2026/6/24

N2 - 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.

AB - 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.

U2 - 10.1093/nar/gkag560

DO - 10.1093/nar/gkag560

M3 - Article

C2 - 42258544

AN - SCOPUS:105041249698

SN - 0305-1048

VL - 54

JO - Nucleic Acids Research

JF - Nucleic Acids Research

IS - 11

M1 - gkag560

ER -