Development of a CRISPR/Cas9D10A Nickase (nCas9)-Mediated Genome Editing Tool in Streptomyces

• DOI: 10.1021/acssynbio.3c00466
• Publication Date: 2023-09-18T19:56:12Z
• Authors: Jia-Xiang Ma; Wen-Yan He; Hui-Min Hua; Qian Zhu; Guo-Song Zheng; Andrei A. Zimin; Wen-Fang Wang; Yin-Hua Lu
• Keywords: Gene Editing; Actinomycetales; Humans; Deoxyribonuclease I; DNA; CRISPR-Cas Systems; RNA, Guide, CRISPR-Cas Systems; Streptomyces

Streptomycetes have a strong ability to produce vast array of bioactive natural products (NPs) widely used in agriculture and veterinary/human medicine. The recently developed CRISPR/Cas9-based genome editing tools greatly facilitated strain improvement for target NP overproduction as well novel discovery Streptomyces. However, CRISPR/Cas9 shows high toxicity the host, limiting its application many Streptomyces strains with low DNA transformation efficiency. In this study, we low-toxicity CRISPR/Cas9D10A nickase (nCas9)-based tool model coelicolor M145. We showed that presence both targeting sgRNA Cas proteins, utilization nCas9 instead Cas9 significantly reduced host enhanced cell survival. Using tool, achieved deletion single genes gene clusters efficiencies 87-100 63-87%, simultaneous two or 47 43%, respectively. efficiency is comparable Cas9-mediated tool. Finally, nCas9-based was successfully applied industrial rapamycin-producing rapamycinicus, which cannot work well. three tested an 27.2-30%. Collectively, CRISPR/nCas9-based offers convenient efficient genetic modification system engineering streptomycetes, particularly those