posted on 2024-03-01, 16:06authored byHui-Min Hua, Jia-Feng Xu, Xue-Shuang Huang, Andrei A. Zimin, Wen-Fang Wang, Yin-Hua Lu
Genome editing tools based on SpCas9
and FnCpf1 have
facilitated
strain improvements for natural product production and novel drug
discovery in Streptomyces. However, due to high toxicity,
their editing requires high DNA transformation efficiency, which is
unavailable in most streptomycetes. The transformation efficiency
of an all-in-one editing tool based on miniature Cas nuclease AsCas12f1
was significantly higher than those of SpCas9 and FnCpf1 in tested
streptomycetes, which is due to its small size and weak DNA cleavage
activity. Using this tool, in Streptomyces coelicolor, we achieved 100% efficiency for single gene or gene cluster deletion
and 46.7 and 40% efficiency for simultaneous deletion of two genes
and two gene clusters, respectively. AsCas12f1 was successfully extended
to Streptomyces hygroscopicus SIPI-054
for efficient genome editing, in which SpCas9/FnCpf1 does not work
well. Collectively, this work offers a low-toxicity, high-efficiency
genome editing tool for streptomycetes, particularly those with low
DNA transformation efficiency.