Fine-Tuning Cas9 Activity with a Cognate Inhibitor AcrIIA4 to Improve Genome Editing in Streptomyces

Efficient enabling technology is required for synthetic biology in Streptomyces due to its natural product reservoir. Though the CRISPR-Cas9 system is powerful for genome editing in this genus, the proposed Cas9 toxicity has limited its application. Here on the basis of previous inducible Cas9 expression at the transcriptional and translational levels coupled with atpD overexpression, a Cas9 cognate inhibitor AcrIIA4 was further introduced to fine-tune the Cas9 activity. In both laboratory and industrial Streptomyces species, we showed that, compared to the constitutively expressed Cas9, incorporating AcrIIA4 increased the conjugation efficiency from 700- to 7000-fold before induction, while a comparable 65%–90% editing efficiency was obtained even on multiple loci for simultaneous deletion after Cas9 expression was induced, along with no significant off-targets. Thus, AcrIIA4 could be a modulator to control Cas9 activity to significantly improve genome editing, and this new toolkit would be widely adaptable and fasten genetic engineering in Streptomyces.鉴于链霉菌属（Streptomyces）拥有丰富的天然产物储备，其合成生物学研究亟需高效的使能技术。尽管CRISPR-Cas9系统在该属微生物的基因组编辑中展现出强大应用潜力，但此前报道的Cas9毒性限制了其实际应用。本研究在既往基于转录与翻译水平的诱导型Cas9表达、并结合atpD过表达的研究基础上，进一步引入了Cas9同源抑制剂AcrIIA4，以精准调控Cas9活性。在实验室菌株与工业用链霉菌菌株中，研究结果表明：相较于组成型表达的Cas9，引入AcrIIA4可在诱导前将接合转移效率提升700至7000倍；而在诱导Cas9表达后，即便针对多个位点同时进行基因敲除，仍可获得65%至90%的可比编辑效率，且未检测到显著的脱靶效应。综上，AcrIIA4可作为调控Cas9活性的调节剂，显著提升基因组编辑效率；这套全新的基因编辑工具包具备广泛适配性，将有效加速链霉菌的基因工程改造进程。