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

Efficient enabling technology is required for synthetic biology in 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 overexpression, a Cas9 cognate inhibitor AcrIIA4 was further introduced to fine-tune the Cas9 activity. In both laboratory and industrial 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 .