Research Center for Clinical Pharmacy, The First Affiliated Hospital & Institute of Pharmaceutical Biotechnology, School of Medicine, Zhejiang University, Hangzhou 310058, China.
Zhejiang Provincial Key Laboratory for Microbial Biochemistry and Metabolic Engineering, Hangzhou 310058, China.
ACS Synth Biol. 2021 Nov 19;10(11):2833-2841. doi: 10.1021/acssynbio.1c00141. Epub 2021 Nov 4.
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 .
由于其天然产物库，合成生物学需要有效的使能技术。尽管 CRISPR-Cas9 系统在该属的基因组编辑中非常强大，但拟议的 Cas9 毒性限制了其应用。在这里，在先前的诱导型 Cas9 在转录和翻译水平上的表达以及 过表达的基础上，进一步引入了 Cas9 同源抑制剂 AcrIIA4 来微调 Cas9 活性。在实验室和工业 物种中，我们表明，与组成型表达的 Cas9 相比，在诱导前，引入 AcrIIA4 将接合效率从 700 倍提高到 7000 倍，而即使在 Cas9 表达诱导后同时对多个基因座进行同时删除，也可以获得相当的 65%-90%编辑效率，并且没有明显的脱靶效应。因此，AcrIIA4 可以作为一种调节剂来控制 Cas9 活性，从而显著提高基因组编辑效率，并且这个新工具包将具有广泛的适应性，并加快 的基因工程。
