College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China.
Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
ACS Synth Biol. 2020 Jul 17;9(7):1781-1789. doi: 10.1021/acssynbio.0c00151. Epub 2020 Jul 1.
Base editing technology based on clustered regularly interspaced short palindromic repeats/associated protein 9 (CRISPR/Cas9) is a recent addition to the family of CRISPR technologies. Compared with the traditional CRISPR/Cas9 technology, it does not rely on DNA double strand break and homologous recombination, and can realize gene inactivation and point mutation more quickly and simply. Herein, we first developed a base editing method for genome editing in utilizing CRISPR/dCas9 (a fully nuclease-deficient mutant of Cas9 from ) and activation-induced cytidine deaminase (AID). This method achieved three and four loci simultaneous editing with editing efficiency up to 100% and 50%, respectively. Our base editing system in has a 5 nt editing window, which is similar to previously reported base editing in other microorganisms. We demonstrated that the plasmid curing rate is almost 100%, which is advantageous for multiple rounds of genome engineering in . Finally, we applied multiplex genome editing to generate a 168 mutant strain with eight inactive extracellular protease genes in just two rounds of base editing and plasmid curing, suggesting that it is a powerful tool for gene manipulation in and industrial applications in the future.
基于成簇规律间隔短回文重复序列/相关蛋白 9（CRISPR/Cas9）的碱基编辑技术是 CRISPR 技术家族的新成员。与传统的 CRISPR/Cas9 技术相比，它不依赖于 DNA 双链断裂和同源重组，能够更快速、更简单地实现基因失活和点突变。在此，我们首次利用 CRISPR/dCas9（来自 的一种完全无核酸酶的 Cas9 突变体）和激活诱导胞嘧啶脱氨酶（AID）开发了用于基因组编辑的碱基编辑方法。该方法可实现三个和四个靶位的同时编辑，编辑效率分别高达 100%和 50%。我们在 中开发的碱基编辑系统具有 5 个核苷酸的编辑窗口，与先前在其他微生物中报道的碱基编辑相似。我们证明了质粒的消除率几乎为 100%，这有利于 在多轮基因组工程中应用。最后，我们应用多重基因组编辑在两轮碱基编辑和质粒消除后，成功生成了一个具有八个失活胞外蛋白酶基因的 168 突变株，这表明它是 在基因操作和未来工业应用中的有力工具。
