摘要

由于Cas9介导的切割需要原型间隔区和原型间隔区相邻基序（PAM）序列，所以不可能使用CRISPR / Cas9系统直接编辑附近没有可用的PAM序列的基因组位点。在此，我们对CRISPR / Cas9系统进行了优化，并开发了一种创新的两步策略，用于对任何站点进行高效基因组编辑，而不依赖于PAM序列的可用性。采用抗生素抗性盒作为阳性和阴性选择标记。通过整合优化的双质粒CRISPR / Cas系统和供体DNA，我们在大肠杆菌中实现了高效的基因插入和点突变，并且重要的是获得了没有其他不需要的突变的干净突变体。此外，使用这种方法成功实现了必需基因的基因组编辑，并进行了一些修改。因此，我们新开发的方法不依赖于PAM，可用于编辑任何基因组位点，我们希望此方法也可用于其他生物体中的高效基因组编辑。

As Cas9-mediated cleavage requires both protospacer and protospacer adjacent motif (PAM) sequences, it is impossible to employ the CRISPR/Cas9 system to directly edit genomic sites without available PAM sequences nearby. Here, we optimized the CRISPR/Cas9 system and developed an innovative two-step strategy for efficient genome editing of any sites, which did not rely on the availability of PAM sequences. An antibiotic resistance cassette was employed as both a positive and a negative selection marker. By integrating the optimized two-plasmid CRISPR/Cas system and donor DNA, we achieved gene insertion and point mutation with high efficiency in Escherichia coli, and importantly, obtained clean mutants with no other unwanted mutations. Moreover, genome editing of essential genes was successfully achieved using this approach with a few modifications. Therefore, our newly developed method is PAM-independent and can be used to edit any genomic loci, and we hope this method can also be used for efficient genome editing in other organisms.

https://www.frontiersin.org/articles/10.3389/fmicb.2017.00812/full