Su Tianyuan, Guo Qi, Zheng Yi, Chang Yizhao, Gu Fei, Lu Xuemei, Qi Qingsheng
State Key Laboratory of Microbial Technology, Shandong university, Qingdao 266237, People's Republic of China.
CAS Key Lab of Biobased Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, People's Republic of China.
iScience. 2021 Jun 24;24(7):102769. doi: 10.1016/j.isci.2021.102769. eCollection 2021 Jul 23.
Base editing (BE) is a promising genome engineering tool for modifying DNA or RNA and has been widely used in various microorganisms as well as eukaryotic cells. Despite the proximal protospacer adjacent motif (PAM) is critical to the targeting range and off-target effect of BE, there is still lack of a specific approach to analyze the PAM pattern in BE systems. Here, we developed a base editing-coupled survival screening method. Using dCas9 from (SpdCas9) and its variants xdCas9 3.7 and dCas9 NG as example, their PAM patterns in BE systems were extensively characterized using the NNNN PAM library with high sensitivity. In addition to the typical PAM recognition features, we observed more unique PAMs exhibiting BE activity. These PAM patterns will boost the finding of potential off-target editing event arising from non-canonical PAMs and provide the guidelines for PAM usage in the BE system.
碱基编辑（BE）是一种用于修饰DNA或RNA的很有前景的基因组工程工具，已在各种微生物以及真核细胞中广泛使用。尽管近端间隔序列相邻基序（PAM）对BE的靶向范围和脱靶效应至关重要，但仍缺乏一种分析BE系统中PAM模式的特定方法。在此，我们开发了一种碱基编辑偶联生存筛选方法。以来自（SpdCas9）的dCas9及其变体xdCas9 3.7和dCas9 NG为例，使用具有高灵敏度的NNNN PAM文库对它们在BE系统中的PAM模式进行了广泛表征。除了典型的PAM识别特征外，我们还观察到更多具有BE活性的独特PAM。这些PAM模式将促进对非规范PAM引起的潜在脱靶编辑事件的发现，并为BE系统中PAM的使用提供指导。
