Cas12a variants designed for lower genome-wide off-target effect through stringent PAM recognition. Cas12a variants designed for lower genome-wide off-target effect through stringent PAM recognition

our findings show that the Cas proteins could be designed to be stringent in PAM recognition to reduce their off-target effects , which points out a promising and practical way for the minimization of the off-targeting in genome editing. Overall design: To minimize off-targeting for gene editing, here we engineered a variant of LbCas12a (termed Lb-K538R) with more stringent PAM recognition, lower off-targeting, and similar editing efficiency in vivo compared with LbCas12a. We also demonstrate that Lb2Cas12a from Lachnospiraceae bacterium MA2020 exhibits extensive gene-editing activities in mammalian cells. Similar to Lb-K538R, the designed Lb2Cas12a variant (termed Lb2-K518R) also has lower off-target effects and is more stringent in PAM recognition from YYN to be TYN (Y is T or C, N is A, T, C, or G), which supplies more possible target site selections with low off-targeting as compared with the common TTTV (V is A, G, or C) PAM.

研究结果显示，可对Cas蛋白（Cas protein）进行设计，使其在PAM（原间隔序列毗邻基序，Protospacer Adjacent Motif）识别过程中具备更高严谨性，从而降低其脱靶效应，这为基因组编辑中脱靶效应的最小化指明了一条颇具前景且切实可行的路径。
总体实验设计：为降低基因编辑的脱靶风险，本研究对LbCas12a开展工程化改造，获得命名为Lb-K538R的变体；相较于野生型LbCas12a，该变体拥有更严谨的PAM识别能力、更低的脱靶效应，且体内编辑效率与之相当。此外，本研究证实，来源于毛螺菌科细菌MA2020（Lachnospiraceae bacterium MA2020）的Lb2Cas12a在哺乳动物细胞中具有广泛的基因编辑活性。与Lb-K538R类似，本研究设计的Lb2Cas12a变体（命名为Lb2-K518R）同样具备更低的脱靶效应，其PAM识别严谨性从YYN收紧为TYN（其中Y代表T或C，N代表A、T、C或G）；相较于常见的TTTV（其中V代表A、G或C）PAM序列，该变体可提供更多低脱靶风险的靶位点选择空间。