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.