Abstract

Cas12a is an RNA-guided endonuclease that has been widely used for convenient multiplex gene editing with low off-target effects. To minimize off-targeting in gene editing, we engineered a variant of LbCas12a (termed Lb-K538R) with more stringent PAM recognition, lower off-targeting capability, and similar editing efficiency in vivo compared with LbCas12a. We also demonstrated that Lb2Cas12a from Lachnospiraceae bacterium MA2020 has extensive gene-editing activities in mammalian cells. Similar to Lb-K538R, the designed Lb2Cas12a variant (termed Lb2-K518R) not only had a more stringent PAM sequence change from YYN to TYN (Y is T or C, N is A, T, C, or G), but also displayed lower off-target effects, thereby enabling more potential target site selections with low off-targeting than the common TTTV (V is A, G, or C) PAM. To determine whether this type of mutation at the homologous position had similar effects in other Cas12a, As-K548R was evaluated. Based on the results of the genome-wide off-target test, As-K548R displayed lower off-target effects. Collectively, our findings indicate that the Cas proteins could be designed to be stringent in PAM recognition to reduce their off-target effects, which suggests a promising and practical approach for minimizing off-targets effects in genome editing.