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.

Cas12a 是一种 RNA 引导的核酸内切酶，已广泛用于方便的多重基因编辑，且脱靶效应低。为了最大限度地减少基因编辑中的脱靶，我们设计了一种 LbCas12a 变体（称为 Lb-K538R），与 LbCas12a 相比，它具有更严格的 PAM 识别、更低的脱靶能力和相似的体内编辑效率。我们还证明了来自毛螺菌科细菌 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，设计的 Lb2Cas12a 变体（称为 Lb2-K518R）不仅具有从 YYN 到 TYN 的更严格的 PAM 序列变化（Y 是 T 或 C，N 是 A、T、C 或 G），而且显示出较低的脱靶效应，从而比常见的 TTTV（V 是 A、G 或 C）PAM 能够以低脱靶率选择更多潜在的目标位点。为了确定同源位置的此类突变是否在其他 Cas12a 中具有相似的效果，对 As-K548R 进行了评估。基于全基因组脱靶测试的结果，As-K548R 表现出较低的脱靶效应。总的来说，我们的研究结果表明，Cas 蛋白可以设计为在 PAM 识别中严格，以减少它们的脱靶效应，