The CRISPR-Cas12a system shows unique features compared with widely used Cas9, making it an attractive and potentially more precise alternative. However, the adoption of this system has been hindered by its relatively low editing efficiency. Guided by physical chemical principles, we covalently conjugated 5′ terminal modified CRISPR RNA (crRNA) to a site-specifically modified Cas12a through biorthogonal chemical reaction. The genome editing efficiency of the resulting conjugated Cas12a complex (cCas12a) was substantially higher than that of the wild-type complex. We also demonstrated that cCas12a could be used for precise gene knockin and multiplex gene editing in a chimeric antigen receptor T cell preparation with efficiency much higher than that of the wild-type system. Overall, our findings indicate that covalently linking Cas nuclease and crRNA is an effective approach to improve the Cas12a-based genome editing system and could potentially provide an insight into engineering other Cas family members with low efficiency as well.

与广泛使用的 Cas9 相比，CRISPR-Cas12a 系统显示出独特的功能，使其成为一种有吸引力且可能更精确的替代方案。然而，该系统的采用因其相对较低的编辑效率而受到阻碍。在物理化学原理的指导下，我们通过双正交化学反应将 5' 末端修饰的 CRISPR RNA (crRNA) 与位点特异性修饰的 Cas12a 共价结合。由此产生的共轭 Cas12a 复合物 (cCas12a) 的基因组编辑效率大大高于野生型复合物的基因组编辑效率。我们还证明了 cCas12a 可用于嵌合抗原受体 T 细胞制剂中的精确基因敲入和多重基因编辑，其效率远高于野生型系统。全面的，