State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, 38 Xueyuan Road, Haidian District, Beijing 100191, China.
The MOE Key Laboratory of Cell Proliferation and Differentiation, Genome Editing Research Center, School of Life Sciences, Peking University, Beijing 100871, China; Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China.
Mol Cell. 2021 Nov 18;81(22):4747-4756.e7. doi: 10.1016/j.molcel.2021.09.021. Epub 2021 Oct 13.
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.
CRISPR-Cas12a 系统与广泛使用的 Cas9 相比具有独特的特点，使其成为一种有吸引力且潜在更精确的替代方案。然而，由于其编辑效率相对较低，该系统的应用受到了阻碍。受物理化学原理的指导，我们通过生物正交化学反应将 5'端修饰的 CRISPR RNA（crRNA）共价连接到经过位点特异性修饰的 Cas12a 上。所得共轭 Cas12a 复合物（cCas12a）的基因组编辑效率明显高于野生型复合物。我们还证明，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，效率远高于野生型系统。总的来说，我们的研究结果表明，将 Cas 核酸酶和 crRNA 共价连接是一种有效提高基于 Cas12a 的基因组编辑系统的方法，并且可能为其他低效率的 Cas 家族成员的工程改造提供思路。
