Improving the efficiency of CRISPR-Cas12a-based genome editing with site-specific covalent Cas12a-crRNA conjugates
Improving the efficiency of CRISPR-Cas12a-based genome editing with site-specific covalent Cas12a-crRNA conjugates
复制标题
利用位点特异性共价 Cas12a-crRNA 缀合物提高基于 CRISPR-Cas12a 的基因组编辑的效率。
DOI：
10.1016/j.molcel.2021.09.021
复制
发表时间：
2021-11-18
期刊：
影响因子：
16
通讯作者：
Liu, Tao
中科院分区：
文献类型：
--
作者：
Ling, Xinyu;Chang, Liying;Liu, Tao
与广泛使用的Cas9相比，CRISPR-Cas 12 a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，这一系统的采用一直受到其相对较低的编辑效率的阻碍。在物理化学原理的指导下，我们通过双正交化学反应将50个末端修饰的CRISPR RNA（crRNA）共价结合到位点特异性修饰的Cas 12 a上。所得缀合的Cas 12 a复合物（cCas 12 a）的基因组编辑效率显著高于野生型复合物的基因组编辑效率。我们还证明了cCas 12 a可以用于嵌合抗原受体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。总的来说，我们的研究结果表明，共价连接Cas核酸酶和crRNA是改善基于Cas 12 a的基因组编辑系统的有效方法，并可能为低效率地改造其他Cas家族成员提供深入了解。
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 50 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.