Improving the efficiency of CRISPR-Cas12a-based genome editing with site-specific covalent Cas12a-crRNA conjugates

被引：53

作者：

Ling, Xinyu ^{[1
]}

Chang, Liying ^{[1
]}

Chen, Heqi ^{[1
]}

Gao, Xiaoqin ^{[1
]}

Yin, Jianhang ^{[2
,3
]}

Zuo, Yi ^{[1
]}

Huang, Yujia ^{[1
]}

Zhang, Bo ^{[4
]}

Hu, Jiazhi ^{[2
,3
]}

Liu, Tao ^{[1
]}

机构：

[1] Peking Univ, Sch Pharmaceut Sci, State Key Lab Nat & Biomimet Drugs, 38 Xueyuan Rd, Beijing 100191, Peoples R China

[2] Peking Univ, Genome Editing Res Ctr, Sch Life Sci, MOE Key Lab Cell Proliferat & Differentiat, Beijing 100871, Peoples R China

[3] Peking Univ, Peking Tsinghua Ctr Life Sci, Beijing 100871, Peoples R China

[4] Chinese Acad Med Sci & Peking Union Med Coll, Peking Union Med Coll Hosp, Dept Med Res Ctr, Beijing 100730, Peoples R China

来源：

MOLECULAR CELL | 2021年 / 81卷 / 22期

基金：

中国国家自然科学基金; 北京市自然科学基金;

关键词：

GUIDE RNA; CPF1; CYCLOADDITION; CRISPR/CAS9; CAR;

D O I：

10.1016/j.molcel.2021.09.021

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

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.

引用

页码：4747 / +

页数：18

共 53 条

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