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Aptamer inhibitor selection of SpyCas9 through CE-SELEX

文献类型： 外文期刊

作者： Yang, Ge 1 ; Wang, Huimin 1 ; Jiang, Guangyu 2 ; Zhao, Liping 3 ; Qu, Feng 1 ;

作者机构： 1.Beijing Inst Technol, Sch Life Sci, Key Lab Mol Med & Biotherapy, 5 South Zhongguancun St, Beijing 100081, Peoples R China

2.Chinese Acad Med Sci & Peking Union Med Coll, Inst Med Biotechnol, Beijing Key Lab Antimicrobial Agents, CAMS Key Lab Antiviral Drug Res,NHC Key Lab Biotec, Beijing 100050, Peoples R China

3.Inst Qual Stand & Testing Technol BAAFS, Dept Agr, Agr Prod Qual & Safety Risk Assessment Lab, Beijing 100097, Peoples R China

关键词： CRISPR/Cas9; Inhibitors; Aptamer; CE-SELEX

期刊名称：TALANTA （ 影响因子：5.6； 五年影响因子：5.0 ）

ISSN： 0039-9140

年卷期： 2024 年 273 卷

页码：

收录情况： SCI

摘要： CRISPR/Cas9 is a natural immune system of archaea and bacteria, which has been widely used in gene editing. In order to better control and improve the accuracy and safety of the system, inhibitors for SpyCas9 as "switches" have been selected for several years. The available inhibitors currently are all natural polypeptides inhibitors derived from phages, except one small molecule inhibitor. These natural inhibitors are challenging to obtain and are available in limited quantities, and the small molecule inhibitor is cytotoxic. Herein, we discover aptamers against the SpyCas9 protein, by coupling CE-SELEX within one-round pressure controllable selection strategy. One of the identified aptamers, Apt2, shows high affinity at the nanomolar level and leads for effective SpyCas9 enzymatic inhibition in vitro. It is predicted that Apt2 interacts with the HNH and RuvC domains of SpyCas9, competitively inhibiting the binding of substrate DNA to SpyCas9. The proposed aptamer inhibitor is the oligonucleotide inhibitor of SpyCas9, which has the potential in construction of the universal, simple and precise CRISPR-Cas9 system activity control strategy. Meanwhile, these aptamers could also be valuable tools for study of the functions of CRISPR/Cas9 and the related functional mechanisms.

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