Definition of CRISPR Cas12a T rans-Cleavage Units to Facilitate CRISPR Diagnostics

Hailong Lv; Jian Wang; Jian Zhang; Yijian Chen; Lei Yin; Dian Jin; Dayong Gu; Huailong Zhao; Yong Xu; Jin Wang

doi:10.3389/fmicb.2021.766464

Definition of CRISPR Cas12a T rans-Cleavage Units to Facilitate CRISPR Diagnostics

Front Microbiol. 2021 Nov 29:12:766464. doi: 10.3389/fmicb.2021.766464. eCollection 2021.

Authors

Hailong Lv^{1

2}, Jian Wang¹, Jian Zhang¹, Yijian Chen^{3

4}, Lei Yin⁵, Dian Jin⁶, Dayong Gu¹, Huailong Zhao⁷, Yong Xu¹, Jin Wang^{1

8}

Affiliations

¹ Department of Clinical Laboratory, Shenzhen Institute of Translational Medicine, The First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital, Shenzhen, China.
² Department of Biomedical Engineering, Guangdong Key Laboratory for Biomedical Measurements and Ultrasound Imaging, School of Medicine, Shenzhen University, Shenzhen, China.
³ Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, China.
⁴ Key Laboratory of Clinical Pharmacology of Antibiotics, National Health Commission, Shanghai, China.
⁵ Tolo Biotechnology Company Limited, Shanghai, China.
⁶ TEDA Campus of Tianjin University of Science and Technology, Binhai New Area Development Zone, Tianjin, China.
⁷ Jinan Center for Disease Control and Prevention, Jinan, China.
⁸ Guangdong Provincial Key Laboratory of Systems Biology and Synthetic Biology for Urogenital Tumors, The First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital (Shenzhen Institute of Translational Medicine), Shenzhen, China.

Abstract

The CRISPR diagnostic (CRISPR-Dx) technology that employs the trans-cleavage activities has shown great potential in diagnostic sensitivity, specificity, convenience, and portability, and has been recognized as the next-generation diagnostic methods. However, due to the lack of standardized definition of Cas trans-cleavage enzymatic units, it is difficult to standardize the present CRISPR-Dx systems, which have undoubtedly impeded the development of the CRISPR-Dx industry. To solve the problem, we here first systematically optimized the reaction systems for Cas12a, and then defined its trans-cleavage units (transU), which we believe will be of great importance and interest to researchers in both molecular diagnostic industry and basic research. Moreover, a simple protocol was provided to facilitate a step-by-step measurement of the Cas12a transU, which can also act as a reference for the definition of the transU for other Cas proteins.

Keywords: CRISPR diagnostics; Cas12; enzymatic units; trans-cleavage; transU.