CRISPR-HOLMES-based NAD+ detection

Songkuan Zhuang; Tianshuai Hu; Hongzhong Zhou; Shiping He; Jie Li; Yuehui Zhang; Dayong Gu; Yong Xu; Yijian Chen; Jin Wang

doi:10.3389/fbioe.2024.1355640

CRISPR-HOLMES-based NAD⁺ detection

Front Bioeng Biotechnol. 2024 Mar 25:12:1355640. doi: 10.3389/fbioe.2024.1355640. eCollection 2024.

Authors

Songkuan Zhuang^#^{1

2}, Tianshuai Hu^#², Hongzhong Zhou², Shiping He², Jie Li², Yuehui Zhang³, Dayong Gu², Yong Xu⁴, Yijian Chen⁵, Jin Wang^{1

2

6}

Affiliations

¹ Guangdong Key Laboratory for Biomedical Measurements and Ultrasound Imaging, National-Regional Key Technology Engineering Laboratory for Medical Ultrasound, School of Biomedical Engineering, Shenzhen University Medical School, Shenzhen, China.
² Department of Clinical Laboratory, Shenzhen Institute of Translational Medicine, The First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital, Shenzhen, China.
³ Shenzhen Bao An Peoples Hospital, Shenzhen, China.
⁴ Department of Clinical Laboratory, Shenzhen Third People's Hospital, The Second Affiliated Hospital, School of Medicine, Southern University of Science and Technology, National Clinical Research Center for Infectious Disease, Shenzhen, China.
⁵ Institute of Antibiotics, Huashan Hospital, Fudan University & Key Laboratory of Clinical Pharmacology of Antibiotics, National Health Commission, Shanghai, China.
⁶ Shanghai Tolo Biotechnology Co Ltd, Shanghai, China.

^# Contributed equally.

Abstract

Studies have indicated that the intracellular nicotinamide adenine dinucleotide (NAD⁺) level is associated with the occurrence and development of many diseases. However, traditional nicotinamide adenine dinucleotide (NAD⁺) detection techniques are time-consuming and may require large and expensive instruments. We recently found that the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a protein can be inactivated by AcrVA5-mediated acetylation and reactivated by CobB, using NAD⁺ as the co-factor. Therefore, in this study, we created a CRISPR-Cas12a-based one-step HOLMES(NAD⁺) system for rapid and convenient NAD⁺ detection with the employment of both acetylated Cas12a and CobB. In HOLMES(NAD⁺), acetylated Cas12a loses its trans-cleavage activities and can be reactivated by CobB in the presence of NAD⁺, cutting ssDNA reporters to generate fluorescence signals. HOLMES(NAD⁺) shows both sensitivity and specificity in NAD⁺ detection and can be used for quantitative determination of intracellular NAD⁺ concentrations. Therefore, HOLMES(NAD⁺) not only provides a convenient and rapid approach for target NAD⁺ quantitation but also expands the application scenarios of HOLMES to non-nucleic acid detection.

Keywords: CRISPR; Cas12a; HOLMES; NAD+; acetylation.

Grants and funding

The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This research was funded by the National Natural Science Foundation of China (31922046), the National Key Research and Development Program of China (2022YFC2302700), the Guangdong Science and Technology Foundation (2021A1515220084 and 2022B1111020001), the Shenzhen Science and Technology Program (JCYJ20230807115116033, ZDSYS20210623092001003, GJHZ20200731095604013, JSGG20220301090003004, 201906133000069, SGLH20180625171602058, JCYJ20200109120205924, and JCYJ20190809160001751), and the Shanghai Science and Technology Innovation action plan of the 2022 in agricultural field (22N31900200).