CRISPR-HOLMES-based NAD + detection.

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.
Competing Interests: Author JW was employed by Shanghai Tolo Biotechnology Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
(Copyright © 2024 Zhuang, Hu, Zhou, He, Li, Zhang, Gu, Xu, Chen and Wang.)