CRISPR-HOLMES-based NAD⁺ detection.

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.

SUBMITTER: Zhuang S 

PROVIDER: S-EPMC10999544 | biostudies-literature | 2024

REPOSITORIES: biostudies-literature