CRISPR-HOLMES-based NAD+ detection

Studies have indicated that intracellular nicotinamide adenine dinucleotide (NAD+) level is associated with the occurrence and development of many diseases. While traditional NAD+ detection techniques are time-consuming and may require large and expensive instruments. We recently found that the CRISPR-Cas12a protein can be inactivated by AcrVA5-mediated acetylation and reactivated by CobB, using NAD+ as the co-factor. Therefore, here 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 losses its trans-cleavage activates and can be reactivated by CobB at 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.