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

TITLE=CRISPR-HOLMES-based NAD+ detection

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=Volume 12 - 2024

YEAR=2024

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2024.1355640

DOI=10.3389/fbioe.2024.1355640

ISSN=2296-4185

ABSTRACT=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-Cas12abased 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.