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.

中文翻译：

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基于 CRISPR-HOLMES 的 NAD+ 检测

研究表明，细胞内烟酰胺腺嘌呤二核苷酸（NAD+)水平与许多疾病的发生、发展有关。然而，传统的烟酰胺腺嘌呤二核苷酸（NAD+）检测技术非常耗时，并且可能需要大型且昂贵的仪器。我们最近发现，使用 NAD，聚集的规则间隔短回文重复 (CRISPR)-Cas12a 蛋白可以通过 AcrVA5 介导的乙酰化作用失活，并通过 CobB 重新激活+作为辅因子。因此，在本研究中，我们创建了基于 CRISPR-Cas12a 的一步 HOLMES（NAD+) 快速便捷的NAD系统+使用乙酰化 Cas12a 和 CobB 进行检测。在福尔摩斯（NAD+), 乙酰化的 Cas12a 失去其反式- 裂解活性，并且在 NAD 存在的情况下可以被 CobB 重新激活+，切割 ssDNA 报告基因以产生荧光信号。福尔摩斯(NAD+) 显示 NAD 的敏感性和特异性+检测并可用于细胞内NAD的定量测定+浓度。因此，福尔摩斯（NAD+）不仅为目标NAD提供了方便快捷的方法+定量的同时也将HOLMES的应用场景扩展到非核酸检测。