Ajibode Elizabeth Toyin, Bender Alexandra R, Yehl Kevin
Department of Chemistry and Biochemistry, Miami University, Oxford, Oxford, OH, USA.
Commun Biol. 2025 Jun 21;8(1):947. doi: 10.1038/s42003-025-08356-6.
CRISPR-Cas diagnostics are revolutionizing point-of-care molecular testing due to the programmability, simplicity, and sensitivity of Cas systems with trans-cleavage activity. CRISPR-Cas12 assays are promising for detecting single nucleotide polymorphisms (SNPs). However, reports vary widely describing Cas12 SNP sensitivity, and an underlying mechanism is lacking. We systematically varied crRNA length and valency to investigate the role of crRNA architectures on Cas12 biosensing in the context of speed-of-detection, sensitivity, and selectivity. Our results demonstrate that crRNAs complementary to 20 base pairs of the target DNA is optimal for rapid and sensitive detection, while a complementary length of 15 base pairs is ideal for robust SNP detection. Additionally, we uncovered a unique periodicity in SNP sensitivity based on nucleotide position and developed a structural model explaining what drives Cas12 SNP sensitivity. Lastly, we showed that bivalent CRISPR-Cas sensors have synergistic and enhanced activity that is distance dependent.
由于具有反式切割活性的Cas系统具有可编程性、简单性和敏感性，CRISPR-Cas诊断技术正在彻底改变即时分子检测。CRISPR-Cas12检测方法在检测单核苷酸多态性（SNP）方面很有前景。然而，关于Cas12 SNP敏感性的报道差异很大，并且缺乏潜在机制。我们系统地改变了crRNA的长度和价态，以研究crRNA结构在检测速度、灵敏度和选择性方面对Cas12生物传感的作用。我们的结果表明，与目标DNA的20个碱基对互补的crRNA最适合快速灵敏的检测，而15个碱基对的互补长度最适合可靠的SNP检测。此外，我们发现了基于核苷酸位置的SNP敏感性的独特周期性，并开发了一个结构模型来解释驱动Cas12 SNP敏感性的因素。最后，我们表明二价CRISPR-Cas传感器具有协同增强的活性，且这种活性与距离有关。
