CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity

作者：Janice S. Chen, Enbo Ma, Lucas B. Harrington, Maria Da Costa, Xinran Tian, Joel M. Palefsky, Jennifer A. Doudna · 发表于：Science · 年份：2018 · DOI：10.1126/science.aar6245 · 被引用次数：4572 · 研究领域：CRISPR and Genetic Engineering、Genetics, Aging, and Longevity in Model Organisms

CRISPR-Cas12a (Cpf1) proteins are RNA-guided enzymes that bind and cut DNA as components of bacterial adaptive immune systems. Like CRISPR-Cas9, Cas12a has been harnessed for genome editing on the basis of its ability to generate targeted, double-stranded DNA breaks. Here we show that RNA-guided DNA binding unleashes indiscriminate single-stranded DNA (ssDNA) cleavage activity by Cas12a that completely degrades ssDNA molecules. We find that target-activated, nonspecific single-stranded deoxyribonuclease (ssDNase) cleavage is also a property of other type V CRISPR-Cas12 enzymes. By combining Cas12a ssDNase activation with isothermal amplification, we create a method termed DNA endonuclease-targeted CRISPR trans reporter (DETECTR), which achieves attomolar sensitivity for DNA detection. DETECTR enables rapid and specific detection of human papillomavirus in patient samples, thereby providing a simple platform for molecular diagnostics.