CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity
CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity
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DOI：
10.1126/science.aar6245
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发表时间：
2018-04-27
期刊：
影响因子：
56.9
通讯作者：
Doudna, Jennifer A.
中科院分区：
文献类型：
--
作者：
Chen, Janice S.;Ma, Enbo;Doudna, Jennifer A.
CRISPR - Cas12a（Cpf1）蛋白是一种RNA引导的酶，作为细菌适应性免疫系统的组成部分，能够结合并切割DNA。与CRISPR - Cas9一样，基于其产生靶向双链DNA断裂的能力，Cas12a已被用于基因组编辑。在此我们表明，RNA引导的DNA结合会释放Cas12a无差别的单链DNA（ssDNA）切割活性，从而使ssDNA分子完全降解。我们发现，靶标激活的非特异性单链脱氧核糖核酸酶（ssDNase）切割也是其他V型CRISPR - Cas12酶的一个特性。通过将Cas12a的ssDNase激活与等温扩增相结合，我们创建了一种称为DNA内切核酸酶靶向CRISPR转录报告系统（DETECTR）的方法，该方法对DNA检测可达到阿摩尔级灵敏度。DETECTR能够快速、特异性地检测患者样本中的人乳头瘤病毒，从而为分子诊断提供了一个简单的平台。
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.