Broad Institute of the Massachusetts Institute of Technology (MIT) and Harvard, Cambridge, MA 02142, USA.
McGovern Institute for Brain Research, MIT, Cambridge, MA 02139, USA.
Science. 2018 Apr 27;360(6387):439-444. doi: 10.1126/science.aaq0179. Epub 2018 Feb 15.
Rapid detection of nucleic acids is integral for clinical diagnostics and biotechnological applications. We recently developed a platform termed SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) that combines isothermal preamplification with Cas13 to detect single molecules of RNA or DNA. Through characterization of CRISPR enzymology and application development, we report here four advances integrated into SHERLOCK version 2 (SHERLOCKv2) (i) four-channel single-reaction multiplexing with orthogonal CRISPR enzymes; (ii) quantitative measurement of input as low as 2 attomolar; (iii) 3.5-fold increase in signal sensitivity by combining Cas13 with Csm6, an auxiliary CRISPR-associated enzyme; and (iv) lateral-flow readout. SHERLOCKv2 can detect Dengue or Zika virus single-stranded RNA as well as mutations in patient liquid biopsy samples via lateral flow, highlighting its potential as a multiplexable, portable, rapid, and quantitative detection platform of nucleic acids.
核酸的快速检测对于临床诊断和生物技术应用至关重要。我们最近开发了一种名为 SHERLOCK（特异性高灵敏度酶报告物解锁）的平台，它将等温扩增与 Cas13 结合起来，以检测单个 RNA 或 DNA 分子。通过对 CRISPR 酶学的表征和应用开发，我们在此报告了四个集成到 SHERLOCK 版本 2（SHERLOCKv2）中的进展：（i）具有正交 CRISPR 酶的四通道单反应多重检测；（ii）输入的定量测量低至 2 飞摩尔；（iii）通过将 Cas13 与 Csm6（一种辅助的 CRISPR 相关酶）结合，将信号灵敏度提高 3.5 倍；以及（iv）侧向流动读取。SHERLOCKv2 可以通过侧向流动检测登革热或寨卡病毒单链 RNA 以及患者液体活检样本中的突变，突出了其作为一种可多重检测、便携、快速和定量检测核酸的平台的潜力。
