Massively multiplexed nucleic acid detection with Cas13
Massively multiplexed nucleic acid detection with Cas13
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DOI：
10.1038/s41586-020-2279-8
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发表时间：
2020-04-29
期刊：
影响因子：
64.8
通讯作者：
Sabeti, Pardis C.
中科院分区：
文献类型：
--
作者：
Ackerman, Cheri M.;Myhrvold, Cameron;Sabeti, Pardis C.
绝大多数全球流行的病原体没有被发现，破坏了患者护理，阻碍了暴发准备和应对。为了实现常规监测和全面的诊断应用，需要检测技术能够规模化地检测许多样本(1-3个)，同时检测许多病原体(4-6个)。在这里，我们开发了用于核酸多路评估的组合阵列反应(Carmen)，这是一个可扩展的多路病原体检测平台。在卡门平台上，含有基于CRISPR的核酸检测试剂(7)的纳升液滴在微孔阵列(8)中自组织，与扩增样品的液滴配对，针对复制中的每个CRISPR RNA(CrRNA)测试每个样品。结合使用Carmen和Cas13检测(Carmen-Cas13)，可以在单个阵列上对超过4,500个crRNA-靶点对进行可靠测试。使用卡门-卡斯13，我们开发了一种多重检测方法，可以同时区分至少10个已公布基因组序列的所有169种人类相关病毒，并迅速加入额外的crRNA来检测2020年新冠肺炎大流行的病原体。Carmen-Cas13进一步实现了对A型流感病毒株的全面亚型划分，并对数十种艾滋病毒耐药性突变进行了多重鉴定。卡门固有的多路复用和吞吐能力使其具有规模化的可行性，因为小型化将每次检测的试剂成本降低了300多倍。基于CRISPR的可扩展、高度多元化的核酸检测将诊断和监测工作从高优先级样本的目标检测转移到大样本集的综合检测，极大地造福于患者和公共卫生(9-11)。
The great majority of globally circulating pathogens go undetected, undermining patient care and hindering outbreak preparedness and response. To enable routine surveillance and comprehensive diagnostic applications, there is a need for detection technologies that can scale to test many samples(1-3)while simultaneously testing for many pathogens(4-6). Here, we develop Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (CARMEN), a platform for scalable, multiplexed pathogen detection. In the CARMEN platform, nanolitre droplets containing CRISPR-based nucleic acid detection reagents(7)self-organize in a microwell array(8)to pair with droplets of amplified samples, testing each sample against each CRISPR RNA (crRNA) in replicate. The combination of CARMEN and Cas13 detection (CARMEN-Cas13) enables robust testing of more than 4,500 crRNA-target pairs on a single array. Using CARMEN-Cas13, we developed a multiplexed assay that simultaneously differentiates all 169 human-associated viruses with at least 10 published genome sequences and rapidly incorporated an additional crRNA to detect the causative agent of the 2020 COVID-19 pandemic. CARMEN-Cas13 further enables comprehensive subtyping of influenza A strains and multiplexed identification of dozens of HIV drug-resistance mutations. The intrinsic multiplexing and throughput capabilities of CARMEN make it practical to scale, as miniaturization decreases reagent cost per test by more than 300-fold. Scalable, highly multiplexed CRISPR-based nucleic acid detection shifts diagnostic and surveillance efforts from targeted testing of high-priority samples to comprehensive testing of large sample sets, greatly benefiting patients and public health(9-11).