Accelerated RNA detection using tandem CRISPR nucleases

Author:

Liu Tina Y.ORCID,Knott Gavin J.ORCID,Smock Dylan C. J.,Desmarais John J.ORCID,Son Sungmin,Bhuiya Abdul,Jakhanwal Shrutee,Prywes Noam,Agrawal Shreeya,de León Derby María Díaz,Switz Neil A.,Armstrong Maxim,Harris Andrew R.,Charles Emeric J.,Thornton Brittney W.,Fozouni ParinazORCID,Shu JeffreyORCID,Stephens Stephanie I.ORCID,Kumar G. RenukaORCID,Zhao ChunyuORCID,Mok AmandaORCID,Iavarone Anthony T.,Escajeda Arturo M.,McIntosh Roger,Kim Shin E.,Dugan Eli J.,Pollard Katherine S.,Tan Ming X.,Ott MelanieORCID,Fletcher Daniel A.ORCID,Lareau Liana F.ORCID,Hsu Patrick D.ORCID,Savage David F.ORCID,Doudna Jennifer A.ORCID,

Abstract

Direct, amplification-free detection of RNA has the potential to transform molecular diagnostics by enabling simple on-site analysis of human or environmental samples. CRISPR-Cas nucleases offer programmable RNA-guided recognition of RNA that triggers cleavage and release of a fluorescent reporter molecule1,2, but long reaction times hamper sensitivity and speed when applied to point-of-care testing. Here we show that unrelated CRISPR nucleases can be deployed in tandem to provide both direct RNA sensing and rapid signal generation, thus enabling robust detection of ∼30 RNA copies/microliter in 20 minutes. Combining RNA-guided Cas13 and Csm6 with a chemically stabilized activator creates a one-step assay that detected SARS-CoV-2 RNA from nasopharyngeal samples with PCR-derived Ct values up to 29 in microfluidic chips, using a compact imaging system. This Fast Integrated Nuclease Detection In Tandem (FIND-IT) approach enables direct RNA detection in a format amenable to point-of-care infection diagnosis, as well as to a wide range of other diagnostic or research applications.

Publisher

Cold Spring Harbor Laboratory

Reference36 articles.

1. Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection

2. Nucleic acid detection with CRISPR-Cas13a/C2c2;Science (New York, N.Y,2017

3. Test sensitivity is secondary to frequency and turnaround time for COVID-19 screening;Science Advances,2021

4. Analytical sensitivity and efficiency comparisons of SARS-CoV-2 RT–qPCR primer–probe sets;Nature Microbiology,2020

5. Rethinking Covid-19 Test Sensitivity — A Strategy for Containment;New England Journal of Medicine,2020

Cited by 5 articles. 订阅此论文施引文献 订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献

同舟云学术

1.学者识别学者识别

2.学术分析学术分析

3.人才评估人才评估

"同舟云学术"是以全球学者为主线,采集、加工和组织学术论文而形成的新型学术文献查询和分析系统,可以对全球学者进行文献检索和人才价值评估。用户可以通过关注某些学科领域的顶尖人物而持续追踪该领域的学科进展和研究前沿。经过近期的数据扩容,当前同舟云学术共收录了国内外主流学术期刊6万余种,收集的期刊论文及会议论文总量共计约1.5亿篇,并以每天添加12000余篇中外论文的速度递增。我们也可以为用户提供个性化、定制化的学者数据。欢迎来电咨询!咨询电话:010-8811{复制后删除}0370

www.globalauthorid.com

TOP

Copyright © 2019-2024 北京同舟云网络信息技术有限公司
京公网安备11010802033243号  京ICP备18003416号-3