TY - JOUR
T1 - Nucleic acid detection with CRISPR-Cas13a/C2c2
AU - Gootenberg, Jonathan S.
AU - Abudayyeh, Omar O.
AU - Lee, Jeong Wook
AU - Essletzbichler, Patrick
AU - Dy, Aaron J.
AU - Joung, Julia
AU - Verdine, Vanessa
AU - Donghia, Nina
AU - Daringer, Nichole M.
AU - Freije, Catherine A.
AU - Myhrvold, Cameron
AU - Bhattacharyya, Roby P.
AU - Livny, Jonathan
AU - Regev, Aviv
AU - Koonin, Eugene V.
AU - Hung, Deborah T.
AU - Sabeti, Pardis C.
AU - Collins, James J.
AU - Zhang, Feng
N1 - Funding Information:
We thank F. Chen, V. Rusu, R. Gupta, D. Daniels, C. Garvie, I. Finkelstein, V. Adalsteinsson, A. Das, E. S. Lander, R. Macrae, and R. Belliveau for discussions and support. Human genotyping data were collected with the informed consent of the subjects and in consent with the guidelines of the approved Massachusetts Institute of Technology (MIT) institutional review board (IRB) protocol IRB-4062. O.O.A. is supported by a Paul and Daisy Soros Fellowship and a National Defense Science and Engineering Fellowship. J.S.G. is supported by a U.S. Department of Energy Computational Science Graduate Fellowship. R.P.B, J.L, and D.T.H. are supported by the NIH through a National Institute of Allergies and Infectious Diseases grant (R01AI117043). A.J.D. is supported by an NSF Graduate Research Fellowship and an Air Force Office of Scientific Research grant (FA9550-14-1-0060). Zika work was partially funded by M. and L. Benioff to P.C.S., and antibiotic resistance work was partially funded by J. and A. Bekenstein to D.T.H. A.R. is supported by the Howard Hughes Medical Institute. J.J.C. is supported by the Defense Threat Reduction Agency grant HDTRA1-14-1-0006, the Paul G. Allen Frontiers Group, and the Wyss Institute. F.Z. is a New York Stem Cell Foundation-Robertson Investigator. F.Z. is supported by the NIH through National Institute of Mental Health grants (5DP1-MH100706 and 1R01-MH110049); the NSF; the Howard Hughes Medical Institute; the New York Stem Cell, Simons, Paul G. Allen Family, and Vallee Foundations; and J. and P. Poitras, R. Metcalfe, and D. Cheng. A.R. is a member of the Scientific Advisory Board for ThermoFisher Scientific. J.S.G., O.O.A., R.P.B., A.R., E.V.K., D.T.H., P.C.S., J.J.C., and F.Z. have filed patent applications relating to the work in this manuscript, including J.S.G., O.O.A., E.V.K., and F.Z. on international application no. PCT/US2016/038258 filed 18 June 2015 (CRISPR-C2c2 systems and uses thereof); J.S.G., O.O.A., and F.Z. on U.S. provisional patent application no. 62/351,662 filed 17 June 2016 (CRISPR-C2c2 systems and diagnostic uses thereof); J.S.G., O.O.A., J.J.C., and F.Z. on U.S. provisional patent application no. 62/432,553 filed 9 December 2016 (SHERLOCK diagnostic); J.S.G., O.O.A., P.C.S., J.J.C., and F.Z. on U.S. provisional patent application no. 62/471,917 filed 15 March 2017 (viral application of SHERLOCK); J.S.G., O.O.A., A.R., J.J.C., and F.Z. on U.S. provisional patent application no. 62/471,931 filed 15 March 2017 (mutation detection with SHERLOCK); J.S.G., O.O.A., R.P.B., D.T.H., J.J.C., and F.Z. on U.S. provisional patent application no. 62/471,936 filed 15 March 2017 (bacterial applications of SHERLOCK); and J.S.G., O.O.A., J.J.C., and F.Z. on U.S. provisional patent application no. 62/471,940 filed 15 March 2017 (devices). Each patent application relates to CRISPR-C2c2 systems, specific uses, and improved uses thereof for diagnostic application filed by Broad, Harvard, Massachusetts General Hospital, MIT, and NIH. Cas13a/C2c2 expression plasmids are available from Addgene under a Uniform Biological Material Transfer Agreement.
Publisher Copyright:
Copyright © 2016 by the American Association for the Advancement of Science; All rights reserved.
PY - 2017/4/28
Y1 - 2017/4/28
N2 - Rapid, inexpensive, and sensitive nucleic acid detection may aid point-of-care pathogen detection, genotyping, and disease monitoring. The RNA-guided, RNA-targeting clustered regularly interspaced short palindromic repeats (CRISPR) effector Cas13a (previously known as C2c2) exhibits a "collateral effect" of promiscuous ribonuclease activity upon target recognition. We combine the collateral effect of Cas13a with isothermal amplification to establish a CRISPR-based diagnostic (CRISPR-Dx), providing rapid DNA or RNA detection with attomolar sensitivity and single-base mismatch specificity. We use this Cas13a-based molecular detection platform, termed Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), to detect specific strains of Zika and Dengue virus, distinguish pathogenic bacteria, genotype human DNA, and identify mutations in cell-free tumor DNA. Furthermore, SHERLOCK reaction reagents can be lyophilized for cold-chain independence and long-term storage and be readily reconstituted on paper for field applications.
AB - Rapid, inexpensive, and sensitive nucleic acid detection may aid point-of-care pathogen detection, genotyping, and disease monitoring. The RNA-guided, RNA-targeting clustered regularly interspaced short palindromic repeats (CRISPR) effector Cas13a (previously known as C2c2) exhibits a "collateral effect" of promiscuous ribonuclease activity upon target recognition. We combine the collateral effect of Cas13a with isothermal amplification to establish a CRISPR-based diagnostic (CRISPR-Dx), providing rapid DNA or RNA detection with attomolar sensitivity and single-base mismatch specificity. We use this Cas13a-based molecular detection platform, termed Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), to detect specific strains of Zika and Dengue virus, distinguish pathogenic bacteria, genotype human DNA, and identify mutations in cell-free tumor DNA. Furthermore, SHERLOCK reaction reagents can be lyophilized for cold-chain independence and long-term storage and be readily reconstituted on paper for field applications.
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U2 - 10.1126/science.aam9321
DO - 10.1126/science.aam9321
M3 - Article
C2 - 28408723
AN - SCOPUS:85017652697
SN - 0036-8075
VL - 356
SP - 438
EP - 442
JO - Science
JF - Science
IS - 6336
ER -