Nucleic acid detection with CRISPR-Cas13a/C2c2

By Jonathan Gootenberg, Omar Abudayyeh, Jeong Wook Lee, Patrick Essletzbichler, A. J. Dy, J. Keith Joung, V. Verdine, Nina M. Donghia, Nichole M. Daringer, C. A. Freije and 9 more. Science, 2017

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Sensitive and specific CRISPR diagnostics Methods are needed that can easily detect nucleic acids that signal the presence of pathogens, even at very low levels. Gootenberg et al. combined the allele-specific sensing ability of CRISPR-Cas13a with recombinase polymerase amplification methods to detect specific RNA and DNA sequences. The method successfully detected attomolar levels of Zika virus, as well as the presence of pathogenic bacteria. It could also be used to perform human genotyping from cell-free DNA. Science, this issue p. 438 An ortholog of CRISPR-Cas13a/C2c2 can be used as a highly sensitive detector of specific RNA and DNA sequences. 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.

Subfields: Genetics, Molecular Biology, Public Health, Environmental and Occupational Health

View Paper: https://www.semanticscholar.org/paper/39eb38459e23964ccd4a4fa07b563c83922aacf3

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Citations in the corpus

RNA virus interference via CRISPR/Cas13a system in plants (2017, background): “match specificity and attomolar sensitivity, demonstrating a wide range of potential utility in diagnostic and basic research applications [16].”
Genome Engineering Using the CRISPR Cas9 System (2019, background): “MIT and Harvard researchers led by Feng Zhang has developed several version of SHERLOCK and HOLMES diagnostic kit which can be used as a biosensor to detect Zika and dengue viruses [11].”
Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6 (2018, positive): “This detection platform, termed SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) (3), can discriminate between inputs that differ by a single nucleotide at very low concentrations and can be lyophilized for portable deployment.”
Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy (2020, neutral): “SHERLOCK had previously reported a limit of detection of 50 fM without pre-amplification (Gootenberg et al., 2017).”
Field-deployable viral diagnostics using CRISPR-Cas13 (2018, positive): “Furthermore, this approach can be easily adapted to detect virtually any virus present in bodily fluids, scaled to enable multiplexed detection(16), and the reagents can be lyophilized for cold-chain independence(13).”