Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6

By Jonathan Gootenberg, Omar Abudayyeh, M. Kellner, J. Keith Joung, James J. Collins, Feng Zhang. Science, 2018

2,264 citations recorded by the source corpus.

Taking CRISPR technology further CRISPR techniques are allowing the development of technologies for nucleic acid detection (see the Perspective by Chertow). Taking advantages of the distinctive enzymatic properties of CRISPR enzymes, Gootenberg et al. developed an improved nucleic acid detection technology for multiplexed quantitative and highly sensitive detection, combined with lateral flow for visual readout. Myhrvold et al. added a sample preparation protocol to create a field-deployable viral diagnostic platform for rapid detection of specific strains of pathogens in clinical samples. Cas12a (also known as Cpf1), a type V CRISPR protein, cleaves double-stranded DNA and has been adapted for genome editing. Chen et al. discovered that Cas12a also processes single-stranded DNA threading activity. A technology platform based on this activity detected human papillomavirus in patient samples with high sensitivity. Science, this issue p. 439, p. 444, p. 436; see also p. 381 A nucleic acid detection platform enables sensitive pathogen genotyping and cancer mutation monitoring with visual readout. 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.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/023ab2866ab27ca1482dc0f7f05ef3572995ed8d

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In Molecular Biology

1. Feng Zhang
2. Shiyuan Li
3. Jonathan Gootenberg
4. Jin Wang
5. Omar Abudayyeh
6. Guozhen Liu
7. Yi Li
8. Pardis C Sabeti

Citations in the corpus

• HOLMESv2: a CRISPR-Cas12b-assisted platform for nucleic acid detection and DNA methylation quantitation. (2019, background): “(4) Detection of urine spike-ins of target DNA or target RNA with HOLMESv2”
• CRISPR/Cas Systems towards Next-Generation Biosensing. (2019, methodology): “POC Capability Most current CRISPR/Cas biosensing systems (e.g., SHERLOCK, SHERLOCKv2, HOLMES, HOLMESv2, DETECTR, RCH, and CAS-EXPAR) rely on fluorescent signals, which could be detected by portable fluorescence readers in the future.”
• Field-deployable viral diagnostics using CRISPR-Cas13 (2018, methodology): “Here, we test the performance of SHERLOCK for ZIKV and DENV detection on patient samples and develop HUDSON (Heating Unextracted Diagnostic Samples to Obliterate Nucleases), a method to enable rapid, sensitive detection of ZIKV and DENV directly from bodily fluids with a colorimetric readout, recent”
• Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy (2020, neutral): “, isothermal amplification, loop-mediated isothermal amplification [LAMP]), and transcription back to RNA for detection by Cas13a or Cas13b, an approach named ‘‘SHERLOCK’’ (Gootenberg et al., 2018; 2017).”
• Towards the Optimization of eDNA/eRNA Sampling Technologies for Marine Biosecurity Surveillance (2021, methodology): “These assays have already been developed and applied to a number of pests, diseases, and ecological monitoring [187,188,191,192], but have not yet been widely applied in the marine biosecurity context.”