Massively Multiplexed Nucleic Acid Detection with Cas13
International Conference on CRISPR Technologies
2019
3rd International Conference on CRISPR Technologies
General Submissions
Foundational Technologies
Monday, September 16, 2019 - 3:15pm to 3:40pm
The overwhelming majority of pathogens that circulate in human and animal populations go undetected, undermining patient care and hindering outbreak preparedness. This is largely due to a lack of inexpensive, scalable technologies for pathogen detection and surveillance. CRISPR-based nucleic acid detection methods such as SHERLOCK and DETECTR are emerging solutions to the problem of pathogen surveillance, but are limited to testing 1-4 targets per assay. Here, we introduce Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (CARMEN), a technology that uses self-organization and miniaturization to enable highly multiplexed nucleic acid detection. Using CARMEN, we demonstrate parallelized CRISPR-Cas13 detection with up to 5,000 crRNA-target pairs tested in a single assay, increasing multiplexing and throughput while simultaneously decreasing the reagent cost per test by >300-fold. Using CARMEN-Cas13, we designed and extensively tested a 169-plex assay that simultaneously differentiates all human-associated viruses with â¥10 available genome sequences. CARMEN-Cas13 also enables comprehensive subtyping of influenza A strains and multiplexed identification of dozens of HIV drug-resistance mutations. Highly multiplexed CRISPR-based nucleic acid detection can catalyze rapid molecular diagnosis and characterization of wide-ranging pathogens, greatly benefiting patients and public health.