Massively Multiplexed Nucleic Acid Detection with Cas13

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 ≥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.