Massively multiplexed nucleic acid detection with Cas13

Abstract

The great majority of globally circulating pathogens go undetected, undermining patient care and hindering outbreak preparedness and response. To enable routine surveillance and comprehensive diagnostic applications, there is a need for detection technologies that can scale to test many samples 1 – 3 while simultaneously testing for many pathogens 4 – 6 . Here, we develop Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (CARMEN), a platform for scalable, multiplexed pathogen detection. In the CARMEN platform, nanolitre droplets containing CRISPR-based nucleic acid detection reagents 7 self-organize in a microwell array 8 to pair with droplets of amplified samples, testing each sample against each CRISPR RNA (crRNA) in replicate. The combination of CARMEN and Cas13 detection (CARMEN–Cas13) enables robust testing of more than 4,500 crRNA–target pairs on a single array. Using CARMEN–Cas13, we developed a multiplexed assay that simultaneously differentiates all 169 human-associated viruses with at least 10 published genome sequences and rapidly incorporated an additional crRNA to detect the causative agent of the 2020 COVID-19 pandemic. CARMEN–Cas13 further enables comprehensive subtyping of influenza A strains and multiplexed identification of dozens of HIV drug-resistance mutations. The intrinsic multiplexing and throughput capabilities of CARMEN make it practical to scale, as miniaturization decreases reagent cost per test by more than 300-fold. Scalable, highly multiplexed CRISPR-based nucleic acid detection shifts diagnostic and surveillance efforts from targeted testing of high-priority samples to comprehensive testing of large sample sets, greatly benefiting patients and public health 9 – 11 .

COVID‐19 Clinical Diagnostics and Testing Technology

“…A single CARMEN-Cas13 chip can be scaled to either detect a single type of virus in more than 1000 samples at a time or 160 different viruses in a small number of samples. 46 Another similar CRISPR-based test uses the DNA endonuclease-targeted CRISPR trans reporter (DETECTR) platform combining RT-LAMP with Cas12. 47 The SARS-CoV-2 DETECTR protocol uses the N and E genes as targets with a similar visual lateral flow strip readout that enables viral RNA detection within 45 minutes.…”

Positive interactions are common among culturable bacteria

“…To gain a broad understanding of how species interact across a wide range of environments, we used a combinatorial screening platform called kChip (33)(34)(35) to measure >150,000 pairwise bacterial cocultures among 20 different soil bacterial strains across 40 environments with differing carbon source identity or concentration. The kChip generates cocultures at an ultra-high throughput scale by rapidly and randomly combining droplets containing microbial cultures and/or medium components within microwells (33) ( Fig.…”

A new role for Biofoundries in rapid prototyping, development, and validation of automated clinical diagnostic tests for SARS-CoV-2

“…The diagnostic output interchangeability is created not only by generating custom protocols for several commercially available kits, but also by adapting the CRISPR Cas13 detection and colorimetric LAMP systems to HT SARS-CoV-2 diagnostic testing. CRISPR-based detection technologies are also currently being developed by Sherlock Biosciences and Mammoth Biosciences to generate at-home point-of-care testing kits [14], [23], [24], as well as in CARMEN-Cas13, a microwell array which multiplexes virus detection [25].…”