Hayden C. Metsky†, Nicole L. Welch, Nicholas J. Haradhvala, Laurie Rumker, Yibin B. Zhang, Priya P. Pillai, David K. Yang, Cheri M. Ackerman, Juliane Weller, Paul C. Blainey, Cameron Myhrvold*, Michael Mitzenmacher*, and Pardis C. Sabeti*. 11/28/2020. “Efficient design of maximally active and specific nucleic acid diagnostics for thousands of viruses.” bioRxiv. Publisher's VersionAbstract
Harnessing genomic data and predictive models will provide activity-informed diagnostic assays for thousands of viruses and offer rapid design for novel ones. Here we develop and extensively validate new algorithms that design nucleic acid assays having maximal predicted detection activity over a virus’s full genomic diversity with stringent specificity. Focusing on CRISPR-Cas13a detection, we test a library of  ~ 19,000 guide-target pairs and construct a convolutional neural network that predicts Cas13a detection activity better than other techniques. We link our methods by building ADAPT, an end-to-end system that automatically leverages the latest viral genome data. We designed optimal species-specific assays for the 1,933 vertebrate-infecting viral species within 2 hours for most species and 24 hours for all but 3. ADAPT’s designs are sensitive and specific down to the lineage-level for the range of taxa we tested, including ones that pose challenges involving genomic diversity and specificity. They also exhibit significantly higher fluorescence and lower limits of detection, across a virus’s full spectrum of genomic diversity, than designs from standard techniques. ADAPT is available in an accessible software package and can be applied to other detection technologies to enhance critically-needed viral diagnostic and surveillance efforts.
Jon Arizti-Sanz*, Catherine A Freije*, Alexandra C Stanton, Chloe K Boehm, Brittany A Petros, Sameed Siddiqui, Bennett M Shaw, Gordon Adams, Tinna-Solveig F Kosoko-Thoroddsen, Molly E Kemball, Jessica N Uwanibe, Jacob E Lemieux, Fehintola V Ajobasile, Philomena E Eromon, Robin Gross, Loni Wronka, Katie Caviness, Lisa E Hensley, Nicholas H Bergman, Bronwyn L MacInnis, Christian T Happi, Pardis C Sabeti*, and Cameron Myhrvold*†. 11/20/2020. “Integrated sample inactivation, amplification, and Cas13-based detection of SARS-CoV-2.” Nature Communications, 11. Publisher's VersionAbstract
The COVID-19 pandemic has highlighted that new diagnostic technologies are essential for controlling disease transmission. Here, we develop SHINE (Streamlined Highlighting of Infections to Navigate Epidemics), a sensitive and specific diagnostic tool that can detect SARS-CoV-2 RNA from unextracted samples. We identify the optimal conditions to allow RPA-based amplification and Cas13-based detection to occur in a single step, simplifying assay preparation and reducing run-time. We improve HUDSON to rapidly inactivate viruses in nasopharyngeal swabs and saliva in 10 min. SHINE’s results can be visualized with an in-tube fluorescent readout — reducing contamination risk as amplification reaction tubes remain sealed — and interpreted by a companion smartphone application. We validate SHINE on 50 nasopharyngeal patient samples, demonstrating 90% sensitivity and 100% specificity compared to RT-qPCR with a sample-to-answer time of 50 min. SHINE has the potential to be used outside of hospitals and clinical laboratories, greatly enhancing diagnostic capabilities.
Cheri M Ackerman*, Cameron Myhrvold*†, Sri Gowtham Thakku, Catherine A Freije, Hayden C Metsky, David K Yang, Simon H Ye, Chloe K Boehm, Tinna-Sólveig F Kosoko-Thoroddsen, Jared Kehe, Tien G Nguyen, Amber Carter, Anthony Kulesa, John R Barnes, Vivien G Dugan, Deborah T Hung, Paul C Blainey*†, and Pardis C Sabeti*. 4/29/2020. “Massively multiplexed nucleic acid detection with Cas13.” Nature, 582, 7811, Pp. 277-282.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 while simultaneously testing for many pathogens. 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 self-organize in a microwell array 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.
Hayden C Metsky*, Catherine A Freije*, Tinna-Solveig F Kosoko-Thoroddsen, Pardis C Sabeti, and Cameron Myhrvold*†. 3/2/2020. “CRISPR-based surveillance for COVID-19 using genomically-comprehensive machine learning design.” bioRxiv. Publisher's VersionAbstract
The emergence and outbreak of SARS-CoV-2, the causative agent of COVID-19, has rapidly become a global concern and has highlighted the need for fast, sensitive, and specific tools to surveil circulating viruses. Here we provide assay designs and experimental resources, for use with CRISPR-based nucleic acid detection, that could be valuable for ongoing surveillance. We provide assay designs for detection of 67 viral species and subspecies, including: SARS-CoV-2, phylogenetically-related viruses, and viruses with similar clinical presentation. The designs are outputs of algorithms that we are developing for rapidly designing nucleic acid detection assays that are comprehensive across genomic diversity and predicted to be highly sensitive and specific. Of our design set, we experimentally screened 4 SARS-CoV-2 designs with a CRISPR-Cas13 detection system and then extensively tested the highest-performing SARS-CoV-2 assay. We demonstrate the sensitivity and speed of this assay using synthetic targets with fluorescent and lateral flow detection. Moreover, our provided protocol can be extended for testing the other 66 provided designs. Assay designs are available at
Catherine A Freije*†, Cameron Myhrvold*†, Chloe K Boehm, Aaron E Lin, Nicole L Welch, Amber Carter, Hayden C Metsky, Cynthia Y Luo, Omar O Abudayyeh, Jonathan S Gootenberg, Nathan L Yozwiak, Feng Zhang, and Pardis C Sabeti†. 2019. “Programmable Inhibition and Detection of RNA Viruses Using Cas13.” Mol Cell, 76, 5, Pp. 826-837.e11.Abstract
The CRISPR effector Cas13 could be an effective antiviral for single-stranded RNA (ssRNA) viruses because it programmably cleaves RNAs complementary to its CRISPR RNA (crRNA). Here, we computationally identify thousands of potential Cas13 crRNA target sites in hundreds of ssRNA viral species that can potentially infect humans. We experimentally demonstrate Cas13's potent activity against three distinct ssRNA viruses: lymphocytic choriomeningitis virus (LCMV); influenza A virus (IAV); and vesicular stomatitis virus (VSV). Combining this antiviral activity with Cas13-based diagnostics, we develop Cas13-assisted restriction of viral expression and readout (CARVER), an end-to-end platform that uses Cas13 to detect and destroy viral RNA. We further screen hundreds of crRNAs along the LCMV genome to evaluate how conservation and target RNA nucleotide content influence Cas13's antiviral activity. Our results demonstrate that Cas13 can be harnessed to target a wide range of ssRNA viruses and CARVER's potential broad utility for rapid diagnostic and antiviral drug development.
Cameron Myhrvold*†, Catherine A Freije*†, Jonathan S Gootenberg, Omar O Abudayyeh, Hayden C Metsky, Ann F Durbin, Max J Kellner, Amanda L Tan, Lauren M Paul, Leda A Parham, Kimberly F Garcia, Kayla G Barnes, Bridget Chak, Adriano Mondini, Mauricio L Nogueira, Sharon Isern, Scott F Michael, Ivette Lorenzana, Nathan L Yozwiak, Bronwyn L MacInnis, Irene Bosch, Lee Gehrke, Feng Zhang, and Pardis C Sabeti†. 2018. “Field-deployable viral diagnostics using CRISPR-Cas13.” Science, 360, 6387, Pp. 444-448.Abstract
Mitigating global infectious disease requires diagnostic tools that are sensitive, specific, and rapidly field deployable. In this study, we demonstrate that the Cas13-based SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) platform can detect Zika virus (ZIKV) and dengue virus (DENV) in patient samples at concentrations as low as 1 copy per microliter. We developed HUDSON (heating unextracted diagnostic samples to obliterate nucleases), a protocol that pairs with SHERLOCK for viral detection directly from bodily fluids, enabling instrument-free DENV detection directly from patient samples in <2 hours. We further demonstrate that SHERLOCK can distinguish the four DENV serotypes, as well as region-specific strains of ZIKV from the 2015-2016 pandemic. Finally, we report the rapid (<1 week) design and testing of instrument-free assays to detect clinically relevant viral single-nucleotide polymorphisms.
Jonathan S Gootenberg*, Omar O Abudayyeh*, Jeong Wook Lee, Patrick Essletzbichler, Aaron J Dy, Julia Joung, Vanessa Verdine, Nina Donghia, Nichole M Daringer, Catherine A Freije, Cameron Myhrvold, Roby P Bhattacharyya, Jonathan Livny, Aviv Regev, Eugene V Koonin, Deborah T Hung, Pardis C Sabeti, James J Collins†, and Feng Zhang†. 2017. “Nucleic acid detection with CRISPR-Cas13a/C2c2.” Science, 356, 6336, Pp. 438-442.Abstract
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.

* denotes equal contribution
† denotes corresponding authorship