Bead-based approaches to CRISPR diagnostics

CRISPR-based diagnostics have emerged as a promising tool for fast, accurate, and portable pathogen detection. There has been rapid progress in areas such as pre-amplification processes and CRISPR-related enzymes, but the development of reporter systems and reaction platforms has lagged behind. In this paper, we develop new bead-based techniques that can help fill both gaps. First, we develop a novel bead-based split-luciferase reporter system with improved sensitivity compared to standard fluorescence-based reporter design in CRISPR diagnostics. Second, we develop a highly deployable, bead-based platform capable of detecting nine distinct viral targets in parallelized, droplet-based reactions. We demonstrate the enhanced performance of both approaches on synthetic and clinical samples. Together, these systems represent new modalities in CRISPR diagnostics with increased sensitivity, speed, multiplexing, and deployability. ### Competing Interest Statement S.M.S., N.L.W, J.A.S, C.A., P.C.B., P.C.S., and C.M. are inventors on pending patent applications related to this work, SHINE, and multiplexed Cas13 diagnostics. P.C.S. is a co-founder of, shareholder in, and consultant to Sherlock Biosciences, Inc. and Delve Bio, as well as a Board member of and shareholder in Danaher Corporation. C.M. is a co-founder of Carver Biosciences, a startup company developing Cas13-based antivirals, and holds equity in Carver Biosciences. P.C.B. is a consultant to or holds equity in 10X Genomics, General Automation Lab Technologies/Isolation Bio, Celsius Therapeutics, Next Gen Diagnostics, Cache DNA, Concerto Biosciences, Stately, Ramona Optics, Bifrost Biosystems, and Amber Bio. His laboratory receives research funding from Calico Life Sciences, Merck, and Genentech for unrelated work. C.A. is the CEO and co-founder of Concerto Biosciences..All other authors declare no competing interests. ### Funding Statement This work was made possible by funding provided by the Defense Advanced Research Projects Agency (no. D18AC00006), the Centers for Disease Control (no. 75D30122C15113), Howard Hughes Medical Institute, the Flu Lab, and a cohort of donors through the Audacious Project, a collaborative funding initiative housed at TED, including The ELMA Foundation, MacKenzie Scott, the Skoll Foundation, and Open Philanthropy. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This study received a non-human subjects research determination from the Broad Institute Office of Research Subject Protections (NHSR-4318). To conduct this research, de-identified nasopharyngeal swab patient samples were purchased from Boca Biolistics (USA). Additional de-identified clinical samples were obtained from studies evaluated and approved at the Center for Disease Control and Prevention (CDC). This activity was reviewed by CDC and was conducted consistent with applicable federal law and CDC policy (See e.g., 45 C.F.R. part 46; 21 definition of research as defined in and was conducted consistent with C.F.R. part 56; 42 U.S.C. 241(d), 46.102(l) but did not involve human applicable federal law and CDC U.S.C. 552a, 44 U.S.C. 3501 et seq.) I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present work are contained in the manuscript