Developing, characterizing and modeling CRISPR-based point-of-use pathogen diagnostics.

Autor:	Jung JK; Department of Chemical and Biological Engineering, Northwestern University (Evanston IL, USA).; Center for Synthetic Biology, Northwestern University (Evanston, IL, USA).; Center for Water Research, Northwestern University (Evanston, IL, USA)., Dreyer KS; Department of Chemical and Biological Engineering, Northwestern University (Evanston IL, USA).; Center for Synthetic Biology, Northwestern University (Evanston, IL, USA)., Dray KE; Department of Chemical and Biological Engineering, Northwestern University (Evanston IL, USA).; Center for Synthetic Biology, Northwestern University (Evanston, IL, USA)., Muldoon JJ; Department of Medicine, University of California, San Francisco (San Francisco, CA, USA).; Gladstone-UCSF Institute of Genomic Immunology (San Francisco, CA, USA)., George J; Center for Synthetic Biology, Northwestern University (Evanston, IL, USA).; Department of Engineering Sciences and Applied Mathematics, Northwestern University (Evanston, IL, USA).; NSF-Simons Center for Quantitative Biology, Northwestern University (Evanston, IL, USA)., Shirman S; Center for Synthetic Biology, Northwestern University (Evanston, IL, USA).; NSF-Simons Center for Quantitative Biology, Northwestern University (Evanston, IL, USA)., Cabezas MD; Center for Synthetic Biology, Northwestern University (Evanston, IL, USA).; Department of Biomedical Engineering, Northwestern University (Evanston, IL, USA)., D'Aquino AE; Center for Synthetic Biology, Northwestern University (Evanston, IL, USA).; Stemloop, Inc. (Evanston, IL, USA).; Interdisciplinary Biological Sciences Program, Northwestern University (Evanston, IL, USA)., Verosloff MS; Center for Synthetic Biology, Northwestern University (Evanston, IL, USA).; Interdisciplinary Biological Sciences Program, Northwestern University (Evanston, IL, USA)., Seki K; Department of Chemical and Biological Engineering, Northwestern University (Evanston IL, USA).; Center for Synthetic Biology, Northwestern University (Evanston, IL, USA)., Rybnicky GA; Center for Synthetic Biology, Northwestern University (Evanston, IL, USA).; Interdisciplinary Biological Sciences Program, Northwestern University (Evanston, IL, USA).; Chemistry of Life Processes Institute, Northwestern University (Evanston, IL, USA)., Alam KK; Stemloop, Inc. (Evanston, IL, USA)., Bagheri N; Department of Chemical and Biological Engineering, Northwestern University (Evanston IL, USA).; Center for Synthetic Biology, Northwestern University (Evanston, IL, USA).; Interdisciplinary Biological Sciences Program, Northwestern University (Evanston, IL, USA).; Departments of Biology and Chemical Engineering, University of Washington (Seattle, WA, USA)., Jewett MC; Department of Chemical and Biological Engineering, Northwestern University (Evanston IL, USA).; Center for Synthetic Biology, Northwestern University (Evanston, IL, USA).; Department of Bioengineering, Stanford University (Stanford, CA)., Leonard JN; Department of Chemical and Biological Engineering, Northwestern University (Evanston IL, USA).; Center for Synthetic Biology, Northwestern University (Evanston, IL, USA).; Interdisciplinary Biological Sciences Program, Northwestern University (Evanston, IL, USA)., Mangan NM; Center for Synthetic Biology, Northwestern University (Evanston, IL, USA).; Department of Engineering Sciences and Applied Mathematics, Northwestern University (Evanston, IL, USA).; NSF-Simons Center for Quantitative Biology, Northwestern University (Evanston, IL, USA)., Lucks JB; Department of Chemical and Biological Engineering, Northwestern University (Evanston IL, USA).; Center for Synthetic Biology, Northwestern University (Evanston, IL, USA).; Center for Water Research, Northwestern University (Evanston, IL, USA).; Chemistry of Life Processes Institute, Northwestern University (Evanston, IL, USA).
Jazyk:	angličtina
Zdroj:	BioRxiv : the preprint server for biology [bioRxiv] 2024 Jul 03. Date of Electronic Publication: 2024 Jul 03.
DOI:	10.1101/2024.07.03.601853
Abstrakt:	Recent years have seen intense interest in the development of point-of-care nucleic acid diagnostic technologies to address the scaling limitations of laboratory-based approaches. Chief among these are combinations of isothermal amplification approaches with CRISPR-based detection and readouts of target products. Here, we contribute to the growing body of rapid, programmable point-of-care pathogen tests by developing and optimizing a one-pot NASBA-Cas13a nucleic acid detection assay. This test uses the isothermal amplification technique NASBA to amplify target viral nucleic acids, followed by Cas13a-based detection of amplified sequences. We first demonstrate an in-house formulation of NASBA that enables optimization of individual NASBA components. We then present design rules for NASBA primer sets and LbuCas13a guide RNAs for fast and sensitive detection of SARS-CoV-2 viral RNA fragments, resulting in 20 - 200 aM sensitivity without any specialized equipment. Finally, we explore the combination of high-throughput assay condition screening with mechanistic ordinary differential equation modeling of the reaction scheme to gain a deeper understanding of the NASBA-Cas13a system. This work presents a framework for developing a mechanistic understanding of reaction performance and optimization that uses both experiments and modeling, which we anticipate will be useful in developing future nucleic acid detection technologies. Competing Interests: CONFLICT OF INTEREST DISCLOSURE K.K.A. M. C. J. & J.B.L. are founders and have financial interest in Stemloop, Inc, and these interests are reviewed and managed by Northwestern University and Stanford University in accordance with their conflict-of-interest policies. All other authors declare no conflicts of interest.
Databáze:	MEDLINE
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