FLASH: a next-generation CRISPR diagnostic for multiplexed detection of antimicrobial resistance sequences

Author:

Quan Jenai12,Langelier Charles3,Kuchta Alison4,Batson Joshua1,Teyssier Noam3,Lyden Amy1,Caldera Saharai3,McGeever Aaron1,Dimitrov Boris5,King Ryan5,Wilheim Jordan6,Murphy Maxwell6,Ares Lara Pesce1,Travisano Katherine A1,Sit Rene1,Amato Roberto7,Mumbengegwi Davis R8,Smith Jennifer L9,Bennett Adam9,Gosling Roly9,Mourani Peter M10,Calfee Carolyn S11,Neff Norma F1,Chow Eric D212,Kim Peter S11314,Greenhouse Bryan16,DeRisi Joseph L12,Crawford Emily D115ORCID

Affiliation:

1. Chan Zuckerberg Biohub, San Francisco, CA 94158, USA

2. Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA 94158, USA

3. Division of Infectious Diseases, Department of Medicine, University of California San Francisco, San Francisco, CA 94158, USA

4. Department of Pediatrics, University of California San Francisco, San Francisco, CA 94158, USA

5. Chan Zuckerberg Initiative, Redwood City, CA 94063, USA

6. Division of HIV, Infectious Diseases and Global Medicine, Department of Medicine, University of California San Francisco, San Francisco, CA 94143, USA

7. Wellcome Sanger Institute, Hinxton CB10 1SA, UK

8. Multidisciplinary Research Centre, University of Namibia, Windhoek 93Q5+48, Namibia

9. Department of Epidemiology and Biostatistics, University of California San Francisco, San Francisco, CA 94158, USA

10. Section of Critical Care Medicine, Department of Pediatrics, University of Colorado School of Medicine and Children's Hospital Colorado, Aurora, CO 80045, USA

11. Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of California San Francisco, San Francisco, CA 94158, USA

12. Center for Advanced Technology, University of California San Francisco, San Francisco, CA 94158, USA

13. Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305, USA

14. Stanford ChEM-H, Stanford, CA 94305, USA

15. Department of Microbiology and Immunology, University of California San Francisco, San Francisco, CA 94158, USA

Abstract

Abstract The growing prevalence of deadly microbes with resistance to previously life-saving drug therapies is a dire threat to human health. Detection of low abundance pathogen sequences remains a challenge for metagenomic Next Generation Sequencing (NGS). We introduce FLASH (Finding Low Abundance Sequences by Hybridization), a next-generation CRISPR/Cas9 diagnostic method that takes advantage of the efficiency, specificity and flexibility of Cas9 to enrich for a programmed set of sequences. FLASH-NGS achieves up to 5 orders of magnitude of enrichment and sub-attomolar gene detection with minimal background. We provide an open-source software tool (FLASHit) for guide RNA design. Here we applied it to detection of antimicrobial resistance genes in respiratory fluid and dried blood spots, but FLASH-NGS is applicable to all areas that rely on multiplex PCR.

Funder

University of California San Francisco

National Institutes of Health

Publisher

Oxford University Press (OUP)

Subject

Genetics

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