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

Jenai Quan(University of California, San Francisco), Charles Langelier(University of California, San Francisco), Alison Kuchta(University of California, San Francisco), Joshua Batson(Chan Zuckerberg Initiative (United States)), Noam Teyssier(University of California, San Francisco), Amy Lyden(Chan Zuckerberg Initiative (United States)), Saharai Caldera(University of California, San Francisco), Aaron McGeever(Chan Zuckerberg Initiative (United States)), Boris Dimitrov(Chan Zuckerberg Initiative (United States)), Ryan J. King(Chan Zuckerberg Initiative (United States)), Jordan Wilheim(University of California, San Francisco), Maxwell Murphy(University of California, San Francisco), Lara Pesce Ares(Chan Zuckerberg Initiative (United States)), Katherine Travisano(Chan Zuckerberg Initiative (United States)), Rene Sit(Chan Zuckerberg Initiative (United States)), Roberto Amato(Wellcome Sanger Institute), Davis Mumbengegwi(University of Namibia), Jennifer L. Smith(University of California, San Francisco), Adam Bennett(University of California, San Francisco), Roly Gosling(University of California, San Francisco), Peter M. Mourani(Children's Hospital Colorado), Carolyn S. Calfee(University of California, San Francisco), Norma Neff(Chan Zuckerberg Initiative (United States)), Eric D. Chow(University of California, San Francisco), Peter S. Kim(Chan Zuckerberg Initiative (United States)), Bryan Greenhouse(University of California, San Francisco), Joseph L. DeRisi(University of California, San Francisco), Emily Crawford(University of California, San Francisco)
Nucleic Acids Research
May 7, 2019
10.1093/nar/gkz418
Cited by 258Open Access
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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.

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