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

  • Author(s): Quan, Jenai
  • Langelier, Charles
  • Kuchta, Alison
  • Batson, Joshua
  • Teyssier, Noam
  • Lyden, Amy
  • Caldera, Saharai
  • McGeever, Aaron
  • Dimitrov, Boris
  • King, Ryan
  • Wilheim, Jordan
  • Murphy, Maxwell
  • Ares, Lara Pesce
  • Travisano, Katherine A
  • Sit, Rene
  • Amato, Roberto
  • Mumbengegwi, Davis R
  • Smith, Jennifer L
  • Bennett, Adam
  • Gosling, Roly
  • Mourani, Peter M
  • Calfee, Carolyn S
  • Neff, Norma F
  • Chow, Eric D
  • Kim, Peter S
  • Greenhouse, Bryan
  • DeRisi, Joseph L
  • Crawford, Emily D
  • et al.
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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