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

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 metagenomicNext Generation Sequencing (NGS). We introduce FLASH (Finding Low Abundance Sequencesby Hybridization), a next-generation CRISPR/ Cas9diagnostic method that takes advantage of the efficiency, specificity and flexibility of Cas9to 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 RNAdesign. 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.