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

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.

Serological and metagenomic interrogation of cerebrospinal fluid implicates enteroviruses in pediatric acute flaccid myelitis

“…Samples were also sequenced after enrichment for EV-A71 and EV-D68 genomes using FLASH (Finding Low Abundance Sequences by Hybridization, Supplemental Table 2). 20 All NGS libraries were depleted of host ribosomal RNA with DASH and spiked-in with External RNA Controls Consortium (ERCC) sequences as previously described. 21,22…”

Harnessing the potential of CRISPR-based platforms to advance the field of hospital medicine

“…This platform also has tremendous potential to identify and eradicate bacterial resistance genes, which enable pathogens to evade or neutralize antibiotics [2]. One such approach, employed by Quan and colleagues, is FLASH (Finding Low Abundance Sequences by Hybridization), a nextgeneration CRISPR-based diagnostic method that leverages the flexibility and specificity of genetic modification [55]. This approach has been shown to antibiotic resistance genes in saliva and serum and may soon replace multiplex PCR [56].…”

CASLFA: CRISPR/Cas9-mediated lateral flow nucleic acid assay

“…Using the CRISPR/Cas9 tool, our laboratory and other groups have developed new methods for genotyping pathogens and discriminating single nucleotide polymorphisms (SNPs) based on CRISPR/Cas9-mediated cleavage [32][33][34][35] . Very recently, two isothermal nucleic acid amplification methods based on the Cas9 nickase have also been developed and require a pair of Cas9/sgRNAs, two or three nucleases, accessory proteins, and expensive fluorescent probes 36,37 .…”

“…Very recently, two isothermal nucleic acid amplification methods based on the Cas9 nickase have also been developed and require a pair of Cas9/sgRNAs, two or three nucleases, accessory proteins, and expensive fluorescent probes 36,37 . Successful detection of dsDNA has also been achieved by binding based on dead Cas9 (dCas9) [32][33][34][35] . Despite this continuous progress, the highly complex the biosensor construction process or cost of this technique hampers its widespread application, particularly at the point of care.…”