FLASH (FINDING LOW ABUNDANCE SEQUENCES BY HYBRIDIZATION)
Researchers at the Chan Zuckerberg Biohub have developed an improved technique, FLASH, which uses a programmable nuclease to enrich target sequences during preparation of nucleotide libraries.
Next generation sequencing (NGS) technology is a powerful tool in diagnostic and functional genomics. However, the detection of low abundance targets from clinical specimens is a central challenge in clinical diagnostics. Current methods for enriching sequences of interest in a complex nucleic acid library can be inefficient, expensive, difficult to optimize, and limited in the number of sequences that can be enriched for in a given sample.
Stage of Research
The inventors have developed an improved method for enriching sequences of interest in a complex nucleic acid library. Their technique, FLASH (Finding Low Abundance Sequences by Hybridization), uses a sequence-specific nuclease, such as CRISPR/Cas9, to cleave sequences of interest into fragments that are compatible with further molecular analysis, such as NGS. Cleaved fragments of interest are enriched over background by amplification and are appropriately sized for NGS sequencing. While FLASH has relevance across many biomedical disciplines, the authors demonstrate the utility of the technique by applying it to the detection of multiple antimicrobial resistance genes in patient-derived biological samples.
Applications
- Detection of low abundance sequences in direct clinical specimens, such as pathogen antimicrobial resistance genes, for metagenomic next generation sequencing (NGS)
- CRISPR-based diagnostics of cancer mutations or rare mosaic alleles
- Targeted transcriptomics from clinical samples
Advantages
- Enrichment and detection of low abundance target genes in DNA or RNA-derived cDNA libraries with minimal background
- Enables high levels of multiplexing (e.g., enrichment of thousands of targets)
- Open-source software (FLASHit) for guide RNA design
Stage of Development
Research – in vitro
Publications
Quan J, Langelier C, Kuchta A, Batson J, Teyssier N, Lyden A, Caldera S, McGeever A, Dimitrov B, King R, Wilheim J, Murphy M, Pesce Ares L, Travisano KA, Sit R, Amato R, Mumbengegwi DR, Smith JL, Bennett A, Gosling R, Mourani PM, Calfee CS, Neff NF, Chow ED, Kim PS, Greenhouse B, DeRisi JL, Crawford ED. FLASH: A next-generation CRISPR diagnostic for multiplexed detection of antimicrobial resistant sequences. bioRxiv doi 10.1101/426338 (2019); also published in Nucleic Acids Research 47, 14: page e83 (2019)
PCT Publication WO/2020/033438
Keywords
CRISPR/Cas9, next generation sequencing (NGS), multiplex PCR, nucleic acid library
Technology Reference
Chan Zuckerberg CZB-114
