FLASH-MS: FINDING LOW ABUNDANCE SEQUENCES BY CAS9 HYBRIDIZATION MASS SPECTROMETRY
Researchers at the Chan Zuckerberg Biohub have developed FLASH-MS, a method for nucleic acid sequence detection by endonuclease digestion and mass spectrometry.
The rapid detection of low abundance targets from clinical specimens is a central challenge in clinical diagnostics. The inventors have previously developed a 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 next-generation sequencing (NGS).
Stage of Research
The inventors have developed a new FLASH application, FLASH-MS, which is a method for low abundance sequence detection by mass spectrometry. In FLASH-MS, Cas9 is used in the same manner, but instead of creating NGS-compatible fragments, the CRISPR guide RNA library is designed to create fragments with unique mass profiles for identifying the gene(s) of interest. FLASH-MS is also amenable to detection of FLASH-derived RNA products or polypeptides following in vitro transcription and translation reactions. For example, adapters ligated during FLASH reactions may contain regulatory elements that direct transcription of the adjacent sequences of interest, and the RNA or polypeptides translated from the RNA are detected and characterized by mass spectrometry.
Applications
- Rapid and economical methods for enrichment and detection of low abundance target genes, with applications for infectious disease or cancer diagnostics
Advantages
- Enables high levels of multiplexing, and sequences can be designed for optimal resolution of signals associated with different transcripts or polypeptides associated with different target sequences
- Sequences of fragments produced can be designed to encode RNAs or polypeptides that produce an optimal signal and unique mass pattern
Stage of Development
Research – in vitro
Publications
Quan J, et al. FLASH: a next-generation CRISPR diagnostic for multiplexed detection of antimicrobial resistance sequences. 2019. Nucleic Acids Research. DOI: 10.1093/nar/gkz418
Keywords
Cas9, CRISPR, nuclease, RNA sequencing, genome dissection
Reference
Chan Zuckerberg Biohub CZB-131
