CRISPR-Based Metabarcoding
Noteworthy
Microbes are abundant and very diverse. But acknowledging their significance in our biosphere has taken time and relied on curiosity, hard work, and the development of technologies to access, identify, and study microbial communities.
One powerful strategy for surveying samples without the need for cultivation, from microbiomes to environmental DNA (eDNA), involves coupling PCR with high-throughput sequencing technologies. In metabarcoding, PCR-generated amplicons of conserved genes or gene regions, such as 16S rRNA or ITS sequences, are subjected to massive sequencing and analyses. While valuable, this approach introduces biases at various steps, one of which is the use of PCR to enrich marker sequences.
In a new study, researchers at the University of Bristol exploited the natural CRISPR defense system found in bacteria and archaea to survey microbial communities. They used a method previously described to enrich regions of interest by harnessing the capacity of the Cas9 nuclease to target and cleave specific DNA sequences. In this work, the authors adapted this methodology to enrich taxonomic marker genes directly from a DNA sample and used these fragments for long-read nanopore sequencing.
Though the technique still needs some tweaking, such as improving nanopore read accuracy and taxonomic classification, it opens the possibility of capturing biodiversity in complex ecosystems without the need for PCR amplification.
("Noteworthy" is the new format for STC's Thursday posts. Please read our Jan 20, 2025 post outlining this and other changes in our blog.)
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