A CRISPR-based strategy for targeted sequencing in biodiversity science

Area of Science:

• Molecular Ecology
• Genomics
• Biotechnology

Background:

• Molecular ecology relies on matching DNA sequences from samples to reference libraries.
• Current DNA barcoding and metabarcoding use PCR and amplicon sequencing.
• CRISPR-Cas systems offer potential for targeted DNA enrichment.

Purpose of the Study:

• To identify and test CRISPR-Cas guide RNAs for enriching plant chloroplast DNA.
• To evaluate the efficacy of CRISPR-Cas enrichment for DNA barcoding and chloroplast assembly.
• To compare CRISPR-Cas enrichment with traditional PCR-based methods for single and mixed-species samples.

Main Methods:

• Identification of 54,837 potential CRISPR-Cas guide RNAs for plant chloroplast DNA.
• In vitro testing of 17 guide RNAs for enriching DNA fragments from 1,428 bp to 121,284 bp.
• Sequencing using Oxford Nanopore technology on single- and mixed-species plant DNA samples.

Main Results:

• CRISPR-Cas enrichment successfully enriched plant DNA strands of various lengths.
• Sequencing yielded mean chloroplast sequence lengths of 2,530-11,367 bp.
• Mixed-species experiments showed a ≥48-fold increase in sequence length and improved abundance estimates compared to trnL-P6 metabarcoding.

Conclusions:

• CRISPR-Cas systems show promise for plant DNA barcoding and chloroplast genome assembly.
• This method offers advantages over PCR-based workflows, particularly for long-read sequencing.
• Further development of in vitro and in silico CRISPR-based methods is needed for complex mixed-species analyses.