CaBagE: A Cas9-based Background Elimination strategy for targeted, long-read DNA sequencing.

Area of Science:

• Genomics
• Molecular Biology
• Biotechnology

Background:

• Short-read DNA sequencing faces limitations in interrogating complex genomic regions like paralogs and tandem repeats.
• Long-read sequencing offers direct measurement of complex loci but has lower throughput.
• Target enrichment strategies are needed to improve efficiency for long-read sequencing of challenging genomic areas.

Purpose of the Study:

• To present CaBagE, an efficient and versatile method for target enrichment of large, structurally complex DNA targets for long-read sequencing.
• To demonstrate CaBagE's utility in enriching specific genomic loci, including cancer genes and repeat expansions associated with diseases.

Main Methods:

• CaBagE utilizes Cas9's DNA-binding ability to protect target fragments from exonuclease digestion, enabling enrichment.
• Enriched DNA fragments are then sequenced using Oxford Nanopore's MinION long-read sequencing technology.
• The method was tested on five genomic targets (4-20kb) and applied to cancer gene panels and C9orf72 repeat expansions in ALS patients.

Main Results:

• CaBagE achieved a median of 116X coverage (range 39-416X) for target loci using healthy donor DNA.
• Four cancer gene targets were successfully enriched in a single reaction and multiplexed on one MinION flow cell.
• Genotype estimates for C9orf72 short tandem repeat expansions in ALS patients were obtained, comparable to repeat-primed PCR.

Conclusions:

• CaBagE provides physical enrichment of on-target DNA, enhancing sequencing depth and accuracy for complex genomic regions.
• The method is adaptable across sequencing platforms and applicable to various research areas, including disease gene discovery.
• CaBagE offers a rapid enrichment strategy to explore previously inaccessible regions of the human genome relevant to disease.