Cas12a-Capture: A Novel, Low-Cost, and Scalable Method for Targeted Sequencing

Taylor L. Mighell, Andrew Nishida, Brendan L. O'connell, Caitlin V. Miller, Sally Grindstaff, Casey A. Thornton, Andrew C. Adey, Daniel Doherty, Brian J. O'roak

Research output: Contribution to journalArticlepeer-review


Targeted sequencing remains a valuable technique for clinical and research applications. However, many existing technologies suffer from pervasive guanine-cytosine (GC) sequence content bias, high input DNA requirements, and high cost for custom panels. We have developed Cas12a-Capture, a low-cost and highly scalable method for targeted sequencing. The method utilizes preprogrammed guide RNAs to direct CRISPR-Cas12a cleavage of double-stranded DNA in vitro and then takes advantage of the resulting four to five nucleotide overhangs for selective ligation with a custom sequencing adapter. Addition of a second sequencing adapter and enrichment for ligation products generates a targeted sequence library. We first performed a pilot experiment with 7176 guides targeting 3.5 Mb of DNA. Using these data, we modeled the sequence determinants of Cas12a-Capture efficiency, then designed an optimized set of 11,438 guides targeting 3.0 Mb. The optimized guide set achieves an average 64-fold enrichment of targeted regions with minimal GC bias. Cas12a-Capture variant calls had strong concordance with Illumina Platinum Genome calls, especially for single nucleotide variants, which could be improved by applying basic variant quality heuristics. We believe Cas12a-Capture has a wide variety of potential clinical and research applications and is amendable for selective enrichment for any double-stranded DNA template or genome.

Original languageEnglish (US)
Pages (from-to)548-557
Number of pages10
JournalCRISPR Journal
Issue number4
StatePublished - Aug 1 2022

ASJC Scopus subject areas

  • Biotechnology
  • Genetics


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