Cas12a-Capture: A Novel, Low-Cost, and Scalable Method for Targeted Sequencing.
CRISPR J
; 5(4): 548-557, 2022 08.
Article
in En
| MEDLINE
| ID: mdl-35833801
ABSTRACT
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.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
CRISPR-Cas Systems
/
Gene Editing
Type of study:
Health_economic_evaluation
Language:
En
Journal:
CRISPR J
Year:
2022
Document type:
Article
Affiliation country: