Rapid on-site genotyping of the ovine prolific FecB^B mutation using a CRISPR/Cas12a-based detection system

BMPR1B is a pivotal gene that influences reproductive performance in sheep. The sheep populations that carry the FecB^B mutation within this gene exhibit significantly higher lambing rates compared to wild-type populations. Therefore, screening for individuals carrying the FecB^B mutation is crucial for effective sheep breeding programs. This study aims to establish a rapid, precise, and visualised on-site detection method for genotyping the prolific FecB^B mutation in sheep. We combined the CRISPR/Cas12a system with the recombinase-polymerase amplification (RPA) technique. We introduced an additional nucleotide mismatch on the amplification primers to form a Cas12a-recognised protospacer adjacent motif (PAM) sequence. In addition, mismatches were introduced in CRISPR-derived RNA (crRNA) to enable naked-eye differentiation of the assay results. Subsequently, we validated the accuracy of the method by examining additional blood samples from 56 sheep representing four breeds. The results of using our developed system were highly consistent with the Sanger sequencing. Overall, the CRISPR/Cas12a-based detection provides a rapid and more versatitle method for FecB^B genotyping. It holds promise in enhancing efficiency in livestock breeding programmes for any single nucleotide mutations.