Rapid Genotyping of Mutation in Sheep Using CRISPR-Cas12a Integrated with DNA Nanotree Biosensing Platform.

The A-to-G mutation () in the gene is strongly linked to fertility in sheep, significantly increasing ovulation rates and litter sizes compared to wild-type populations. The rapid and reliable screening of the gene is therefore critical for advancing sheep breeding programs. This study aimed to develop a fast and accurate method for detecting the mutation and genotyping the gene to enhance sheep reproduction and productivity. To achieve this, we integrated the CRISPR-Cas12a system with an optimized amplification refractory mutation system (ARMS). A similar DNA origami technique-based fluorescence reporter nanotree structure was synthesized using gold nanomagnetic beads as carriers to amplify the fluorescence signal further. The resulting biosensing platform, termed CRISPR-ARMS, demonstrated excellent sensitivity for detecting mutations, with a detection limit as low as 0.02 pmol. Therefore, this innovative approach shows great promise for single-base mutation detection and represents a pioneering tool for high-yield genetic screening.