Effect of <scp>BRD0539</scp> on Gene Editing and Mosaicism Rate in Porcine Gene Editing Embryos by <scp>CRISPR</scp> /Cas9

Microinjection is a common method for generating gene-edited animals; however, persistent Cas9 activity post-cleavage often results in mosaic embryos due to editing occurring in different blastomeres. This study investigated whether co-injecting the CRISPR/Cas9 system with the small-molecule Cas9 inhibitor BRD0539, or supplementing it in the culture medium, could reduce mosaicism while maintaining editing efficiency in porcine parthenogenetic activation embryos targeting the myostatin (MSTN) gene. The findings are as follows: Co-injection of 10 or 100 μM BRD0539 with Cas9 mRNA: sgRNA significantly reduced gene editing efficiency (28.5% ± 11.6% and 33.8% ± 4.1%, respectively, vs. 86.9% ± 4.5% in control, p < 0.05). Supplementing the culture medium with 10 or 50 μM BRD0539 also reduced both editing efficiency (20.8% ± 12.4% and 47.7% ± 14.6%, respectively, vs. 85.6% ± 4.8%) and mosaicism rate (25.0% ± 15.9% and 12.5% ± 12.5%, respectively, vs. 87.1% ± 7.8%, p < 0.05). Immunofluorescence revealed sustained Cas9 protein expression up to 48 h post-injection. Crucially, short-term addition of 10 μM BRD0539 to the culture medium between 24 and 48 h post-activation significantly reduced mosaicism (32.6% ± 7.5% vs. 78.7% ± 9.6%, p < 0.05) without compromising editing efficiency. Furthermore, this treatment did not adversely affect cleavage rates, blastocyst development, total cell number. These results demonstrate that transient inhibition of Cas9 activity with 10 μM BRD0539 during a critical window effectively reduces mosaicism in microinjected porcine embryos, offering a promising strategy to enhance the efficiency of generating non-mosaic gene-edited livestock.