BRD0539 transiently cuts CRISPR/Cas9 mosaicism by 59% in porcine embryos without losing editing efficiency

CRISPR/Cas9 gene editing is a powerful tool for generating genetically modified animals, but a major challenge is the high rate of mosaicism. This occurs when Cas9 activity persists after the initial cleavage, leading to different genetic edits in various blastomeres. Current methods struggle to precisely control Cas9 activity, resulting in non-uniform gene edits across cells and tissues, which can complicate research and therapeutic applications. Reducing mosaicism is crucial for producing high-quality, uniformly edited animals.

Researchers investigated the small-molecule Cas9 inhibitor BRD0539 in porcine parthenogenetic activation embryos targeting the myostatin (MSTN) gene. They tested two delivery methods: co-injection of 10 or 100 μM BRD0539 with Cas9 mRNA:sgRNA, and supplementation of the culture medium with 10 or 50 μM BRD0539. A key experiment involved short-term addition of 10 μM BRD0539 to the culture medium between 24 and 48 h post-activation. Primary endpoints included gene editing efficiency, mosaicism rate, cleavage rates, blastocyst development, and total cell number, with immunofluorescence used to track Cas9 protein expression.

Immunofluorescence confirmed sustained Cas9 protein expression up to 48 h post-injection. Co-injection of 10 or 100 μM BRD0539 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). Similarly, supplementing the culture medium with 10 or 50 μM BRD0539 also reduced editing efficiency (20.8% ± 12.4% and 47.7% ± 14.6% 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).