ATM inhibition enhances AAV-mediated targeted integration in pig zygotes

A global shortage of transplantable organs has renewed interest in pigs as donors for xenotransplantation and hosts for interspecies organogenesis. Such uses require pigs carrying multiple genetic modifications. Although sequential editing of somatic donor cells followed by somatic cell nuclear transfer can assemble such modifications, this approach is often associated with developmental abnormalities caused by epigenetic errors. Direct genome editing of pig zygotes offers an alternative, yet long-fragment knock-in remains challenging. Here, we evaluated adeno-associated virus (AAV) donors in pig zygotes and tested whether transient inhibition of ataxia-telangiectasia mutated (ATM), which is overactivated during AAV transduction, improved the knock-in efficiency. AAV donors achieve single knock-in in 33% of blastocysts, and this increases to 66% with short-term ATM inhibition. ATM inhibition also improves double knock-in efficiency, increasing double-positive blastocysts from 19% to 52% without compromising blastocyst formation or quality. Embryo transfer yields double knock-in fetuses (33%), including an F0 fetus composed predominantly of edited cells. These results demonstrate that combining AAV donors with transient ATM inhibition is an efficient strategy for long-fragment knock-in in pigs. The authors evaluated adeno-associated virus (AAV) donors in pig zygotes and tested whether transient inhibition of ataxia-telangiectasia mutated (ATM), which is overactivated during AAV transduction, improved the knock-in efficiency.