Ataxia-Telangiectasia Mutated Inhibition Enhances Adeno-Associated Virus-Mediated Knock-in Efficiency in Pig Zygotes

A persistent global shortage of transplantable organs has renewed interest in pigs as donors for xenotransplantation and hosts for interspecies organogenesis considering their physiology closely resembles that of humans. Such applications require pigs harboring genetic modifications, including knockouts, human transgenes, and conditional alleles. Although such complex allele combinations can be assembled by sequential editing of fetal fibroblasts, followed by somatic cell nuclear transfer (SCNT), SCNT is often associated with developmental abnormalities. Direct genome editing of pig zygotes offers an alternative route; however, achieving long-fragment knock-in, particularly at multiple loci, remains challenging. Here, adeno-associated virus (AAV) donor vectors for long-fragment knock-in in pig zygotes were evaluated, and tested whether transient inhibition of ataxia-telangiectasia mutated (ATM), which becomes overactivated during AAV transduction, could enhance knock-in efficiency. AAV6 donors supported efficient ACTB-mEGFP knock-in (33%), whereas short-term treatment with the ATM inhibitor AZ32 increased the efficiency to 66%. In addition, AZ32 enhanced simultaneous double knock-in at ACTB and UBB, generating a higher proportion of double-positive blastocysts (52% vs. 19% in controls), without affecting blastocyst formation rates or total cell numbers. Embryo transfer yielded ACTB-mEGFP and UBB-mScarlet-I3 double knock-in fetuses (33%), including an F0 fetus composed almost entirely of double knock-in cells. These results demonstrate that combining AAV donors with transient ATM inhibition is an efficient strategy for long-fragment knock-in at multiple loci in pigs.