High-efficiency base editing enables multiplex inactivation of retroviral and immunologic barriers in porcine xenotransplantation
Xenotransplantation is constrained by persistent molecular incompatibilities between pigs and humans, including xenoantigens, major histocompatibility complexes, and porcine endogenous retroviruses (PERVs). Existing genome engineering strategies are limited by inefficiency, mosaicism, and genomic instability, hindering scalable multiplex editing. Here, we establish a high-efficiency platform for multiplex genome rewriting in pigs using a fourth-generation cytosine base editor (BE4) that enables precise, double-strand break-free editing in zygotes. We achieve simultaneous inactivation of swine leukocyte antigen loci, resulting in loss of class I and marked reduction of class II expression, and near-complete disruption of replication-competent PERVs, eliminating detectable viral transmission. In parallel, multiplex editing of major carbohydrate xenoantigens (GGTA1, CMAH, B4GALNT2) and GHR yields viable multi-gene knockout pigs with reduced immunogenicity and controlled growth. Functional assays demonstrate improved compatibility in ex vivo human blood perfusion. This platform enables rapid, scalable engineering of clinically relevant donor pigs for xenotransplantation.