A modular genome writing platform enables iterative, scar-minimized installation of synthetic constructs exceeding 100 kb in human pluripotent stem cells, which when applied to engineer synthetic HLA haplotypes reveals that complete excision of active regulatory elements leaves a persistent, heritable epigenetic memory whose resolution to a silenced state requires native intergenic DNA, and that these synthetic loci restore functional immune recognition including NK cell sparing and T cell tolerance required for off-the-shelf cell therapies.

Human leukocyte antigen (HLA) polymorphism hinders off-the-shelf cell therapies. We developed REWRITE, a modular platform for iterative, scar-minimized genome writing of synthetic constructs >100 kb in human pluripotent stem cells (hPSCs). Using REWRITE, we deleted 105–209 kb of the HLA locus and installed synthetic 24 kb or 100 kb HLA haplotypes, and a 62 kb antigen-processing locus. This uncovered a persistent, heritable “epigenetic ghost” — an active state lingering despite genetic removal — whose resolution to a silenced default state is driven by native intergenic DNA. These loci restored inducible expression in key lineages, sparing cells from NK-mediated killing and establishing HLA-matched T-cell tolerance, enabling off-the-shelf cell therapies. REWRITE facilitates extensible programming of multigenic functions in allogeneic human cells — from immune design to genome architecture discovery.