CRISPR-Cas9-mediated knock-in of cytomegalovirus US2 provides an alternative strategy for generating hypoimmunogenic hiPSC lines.

Mismatches in HLA haplotypes between donors and recipients significantly increase the risk of graft failure due to immune rejection. Knockout (KO) of beta-2 microglobulin (B2M) is the current standard for eliminating HLA class I (HLA-I) surface expression and protecting allogeneic products from T-cell-mediated rejection; however, complete HLA-I ablation can trigger natural killer (NK) cell "missing-self" responses and disrupt critical immune-regulatory interactions. To address these limitations, we introduced a cytomegalovirus-derived US2 encoding sequence into the AAVS1 safe-harbor locus of a human induced pluripotent stem cell (hiPSC) line. Flow cytometry showed that US2 selectively abrogates HLA-A2 surface expression while retaining low levels of total HLA-I. In coculture assays, US2 expression abolished HLA-A2 alloreactive T-cell activation without increasing NK cell degranulation, indicating preserved inhibitory signaling. Together, these findings establish US2-mediated immune evasion as a refined single-edit alternative to B2M KO, enabling selective HLA-I modulation while preserving critical immune-regulatory interactions.