Development of a baculoviral CRISPR/Cas9 vector system for beta-2-microglobulin knockout in human pluripotent stem cells.

Derivation of hypoimmunogenic human cells from genetically manipulated pluripotent stem cells holds great promise for future transplantation medicine and adoptive immunotherapy. Disruption of beta-2-microglobulin (B2M) in pluripotent stem cells followed by differentiation into specialized cell types is a promising approach to derive hypoimmunogenic cells. Given the attractive features of CRISPR/Cas9-based gene editing tool and baculoviral delivery system, baculovirus can deliver CRISPR/Cas9 components for site-specific gene editing of B2M. Herein, we report the development of a baculoviral CRISPR/Cas9 vector system for the B2M locus disruption in human cells. When tested in human embryonic stem cells (hESCs), the B2M gene knockdown/out was successfully achieved, leading to the stable down-regulation of human leukocyte antigen class I expression on the cell surface. Fibroblasts derived from the B2M gene-disrupted hESCs were then used as stimulator cells in the co-cultures with human peripheral blood mononuclear cells. These fibroblasts triggered significantly reduced alloimmune responses as assessed by sensitive Elispot assays. The B2M-negative hESCs maintained the pluripotency and the ability to differentiate into three germ lineages in vitro and in vivo. These findings demonstrated the feasibility of using the baculoviral-CRISPR/Cas9 system to establish B2M-disrupted pluripotent stem cells. B2M knockdown/out sufficiently leads to hypoimmunogenic conditions, thereby supporting the potential use of B2M-negative cells as universal donor cells for allogeneic cell therapy.