This invention provides a method for efficient multi-
gene editing and CAR site-directed integration based on BaEV envelope
glycoprotein truncated viral particles and its application in immune
cell engineering. The invention provides an MLV-VLP
system utilizing BaEV envelope
glycoprotein truncated particles to achieve efficient
CRISPR-Cas9 delivery without
electroporation or the use of randomly integrating viruses. Combined with adeno-associated
virus (AAV6) delivery of HDR templates, it enables efficient site-directed integration of CAR sequences at target sites (such as TRAC, PD1, and B2M) in primary human T cells and NK cells. The prepared CAR-T or CAR-NK cells exhibit excellent
cell killing, proliferation, and low exhaustion characteristics
in vitro, achieving 100% tumor clearance in an
in vivo mouse model. It can simultaneously achieve multi-
gene knockout with high efficiency, supporting large-scale allogeneic CAR-T and CAR-NK
cell production.