;Objective To establish an integrated feeder-free platform for in vitro expansion and gene editing to tack-le the major challenges in clinical applications of cryopreserved primary human natural killer(NK)cells in terms of low expansion efficiency,technical difficulty in genetic modification and safety concerns.Methods A non-viral CRISPR-Cas9 ribonucleoprotein(RNP)-based multiplex gene editing system was developed through systematic op-timization of culture medium and nucleofection conditions.Cell phenotype(CD56+CD3-),viability,editing effi-ciency,and tumor-killing activity were evaluated via flow cytometry and cytotoxicity assays.Results The number of NK cells achieved 5 000-fold expansion over 25 days while maintaining high purity(CD56+CD3-＞95％)and viability(＞90％).Post-thawing viability(＞80％)and tumor-killing capacity were preserved.Cas9 RNP delivery enabled efficient dual knockout of NKG2A and CISH immune checkpoint genes(＞80％),significantly enhanced cytotoxicity against K562 tumor cells(P＜0.05).Conclusions Compared to viral vectors,the non-viral strategy eliminates genomic integration risks and reduces off-target effects.This result may provide a safe and efficient tech-nical platform for clinical application of NK cell immunotherapy and potentially encourage application of multiplex gene editing in cancer therapy.

Objective To explore the design of CRISPR interference(CRISPRi)tools based on the deactivated CasMINI(dCasMINI)protein and to evaluate their transcriptional inhibition effects.Methods The tetracycline-on(tet-on)system,flow cytometry,and quantitative reverse transcription polymerase chain reaction(RT-qPCR)were used to evaluate the transcriptional inhibition effects of dCasMINI system in mammalian cells at three level-plasmid genes,exogenous genomic loci,and endogenous genomic loci.Additionally,six dCasMINI-CRISPRi tools(dCasMINI,dCasMINI-ZIM3 KRAB,dCasMINI-KRAB-MeCP2,dCasMINI-ZNF324 KRAB,dCasMINI-3x KRAB,and dCasMINI-Com-KRAB-MECP2)were designed and compared for their transcriptional inhibition effects along with single guide RNA(sgRNA)at different positions.Results dCasMINI,dCasMINI-ZIM3 KRAB,dCasMINI-KRAB-MeCP2,dCasMINI-ZNF324 KRAB,dCasMINI-3x KRAB,and dCasMINI-Com-KRAB-MeCP2 exhibited varying degrees of transcriptional inhibition on plasmids genes and exogenous genomic genes(P＜0.05).Additionally,dCasMINI-ZIM3 KRAB,dCasMINI-KRAB-MeCP2,dCasMINI-ZNF324 KRAB,and dCasMINI-Com-KRAB-MeCP2 demonstrated different levels of transcriptional inhibition on endogenous genes(P＜0.05).Different positions of sgRNAs showed distinct transcriptional inhibition effects(P＜0.05).Conclusions The CasMINI system can be adapted into various CRISPRi tools for gene knockdown studies,with potential applications in various scenarios such as epigenetic gene editing in primary cells,in vivo screening,and clinical therapy in the future.