;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 examine the prevalence of chikungunya virus in brain tissue samples from rat?like animals in Xiamen, Shenzhen and Guangzhou, and to explore whether the rat?like animals are potential sources of human chikungunya fever infections and the host of the virus. Methods Rat?like animals were trapped in residential areas, city parks, hospitals, markets and schools in Xiamen, Shenzhen and Guangzhou (Yuexiu and Baiyun districts) between January 2013 and June 2016. Brain tissue samples of the trapped animals were collected under sterile. Chikungunya virus was detected by using reverse transcription polymerase chain reaction (RT?PCR). Results Totally 1092 rat?like animals were trapped, which belonged to 7 species, 3 genera, 2 families, 2 orders. Rattus norvegicus was the dominant species in the indoor environment, Rattus losea was dominant in wild environment, and 1092 brain tissue samples were collected. No detectable chikungunya virus was found in the brain tissue samples by RT?PCR. Conclusion There is a low possibility that rat?like animals act infectious sources of human chikungunya fever infections and the host of the virus.