ArticleBeijing da xue xue bao. Yi xue ban = Journal of Peking University. Health sciences2026
[CRISPR-Cas9 activation screening identifies candidate chemokine regulators of ter-tiary lymphoid structure formation in bladder cancer].
Article in Beijing da xue xue bao. Yi xue ban = Journal of Peking University. Health sciences, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
objectiveTo identify the cytokine genes influencing the formation of tertiary lymphoid structures (TLS) through CRISPR-Cas9 library screening, and to discover potential key regulatory molecules, providing new targets for enhancing the efficacy of bladder cancer immunotherapy.
methodsBased on a mouse whole-genome library, 44 chemokine-related genes were identified, and an single-guide RNA (sgRNA) library targeting these genes was designed and constructed, with three sgRNAs assigned to each gene. Using a lentiviral packaging system, the library plasmids were used to transfect HEK293T cells to generate a lentiviral library, which was then used to infect the mouse bladder cancer cell line MB49. Purinomycin selection was performed to obtain the MB49-mCherry cell line stably over-expressing chemokines. The cells were inoculated into the peritoneal cavity of C57BL/6 mice to establish a bladder cancer xenograft model, and tumor growth was monitored. Three weeks later, tumor tissue was excised, genomic DNA was extracted for high-throughput sequencing, and sgRNA enrichment was analyzed to screen for differentially expressed cytokine genes. Concurrently, immunohistochemical staining was performed to detect TLS markers CD20 and CD3, and the number, distribution, and maturity of TLS were assessed. The selected candidate genes were validated individually
resultsWe successfully constructed a cytokine gene library containing 132 sgRNAs, covering 44 chemokine genes. Following lentiviral infection, we obtained the MB49-mCherry cell line, which stably expressed the library, and isolated dead Cas9-positive monoclonal cell lines
conclusionThrough CRISPR-Cas9 library screening combined with
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42493436PMC13402049What OpenQuestion holds
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