：基于 CRISPR/Cas12a 的组合筛选在识别遗传相互作用方面显示出巨大的潜力。在这里，我们描述了一种组合遗传筛选的创新方法，通过重叠延伸 PCR (SOE PCR) 进行基因剪接，快速构建双 CRISPR RNA (crRNA) 文库，并采用 CeCas12a，我们之前通过严格的 PAM 识别和识别来识别 CeCas12a。低脱靶，保证保真度和效率。用于双敲除筛选的定制SOE crRNA阵列（SOCA）文库可以在实验室中方便地构建并广泛使用，并且CeCas12a介导的高保真筛选即使在负选择筛选下也显示出良好的性能。通过设计一个 SOCA 双 crRNA 文库，该文库涵盖了 FDA 批准的与肝细胞癌 (HCC) 肿瘤发生有关的药物的大部分激酶和代谢相关基因靶点，两个基因组之间的新型串扰被负选择以抑制 HCC 细胞体外和体内生长，并使用基于 TCGA 数据库的虚拟双敲低筛选进行验证。因此，这种快速、高效和高保真的双敲除筛选系统有望在未来系统地识别多个基因组之间或 FDA 批准的临床转化医学药物组合之间潜在的遗传相互作用。© 2024 作者。约翰·威利出版的《临床与转化医学》

 : CRISPR/Cas12a-based combinational screening has shown remarkable potential for identifying genetic interactions. Here, we describe an innovative method for combinational genetic screening with rapid construction of a dual-CRISPR RNA (crRNA) library using gene splicing through overlap extension PCR (SOE PCR) and the adoption of CeCas12a, which we previously identified with strict PAM recognition and low off-targeting to guarantee fidelity and efficiency. The custom-pooled SOE crRNA array (SOCA) library for double-knockout screening could be conveniently constructed in the laboratory for widespread use, and the CeCas12a-mediated high-fidelity screen displayed good performance even under a negative selection screen. By designing a SOCA dual-crRNA library that covered most of the kinase and metabolism-associated gene targets of FDA-approved drugs implicated in hepatocellular carcinoma (HCC) tumourigenesis, novel cross-talk between the two gene sets was negatively selected to inhibit HCC cell growth in vitro and in vivo and was validated using virtual double-knockdown screening based on TCGA databases. Thus, this rapid, efficient and high-fidelity double-knockout screening system is promising for systemically identifying potential genetic interactions between multiple gene sets or combinations of FDA- approved drugs for clinical translational medicine in the future.© 2024 The Author(s). Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.