Genome editing tools have simplified the generation of knock-in gene fusions, which are widely used to study proteins in their natural context. However, strategies for tagging endogenous genes in primary cells are few and inefficient. In this study, we developed a one-step endogenous gene-tagging strategy by co-delivery of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 ribonucleoprotein complexes and chemically modified donor DNA into cells. Upon CRISPR-Cas9 blunt-end double-strand breaks, highly efficient site-specific insertion of genetic materials (3 × FLAG or eGFP) was achieved in both cell lines and primary cells. We further optimized the gene-tagging efficiency and precision by using CRISPR-Cas12a, which produces a staggered cut with a 5′ overhang and thus enables precise ligation of DNA donors with a complementary 3′ overhang. With high efficiency and flexibility, this platform would be extremely useful for multiplex endogenous genes tagging and further exploration of protein functions in various cell types.

中文翻译：

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使用 CRISPR-Cas 核糖核蛋白对原代细胞中的内源基因进行高效一步标记

基因组编辑工具简化了敲入基因融合的产生，这些融合被广泛用于研究自然环境中的蛋白质。然而，在原代细胞中标记内源基因的策略很少且效率低下。在这项研究中，我们通过将成簇的规则间隔短回文重复序列 (CRISPR)-Cas9 核糖核蛋白复合物和化学修饰的供体 DNA 共同递送到细胞中，开发了一种一步内源基因标记策略。在 CRISPR-Cas9 平末端双链断裂后，在细胞系和原代细胞中都实现了遗传物质（3×FLAG 或 eGFP）的高效位点特异性插入。我们通过使用 CRISPR-Cas12a 进一步优化了基因标记的效率和精度，其产生具有 5' 悬垂的交错切割，从而能够精确连接具有互补 3' 悬垂的 DNA 供体。该平台具有高效率和灵活性，对于多重内源基因标记和进一步探索各种细胞类型中的蛋白质功能非常有用。