在哺乳动物细胞中进行基因敲入通常采用同源定向修复(homology-directed repair, HDR)机制将外源DNA模板整合到目标基因组靶点中。然而HDR效率往往较低，其中外源DNA模板与目标基因组靶点的共定位是关键限制因素之一。为提高CRISPR/Cas9系统介导的HDR效率，本团队及前人研究将不同接头蛋白与SpCas9蛋白融合表达，利用其与特异性DNA序列结合的特性，构建了多种CRISPR/SpCas9供体适配基因编辑系统。为了便于比较、优化不同CRISPR/Cas9供体适配系统，本研究利用这些系统在HEK293T细胞GAPDH和ACTB基因末位外显子3′-端进行了eGFP基因敲入，并采用了优化的供体DNA模板设计方式，通过PCR和Sanger测序检测敲入的准确性，流式细胞分析进行敲入效率的检测。结果表明，将yGal4BD、hGal4BD、hLacI、hTHAP11和N57等接头蛋白与SpCas9蛋白C-端融合对其活性均无显著性影响；在GAPDH位点上，SpCas9融合yGal4BD、hGal4BD、hLacI和hTHAP11的供体适配系统等均能显著提高敲入效率；在ACTB位点上，SpCas9融合yGal4BD和hGal4BD能显著提高敲入效率；且增加供体DNA模板中的结合序列(binding sequence, BS)数量，有利于提高SpCas9-hTHAP11系统介导的敲入效率。总之，本研究比较并优化了不同的CRISPR/Cas9供体适配基因编辑系统，为后续相关的基因编辑应用研究提供了参考和借鉴。

Gene knock-in in mammalian cells usually uses homology-directed repair (HDR) mechanism to integrate exogenous DNA template into the target genome site. However, HDR efficiency is often low, and the co-localization of exogenous DNA template and target genome site is one of the key limiting factors. To improve the efficiency of HDR mediated by CRISPR/Cas9 system, our team and previous studies fused different adaptor proteins with SpCas9 protein and expressed them. By using their characteristics of binding to specific DNA sequences, many different CRISPR/SpCas9 donor adapter gene editing systems were constructed. In this study, we used them to knock-in eGFP gene at the 3′-end of the terminal exon of GAPDH and ACTB genes in HEK293T cells to facilitate a comparison and optimization of these systems. We utilized an optimized donor DNA template design method, validated the knock-in accuracy via PCR and Sanger sequencing, and assessed the efficiency using flow cytometry. The results showed that the fusion of yGal4BD, hGal4BD, hLacI, hTHAP11 as well as N57 and other adaptor proteins with the C-terminus of SpCas9 protein had no significant effect on its activity. At the GAPDH site, the donor adapter systems of SpCas9 fused with yGal4BD, hGal4BD, hLacI and hTHAP11 significantly improved the knock-in efficiency. At the ACTB site, SpCas9 fused with yGal4BD and hGal4BD significantly improved the knock-in efficiency. Furthermore, increasing the number of BS in the donor DNA template was beneficial to enhance the knock-in efficiency mediated by SpCas9-hTHAP11 system. In conclusion, this study compares and optimizes multiple CRISPR/Cas9 donor adapter gene editing systems, providing valuable insights for future gene editing applications.