不同CRISPR/Cas9供体适配基因编辑系统的比较及优化研究  

Comparison and optimization of different CRISPR/Cas9 donor-adapting systems for gene editing

作　　者：马宝霞 杨森 吕明 王昱人 常立业 韩艺帆 王建刚[1] 郭杨 徐坤[1] Baoxia Ma;Sen Yang;Ming Lyu;Yuren Wang;Liye Chang;Yifan Han;Jiangang Wang;Yang Guo;Kun Xu(College of Animal Science and Technology,Northwest A&F University,Yangling 71200,China;Xinjiang Uygur Autonomous Region Animal Husbandry General Station,Urumchi 830002,China)

机构地区：[1]西北农林科技大学动物科技学院,杨凌712100 [2]新疆维吾尔自治区畜牧总站,乌鲁木齐830002

出　　处：《遗传》2024年第6期466-477,共12页Hereditas（Beijing)

基　　金：科技创新2030−农业生物育种重大项目(编号:2023ZD04074,2023ZD04051)资助。

摘　　要：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.

关 键 词：基因编辑 基因敲入 CRISPR/Cas9 供体适配 同源定向修复 

分 类 号：Q78[生物学—分子生物学]