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2025年4月5日 星期六
农业生物技术学报  2024, Vol. 32 Issue (10): 2306-2323    DOI: 10.3969/j.issn.1674-7968.2024.10.010
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
HDACi和RS-1提高CRISPR/Cas12i介导的HDR编辑效率
陈秋崇, 李尚朴, 苗洱钰, 周冰倩, 王旭, 孟祥宇, 王小龙*, 徐坤*
西北农林科技大学 动物科技学院/农业农村部动物生物育种国际联合研究中心/陕西省动物遗传育种与繁殖重点实验室,杨凌 712100
HDACi and RS-1 Enhance CRISPR/Cas12i-mediated HDR Editing Efficiency
CHEN Qiu-Chong, LI Shang-Pu, MIAO Er-Yu, ZHOU Bing-Qian, WANG Xu, MENG Xiang-Yu, WANG Xiao-Long*, XU Kun*
College of Animal Science and Technology/International Joint Research Center of Animal Biobreeding, Ministry of Agriculture and Rural Affairs/Shaanxi Key Laboratory of Animal Genetic Breeding and Reproduction, Northwest A&F University, Yangling 712100, China
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摘要 CRISPR/Cas12i是我国学者新近开发的、具有自主知识产权的CRISPR基因编辑系统,被证实具有不亚于CRISPR/Cas9系统的打靶效率。同源定向修复(homology-directed repair, HDR)是DNA双链断裂(double-stranded break, DSB)的主要修复方式之一。基于HDR机制的基因编辑可用于纠正基因组中任何形式的突变,但受限于哺乳动物细胞中普遍较低的HDR效率。本研究首先通过单链复性(single-strand annealing, SSA)报告试验、剂量梯度和浓度梯度实验分别验证CRISPR/Cas12i系统在人(Homo sapiens)胚肾细胞系HEK293T中不同靶点的活性、介导HDR编辑的单链寡核苷酸(single-stranded oligonucleotides, ssODN)供体模板剂量及添加小分子药物的适宜浓度,进而通过流式细胞分选、基因组PCR、Sanger测序和在线预测等手段,检测添加不同小分子药物对HEK293T细胞和绵羊(Ovis aries)胎儿成纤维细胞中CRISPR/Cas12i系统介导的HDR编辑效率的影响。结果显示,CRISPR/Cas12i系统在HEK293T细胞18个不同靶点均表现出较高编辑活性,除2个位点稍低外,其余均在80%左右;不同剂量及长度的ssODN对HDR效率有一定影响,且小分子药物适宜浓度在不同物种和不同类型细胞中略有不同;组蛋白去乙酰化酶抑制剂(histone deacetylase inhibitor, HDACi)和RS-1 (C20H16Br2N2O3S)对HEK293T细胞和绵羊胎儿成纤维细胞中CRISPR/Cas12i系统介导的HDR编辑效率均有显著提升,其中RS-1细胞毒性最小,且在提升HDR效率同时未显著降低插入/缺失突变(insertion/deletion mutation, InDel)效率;此外,Entinostat作为HDACi之一,在绵羊胎儿成纤维细胞的骨形态发生蛋白受体1B (bone morphogenetic protein receptor 1B, BMPR1B)基因相关位点提升HDR编辑效率约148倍。总之,CRISPR/Cas12i系统具有较高编辑活性,能够在模式细胞和绵羊原代细胞中介导有效的、以ssODN为供体的HDR精确编辑,且适宜浓度的HDACi和RS-1可以有效提高HDR编辑效率。本研究为CRISPR/Cas12i基因编辑系统的推广与应用提供了参考和借鉴。
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陈秋崇
李尚朴
苗洱钰
周冰倩
王旭
孟祥宇
王小龙
徐坤
关键词 CPRISPR/Cas12i同源重组RS-1 (C20H16Br2N2O3S)组蛋白去乙酰化酶抑制剂(HDACi)绵羊    
Abstract:CRISPR/Cas12i is a recently developed CRISPR gene editing system by Chinese scholars, which has independent intellectual property rights and has been proven to have targeting efficiency comparable to the CRISPR/Cas9 system. Homology-directed repair (HDR) is one of the main repair mechanisms for double-stranded DNA breaks (DSBs). Gene editing based on the HDR mechanism can be used to correct any form of mutation in the genome, but it is often limited by the generally lower HDR efficiency in mammalian cells. In this study, the activity of the CRISPR/Cas12i system at different target sites in Homo sapiens embryonic kidney cell line HEK293T, the optimal dosage of single-stranded oligonucleotides (ssODN) donor templates for mediating HDR editing, and the appropriate concentration of small molecule drugs were verified through single-strand annealing (SSA) reporter experiment, dose gradient and concentration gradient experiment. The effects of adding different small molecule drugs on the HDR editing efficiency mediated by the CRISPR/Cas12i system in HEK293T cells and sheep (Ovis aries) fetal fibroblasts were then evaluated using flow cytometry sorting, genomic PCR, Sanger sequencing, and online prediction tools. The results showed that the CRISPR/Cas12i system exhibited high activity at 18 different target sites in HEK293T cells, with efficiencies around 80%, except 2 slightly lower sites. Different dosages and lengths of ssODN had some influence on HDR efficiency, and the appropriate concentrations of small molecule drugs varied slightly in different species and cell types. The addition of histone deacetylase inhibitor (HDACi) and RS-1 (C20H16Br2N2O3S) significantly enhanced the HDR editing efficiency mediated by the CRISPR/Cas12i system in both HEK293T cells and sheep fetal fibroblasts. Among them, RS-1 showed the least cytotoxicity and did not significantly reduce the insertion/deletion mutation (InDel) efficiency while improving HDR efficiency. Furthermore, Entinostat increased HDR editing efficiency by approximately 148 fold at the bone morphogenetic protein receptor 1B (BMPR1B) site in sheep fetal fibroblasts. In conclusion, the CRISPR/Cas12i system exhibited high activity and could mediate efficient and precise HDR editing using ssODN as a donor in model cells and primary sheep cells. The appropriate concentrations of HDACi and RS-1 could effectively improve HDR editing efficiency. This study provides reference and guidance for the application and popularization of the CRISPR/Cas12i gene editing system.
Key wordsCPRISPR/Cas12i    Homologous recombination    RS-1 (C20H16Br2N2O3S)    Histone deacetylase inhibitor (HDACi)    Sheep
收稿日期: 2023-12-11     
中图分类号: S813.3
基金资助:国家自然科学基金(32301251; 32272848); 现代农业产业体系(CARS-39-03); 陕西省地方助学金项目(2022GD-TSLD-46); 国家级大学生创新创业训练计划(202210712099)
通讯作者: * xiaolongwang@nwafu.edu.cn;xukunas@nwafu.edu.cn   
引用本文:   
陈秋崇, 李尚朴, 苗洱钰, 周冰倩, 王旭, 孟祥宇, 王小龙, 徐坤. HDACi和RS-1提高CRISPR/Cas12i介导的HDR编辑效率[J]. 农业生物技术学报, 2024, 32(10): 2306-2323.
CHEN Qiu-Chong, LI Shang-Pu, MIAO Er-Yu, ZHOU Bing-Qian, WANG Xu, MENG Xiang-Yu, WANG Xiao-Long, XU Kun. HDACi and RS-1 Enhance CRISPR/Cas12i-mediated HDR Editing Efficiency. 农业生物技术学报, 2024, 32(10): 2306-2323.
链接本文:  
https://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2024.10.010     或     https://journal05.magtech.org.cn/Jwk_ny/CN/Y2024/V32/I10/2306
 
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