生物技术通报 ›› 2023, Vol. 39 ›› Issue (10): 68-79.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0248

• 技术与方法 • 上一篇    下一篇

基于Csy4与MCP的新型迷你基因组编辑系统的构建

邓嘉辉1(), 雷建峰2, 赵燚1, 刘敏1, 胡子曜1, 尤扬子1, 邵武奎1, 柳建飞1, 刘晓东1()   

  1. 1.新疆农业大学生命科学学院,乌鲁木齐 830052
    2.新疆农业大学农学院 教育部棉花工程研究中心 农业生物技术重点实验室,乌鲁木齐 830052
  • 收稿日期:2023-03-21 出版日期:2023-10-26 发布日期:2023-11-28
  • 通讯作者: 刘晓东,男,博士,教授,研究方向:作物逆境分子生物学;E-mail: xiaodongliu75@aliyun.com
  • 作者简介:邓嘉辉,男,硕士,研究方向:作物逆境分子生物学;E-mail: 15754842489@126.com
  • 基金资助:
    新疆维吾尔自治区重大科技专项计划项目(2021A02001-3)

Construction of a New Mini Genome Editing System Based on Csy4 and MCP

DENG Jia-hui1(), LEI Jian-feng2, ZHAO Yi1, LIU Min1, HU Zi-yao1, YOU Yang-zi1, SHAO Wu-kui1, LIU Jian-fei1, LIU Xiao-dong1()   

  1. 1. College of Life Sciences, Xinjiang Agricultural University, Urumqi 830052
    2. College of Agronomy, Xinjiang Agricultural University, Research Center of Cotton Engineering, Ministry of Education, Laboratory of Agricultural Biotechnology, Urumqi 830052
  • Received:2023-03-21 Published:2023-10-26 Online:2023-11-28

摘要:

MCP是MS2噬菌体的外壳蛋白,Csy4是一种参与CRISPR 1-F系统crRNA生成的小型蛋白,能够以较高的特异性识别并结合RNA。目前CRISPR/Cas等基因组编辑技术存在靶向核酸酶分子量大、脱靶率高、受PAM位点限制等问题,为解决上述问题,构建基于上述两种小型蛋白的新型迷你基因组编辑系统。本研究采用AlphaFold2预测MCP-FokI、FokI-MCP、Csy4-FokI和FokI-Csy4融合蛋白的结构,通过浸花法将MCP-FokI和FokI-MCP编辑载体分别转化拟南芥,利用拟南芥叶片注射的方法投送CLCrV介导的Csy4-FokI与FokI-Csy4编辑系统,提取拟南芥基因组DNA,通过HI-TOM高通量测序检测新型迷你基因组编辑系统的编辑能力。结果显示,融合蛋白中MCP、FokI和Csy4都各自保持着自身原有的三维结构,预示它们都能正常发挥彼此的功能。构建靶向敲除拟南芥CLA1基因的4个不同中间间隔区的双靶位点MCP-FokI和MCP-FokI植物表达载体,初步证明MCP-FokI和FokI-MCP均不能实现对靶基因的靶向编辑。构建靶向敲除拟南芥CLA1基因的7个CLCrV介导的不同中间间隔区的双靶位点Csy4-FokI编辑载体,其中CLCrV介导的Csy4-FokI编辑系统能够实现对靶基因的靶向编辑,但是突变类型均为碱基置换类型且编辑效率很低,而FokI-Csy4基因组编辑体系并未检测到编辑的发生。成功构建了Csy4-FokI新型迷你基因组编辑系统,为克服CRISPR/Cas基因组编辑技术存在的问题提供了一种新的解决方案。

关键词: Csy4, MCP, 棉花 叶皱缩病毒, VIGE

Abstract:

MCP is the coat protein of MS2 phage, and Csy4 is a small protein involved in crRNA generation of CRISPR 1-F system, which can recognize and bind RNA with high specificity. CRISPR/Cas and other genome editing technologies have some problems, such as large molecular weight of targeted nuclease, high off-target rate, and limited by PAM site. To solve these problems and construct new mini genome editing system based on Csy4 and MCP, AlphaFold2 was used to predict the structures of MCP-FokI, FokI-MCP, Csy4-FokI and FokI-Csy4 fusion proteins. The MCP-FokI and FokI-MCP editing vectors were transformed into Arabidopsis thaliana by the flower dip method, respectively, and the CLCrV-mediated Csy4-FokI and FokI-Csy4 editing systems were delivered using A. thaliana leaf injection. The A. thaliana genomic DNA was extracted and the editing ability of the novel mini-genome editing system was tested by HI-TOM high-throughput sequencing. The results showed that each domain of MCP, FokI and Csy4 maintained their original three-dimensional structures in the fusion proteins, suggesting that they all functioned properly. The plant expression vectors of MCP-FokI and MCP-FokI binding dual target sites with four different intermediate spacer regions were constructed to target the AtCLA1 genes. Sequencing results demonstrated that neither MCP-FokI nor FokI-MCP achieved the targeted editing of the target genes. Seven CLCrV-mediated Csy4-FokI and FokI-Csy4 editing vectors with different intermediate spacer regions were constructed to target the AtCLA1 genes. Among them the CLCrV-mediated Csy4-FokI editing system was able to achieve targeted editing of the target gene, but the mutation types were all base substitution types and the editing efficiency was low. No genome editing was detected in the FokI-Csy4 system. Taken together, a novel Csy4-FokI mini-genome editing system was successfully constructed, which may provide an innovative solution to overcome the problems of CRISPR/Cas genome editing technology.

Key words: Csy4, MCP, cotton leaf crumple virus, VIGE