优化CRISPR基因替换编辑技术体系及其玉米基因功能鉴定

Gene replacement editing is a kind of technology that could target a fairy long target DNA region. Due to their feasibility of introducing any types of desired mutation at the target chromosome location, they are of theoretical and practical valuable in gene function identification and crop genetic improvement. Compared with technologies between non-homologous end joining (NHEJ) repair dependent with homologous directed repair (HDR) dependent genome editing, the gene replacement editing technique based on endogenous HDR pathway has the advantage of high accuracy but low efficiency due to the short duration of HDR activity during the whole cell cycle. However, NHEJ dependent genome editing could have much higher frequencies but along with lower accuracy. Therefore, R & D on optimizing both efficiency and accuracy for gene replacement editing technology is of great significance to break through. Based on the previous research, the use of CRISPR / Cpf1 multiple editing activity, cohesive end protection and other characteristics, our objective of this application grant is to develop a higher accurate NHEJ dependent CRISPR / Cpf1 gene replacement editing technology system. And as a usefulness evaluation system, we would use it into functional characterization on the interaction between MiRx gene and its’ target gene, GRFx, by fine modification of the target sequence with the synonymous mutation. The different DNA sequence would modify the microRNA interaction pattern with their target. The refine modifications would help us to clarify the mechanism how the regulatory between MiRx gene and its’ target gene play a role in kernel development. This grant would found the basis for both genome technologies and theory of maize grain yield.

基因替换是一类较长DNA精确定向突变技术，因其可引入任意设计的突变，具有重大理论与应用价值。依赖非同源重组末端连接（NHEJ，Non-Homologous End Joining）与同源重组修复（HDR, Homologous Directed Repair）活性的基因替换编辑技术相比，具有突变率高但准确性低的特点，优化依赖NHEJ基因替换编辑技术准确性具有重要意义。本申请在前期基础上，利用CRISPR/Cpf1多重编辑活性、粘性末端保护等特点，依赖全胞周期存在稳定活性NHEJ连接修复活性，优化基于CRISPR/Cpf1基因替换编辑技术，提高突变准确性，应用于玉米产量MiRx基因对其目标基因GRFx调控区段的精确基因替换编辑。该项目的实施将为基因功能鉴定与遗传改良提供高效而准确的基因替换编辑技术，并阐明籽粒发育重要基因的功能，为玉米遗传改良与基因功能鉴定提供重要的技术与理论基础。

玉米等高等植物基因编辑技术在农作物遗传改良等方面具有重要应用潜力，优化CRISPR基因替换等基因编辑技术体系及其玉米基因功能鉴定具有重要意义。本项目在前期工作的基础上，针对玉米CRISPR/Cas基因编辑系统的向导RNA表达进行了优化，系统鉴定了基因编辑技术两个关键组份之一“向导RNA核内高效表达元件”，基于此建立的系列玉米基因编辑技术体系被国内外同行广泛采用，对推动玉米基因编辑技术及其应用起到了重要作用。还通过建立基于玉米种子荧光报告系统，简化了玉米基因编辑技术突变体检测与分析、转基因阳性与阴性系筛选流程，显著提高了工作效率。在此基础之上，利用CRISPR/Cpf1多重编辑活性、粘性末端保护等特点，依赖NHEJ连接修复活性或者非DNA双链断裂替换活性，优化了基于系列CRISPR/Cas基因替换编辑技术，提高突变准确性，并应用于玉米产量MiRx基因对其目标基因GRFx调控区段的精确基因替换编辑。基于上述技术还开发了玉米磺酰脲类除草剂抗性突变、雄性核不育第三代杂交种制种技术、单倍体诱导技术和灵敏核酸检测技术。该项目的顺利实施推动了玉米内源基因精准遗传改良技术，创制的技术具有重大产业应用潜力与价值。发表论文9篇，其中国际主流期刊SCI论文8篇；培养研究生4名，青年人才1名。