利用微同源末端连接DNA修复机制建立精准高效的鱼类基因敲入技术的研究

The technique of fish genome precise knock-in is the key of fish genetic engineering breeding for conditional knockout and "targeted integration and controllable expression". Compared with the proven technique of genome editing established in a variety of fish, genome-targeted knock-in is only successful on a very few target sites in a model animal zebrafish. One of the most important reasons is that we do not fully understand the repair mechanism of DNA double strand breaks (DSBs) in fish embryo. Recently, the studies from other labs and us have revealed that the DSBs are repaired mainly through microhomology mediated end joining (MMEJ) mediated DNA repair pathway during early embryogenesis. Based on the preliminary investigation of highly efficient and precise knock-in technique using zebrafish, we intend to further optimize the design of the knock-in vector and the knock-in strategy by deeply characterize the sequences of both endogenous and exogenous targets in the events of highly efficient knock-in. This proposal aims to establish a highly efficient and precise targeted knock-in technology based on MMEJ mediated DNA repair mechanisms using a model animal zebrafish and an aquaculture fish common carp. The achievement of this aim will not only help to realize conditional knockout technique in aquaculture fish but also be benefit to rapid directional fish breeding with integration of high yield and high quality characters.

鱼类基因组精细敲入技术是进行条件性基因敲除和“定点整合、可控表达”的鱼类基因工程育种的关键。相较于多种鱼类中建立的成熟的基因组编辑技术，基因组定点敲入技术仍然只在模式动物斑马鱼的极少数靶位中取得成功，其重要原因之一是对鱼类胚胎期DNA双链断裂（DSBs）的修复机制认识和利用不够。最近，我们和他人的研究发现，微同源末端连接（MMEJ）介导的DSBs修复是鱼类早期胚胎发育中DSBs的主要修复方式。本申请项目拟利用模式动物斑马鱼和经济鱼类鲤鱼，在前期初步实现斑马鱼中精准高效的敲入技术的基础之上，进一步优化设计敲入载体和敲入策略，深入剖析高效敲入事件中的内、外源靶点的序列特征，建立基于MMEJ介导的DSBs修复机制的鱼类精准高效的基因定点敲入技术，实现鱼类条件性基因敲除和高产优质性状聚合的快速定向育种。

随着CRISPR/Cas9系统应用的日趋成熟，基因标记和条件性基因功能研究已成为精细剖析基因功能的重要手段，而基因组精细敲入技术正是实现这一遗传操作的关键技术。目前在鱼类中，基因组精细敲入技术仍是该领域研究的 “瓶颈”技术。因此，在鱼类中亟需优化建立一种普适高效精准的基因组敲入技术。微同源末端连接（Microhomology mediated end joining，MMEJ）介导的DNA双链断裂（Double strand breaks, DSBs）修复是鱼类早期胚胎发育中DSBs的主要修复方式。虽然，MMEJ介导的DSBs修复在基因敲除中是一种可预期的缺失突变，但是，MMEJ介导的外源载体同基因组之间的修复是一种“无缝”连接。本项目研究中，我们优化并建立了基于MMEJ修复机制的普适高效精确的基因组靶向敲入技术策略，包括靶点内微同源序列介导基因敲入技术、靶点内 “种子” 序列微同源介导的敲入技术和单一靶点内回文序列介导的基因组靶向敲入技术。更进一步，我们利用该技术策略在斑马鱼和鲤鱼中分别创建了条件性基因敲除品系和多基因聚合定向设计育种的新材料。通过本项目的研究，为突破鱼类基因敲入技术“瓶颈”和在鱼类中实现定向快速精准育种提供理论基础和技术保障。

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