斑马鱼作为一种重要的模式生物，在研究脊椎动物发育、器官形成以及再生等领域具有其独特的优势。受制于高效核酸酶的发现，斑马鱼的基因组定点编辑技术近年来才刚刚兴起。同源重组修复（HR）能够很好地实现基因组的定点定向改造，但是目前并不能很好地运用在斑马鱼上。此前，在斑马鱼中有研究报导的HR都是基于DNA双链缺口（DSBs）实现的。然而，DSBs的产生容易使基因组发生易于突变的NHEJ修复以及易位或者重排等问题。哺乳动物的研究发现DNA单链缺口（nicks）也能促进HR并防止NHEJ的产生，但是效率低于DSB。本项目引入HR的限速因素—同源重组因子，协同DNA单链缺口酶（nickase）的nicks生成技术，实现便捷、高效、精确的斑马鱼基因组定点定向改造。本项目中的转基因方法简称为"NEO"，预期通过NEO把斑马鱼基因组准确敲入的整合效率提高到50%以上，并且把可遗传率提高到10%以上。

Zebrafish (Danio rerio) is becoming more and more popular as a model organism to study vertebrate organogenesis and regeneration, taking the advantage of its extra-uterine development, transparency, strong regeneration ability and suitability for the large scale genetic screen. Subject to the lacking of efficient nucleases, genome editing is just spring up in recent years in zebarfish. Gene targeting by homologous recombination (HR) can precisely modify the genome. However, it can't work well on zebrafish at the moment. Furthermore, studies reported were all based on the induction of the DNA double strand breaks (DSBs) to trigger HR in zebrafish. However, nucleases-induced DSBs were mainly repaired by error-prone non homologous end joining (NHEJ) which ends with mutation, and the production of DSB would cause genomic instability and thus translocation or rearrangement. Several studies in mammalian have found that DNA nicks could promote HR and prevent the occurrence of NHEJ, but the efficiency is lower than DSBs. By introducing sequence-specific nickase supplied with ectopically expressed key homologous recombination factors, which might be the rate-limiting factor of HR, our methods is expected to achieve knock-in efficiency higher than 50% at average in zebrafish larve and the germling transmission would be higher than 10%. The transgenic technique would be named as NEO (Nickase-based homologous recombination Enhanced by recOmbinant enzymes).