Epitope Tagging by Prime Editing in Zebrafish

项目摘要 最近发布的搜索和替换基因组编辑工具利用Cas9 Nickase来制作 在目标位置单链断裂，然后通过反向引入所需的编辑 以特定修饰的引导RNA(PegRNA)的3‘端为模板的转录酶。 这些名为Prime Edtors的工具似乎远远优于当前最先进的方法 例如使用寡核苷酸模板的同源定向修复。重要的是要测试这一点是否 卓越的性能将在包括斑马鱼在内的其他模型系统中重现。我们建议 使用Prime Edtors对几个起重要作用的斑马鱼基因进行表位标记 在各种生物学过程中，从早期胚胎模式到轴突引导和 再生。在这项工作的过程中，我们将确定哪个Prime编辑具有最佳活动 在斑马鱼中，测试pegRNA设计的几个重要变量，如引物的长度 结合位点，并产生母性表达的转基因系(S)，从而促进 这一方法得到了研究界的广泛应用。

Project Summary Recently published search-and-replace genome editing tools utilize the Cas9 nickase to make a single strand break at the target site, followed by introduction of the desired edit by reverse transcriptase using the 3' end of a specifically modified guide RNA (pegRNA) as the template. These tools, named Prime Editors, appear far superior to current state-of-the-art methodologies such as homology directed repair using oligonucleotide templates. It is important to test if this superior performance will reproduce in other model systems including the zebrafish. We propose to use Prime Editors to carry out epitope tagging of several zebrafish genes which play important roles in a variety of biological processes from early embryo patterning to axon guidance and regeneration. Over the course of this work we will determine which Prime Editor has optimal activity in zebrafish, test several important variables of pegRNA design such as the length of the Primer Binding Site, and produce transgenic lines maternally expressing Prime Editor(s), thus facilitating broad application of this method by the research community.