Epitope Tagging by Prime Editing in Zebrafish

通过 Prime 编辑在斑马鱼中进行表位标记

基本信息

批准号：
10303928
负责人：
Darius Balciunas
金额：
$ 23.78万
依托单位：
TEMPLE UNIV OF THE COMMONWEALTH
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-08-01 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10303928
关键词：
Alleles Antibodies Binding Sites Biological Biological Models Biological Process CRISPR/Cas technology Categories Chimeric Proteins Code Communities DNA-Protein Interaction Development Double Strand Break Repair ETV3 gene Embryo Endonuclease I Engineering Ensure Epitopes Genes Genetic Complementation Test Genetic Transcription Genome Guide RNA In Vitro Injections International Laboratories Lead Length Mammalian Cell Mediating Messenger RNA Methodology Methods Morphologic artifacts Names Oligonucleotides Pattern Performance Physiological Play Proteins Protocols documentation Publishing RNA-Directed DNA Polymerase Reagent Reporting Research Methodology Research Personnel Resources Role Scientist Site Techniques Testing Time Tissues Transgenic Organisms Variant Work Zebrafish axon guidance axon regeneration design experimental study feasibility testing genome editing high reward high risk insertion/deletion mutation interest mutant promoter repaired tool transmission process zebrafish genome

项目摘要

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.

项目摘要最近出版的搜索和重建基因组编辑工具利用Cas9 nickase做一个目标站点的单链断裂，然后通过反向引入所需的编辑使用特定修改的导向RNA（PEGRNA）作为模板的3'端转录酶。这些工具，名为Prime编辑器，似乎远远超过了当前的最新方法论例如使用寡核苷酸模板的同源性修复。重要的是要测试这是否卓越的性能将在包括斑马鱼在内的其他模型系统中再现。我们建议使用Prime编辑来执行几个斑马鱼基因的表位标签，这些基因起着重要作用在各种生物学过程中，从早期胚胎构图到轴突指导和再生。在这项工作的过程中，我们将确定哪个主要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，例如底漆的长度结合位点，并产生具有母体表达的主要编辑器的转基因线，从而促进研究社区对这种方法的广泛应用。