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：
10455023
负责人：
Darius Balciunas
金额：
$ 19.81万
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 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10455023
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 prime editing prime editor 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.

项目概要最近发布的搜索和替换基因组编辑工具利用 Cas9 切口酶来制造在目标位点处单链断裂，然后通过反向引入所需的编辑使用经过特殊修饰的指导 RNA (pegRNA) 3' 端作为模板的转录酶。这些名为 Prime Editors 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。测试这是否很重要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 Editors对几个发挥重要作用的斑马鱼基因进行表位标记从早期胚胎模式到轴突引导的各种生物过程再生。在这项工作的过程中，我们将确定哪个 Prime Editor 具有最佳活动在斑马鱼中，测试 pegRNA 设计的几个重要变量，例如引物的长度结合位点，并产生母系表达 Prime Editor 的转基因系，从而促进研究界广泛应用这种方法。