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CRISPR-Cas13d: Transgenic zebrafish lines toknockdown mRNA

CRISPR-Cas13d：转基因斑马鱼系敲除 mRNA

基本信息

批准号：
10598232
负责人：
Ariel Bazzini
金额：
$ 20.63万
依托单位：
STOWERS INSTITUTE FOR MEDICAL RESEARCH
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-12-15 至 2024-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10598232
关键词：
Address Alternative Splicing Animal Model Base Sequence Biological Biological Models Biological Process CRISPR/Cas technology Chronic Clustered Regularly Interspaced Short Palindromic Repeats Codon Nucleotides Collection Communities Complex Consumption DNA DNA Sequence Data Development Disease Effectiveness Embryo Endoribonucleases Enzymes Essential Genes Exons Foundations Free Will Gene Expression Gene Mutation Gene Transfer Techniques Genes Genetic Transcription Genome engineering Genomic DNA Genotype Goals Guide RNA Guidelines In Situ Hybridization Incidence Individual Injections Investigation Killifishes Knock-out Link Masks Messenger RNA Methodology Methods Mus Nuclear Localization Signal Organism Oryziinae Outcome Phenotype Publications RNA RNA Degradation RNA Interference Reagent Research Research Personnel Reverse Transcriptase Polymerase Chain Reaction Role Series Specificity System Techniques Technology Testing Time Tissues Transgenic Organisms Untranslated RNA Validation Variant Vertebrates Western Blotting Work Xenopus Zebrafish cost effective cost efficient gene discovery gene function genome editing genomic locus human disease innovation knock-down knockout gene mRNA Expression model organism mutant new technology novel promoter scaffold teleost fish tool transcriptome sequencing transgene expression vertebrate embryos

项目摘要

Project Summary Determining the function of genes is fundamental for understanding vertebrate development, regulatory mechanisms and human diseases. Genome editing technologies, such CRISPR-Cas9, have allowed associating specific phenotypes to permanent gene alteration. However, some key technical and conceptual issues remain problematic in vertebrates, particularly in aquatic model organisms such zebrafish. For example, the maternally provided mRNA can rescue the phenotype of homozygous mutants; genotyping steps are tedious; and long non- coding RNA, lethal or tissue/temporal genes are difficult to study using DNA manipulation as well as complex genomic loci. Complementary ‘knock-down’ approaches are invaluable tools to circumvent some of these problems, however, there was no systematic tool to knockdown mRNAs in zebrafish or other teleost fish. Our long-term goal is to unravel the function of genes related to regulatory mechanisms, development, and human diseases. Our recent publication demonstrates that injection of the CRISPR-RfxCas13d system into vertebrate embryos provides a robust and cost-effective technology to systematically disrupt gene function. However, the injection of this system only provides transient knockdown for ~3 days. Therefore, the central goal of the proposal is to transfer the CRISPR-RfxCas13d technology from being injected into vertebrate embryos, to being endogenously expressed through transgenesis. We propose that the development of a repertoire transgenic zebrafish expressing the RfxCas13d enzyme (ubiquitous and tissues-specific) and guideRNA would facilitate rapid and vigorous investigation into gene functions. Our preliminary data indicate that transgenic expression of RfxCas13d in zebrafish is functional and not toxic. The objectives are: 1) Define the optimal system to knockdown mRNA expression in CRISPR-RfxCas13d transgenic zebrafish. 2) Knockdown mRNA expression in a tissue-specific manner using specific transgenic lines. This proposal is conceptually innovative as it is based on the exploration of a novel technique, CRISPR-RfxCas13d, to knockdown mRNA in a tissue-specific manner in zebrafish. This approach has never been done in vertebrate model systems where RNAi does not work. The outcomes of this project will be the first transgenic system to dissect and study gene function by knocking down mRNA expression in zebrafish. Our approach will help the scientific community investigate gene function in a faster and tissue-temporal specific manner, as well as answering questions that are very challenging to address by current methodologies, such as the function of non-coding RNAs or phenotypes caused by multiple genes. Moreover, by following the guidelines we will optimize through this project, researchers may produce a whole guideRNA collection, targeting all genes, which should be available for anyone to order to address their individual research questions. Finally, as we have successfully implemented the CRISPR-Cas13d system by injection in other organisms such as medaka, killifish, and mouse embryos, our work described in this proposal may serve as the foundation for transferring this efficient knockdown technology into a range of other species.

项目总结