CRISPR-Cas13d: Transgenic zebrafish lines toknockdown mRNA

CRISPR-Cas13d：转基因斑马鱼系敲除 mRNA

• 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
• 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

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.

项目摘要 确定基因的功能是理解脊椎动物发展，调节性的基础 机制和人类疾病。基因组编辑技术，例如CRISPR-CAS9，允许关联 特定的表型与永久基因改变。但是，仍然存在一些关键的技术和概念问题 脊椎动物中的问题，特别是在水生生物中，斑马鱼。例如，主要 如果mRNA可以拯救纯合突变体的表型；基因分型步骤很乏味。和长期的 编码RNA，致命或组织/时间基因很难使用DNA操纵以及复合物研究 基因组基因座。互补的“淘汰”方法是绕过其中一些的宝贵工具 但是，没有系统的工具可以在斑马鱼或其他硬骨鱼中敲除mRNA。 我们的长期目标是揭示与调节机制，发展和 人类疾病。我们最近的出版物表明，将CRISPR-RFXCAS13D系统注入 脊椎动物的胚胎为系统破坏基因功能提供了强大且具有成本效益的技术。 但是，该系统的注入仅提供约3天的瞬时敲低。因此，中心目标 该提案的内容是将CRISPR-RFXCAS13D技术从注入脊椎动物胚胎到 通过转基因表达内源。我们建议发展曲目 表达RFXCAS13D酶的转基因斑马鱼（普遍和组织特异性）和Guiderna将 促进对基因功能的快速和有力研究。我们的初步数据表明转基因 RFXCAS13D在斑马鱼中的表达是功能性且无毒的。目标是：1）定义最佳系统 在CRISPR-RFXCAS13D转基因斑马鱼中敲低mRNA表达。 2）敲低mRNA表达 使用特定的转基因线以组织特异性方式。该建议在概念上是创新的，因为它是基于的 在探索一种新型技术的CRISPR-RFXCAS13D上，以组织特异性的方式敲除mRNA 在斑马鱼。这种方法从未在RNAi不起作用的脊椎动物模型系统中采用。 该项目的结果将是第一个通过敲门来剖析和研究基因功能的转基因系统 斑马鱼中的mRNA表达。我们的方法将帮助科学界调查基因功能 以更快和组织的特定方式，以及回答非常挑战的问题 通过当前方法的解决 基因。此外，通过遵循该项目的准则，研究人员可能会产生一个 整个Guiderna收集，以所有基因为目标，任何人都应该订购以解决 他们的个人研究问题。最后，随着我们成功实施了CRISPR-CAS13D系统 通过注射Medaka，Killifish和Mouse Embryos等其他生物，我们在此提案中描述了我们的工作 可以作为将这种有效的敲低技术转移到其他物种的基础。