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.

项目概要 确定基因的功能是了解脊椎动物发育、调控的基础 机制和人类疾病。基因组编辑技术，例如 CRISPR-Cas9，已经允许关联 永久性基因改变的特定表型。然而，一些关键的技术和概念问题仍然存在 在脊椎动物中存在问题，特别是在斑马鱼等水生模式生物中。例如，母性 提供的mRNA可以挽救纯合突变体的表型；基因分型步骤繁琐；和长非 编码 RNA、致死基因或组织/时间基因很难使用 DNA 操作进行研究，而且也很复杂 基因组位点。互补的“击倒”方法是规避其中一些问题的宝贵工具 然而，问题是，没有系统的工具来敲除斑马鱼或其他硬骨鱼中的 mRNA。 我们的长期目标是揭示与调控机制、发育和发育相关的基因功能。 人类疾病。我们最近发表的文章表明，将 CRISPR-RfxCas13d 系统注入 脊椎动物胚胎提供了一种强大且具有成本效益的技术来系统地破坏基因功能。 然而，该系统的注射仅提供约 3 天的短暂击倒。因此，中心目标 该提案的目的是将 CRISPR-RfxCas13d 技术从注射到脊椎动物胚胎中转移到 通过转基因内源表达。我们建议开发一套曲目 表达 RfxCas13d 酶（普遍存在且组织特异性）和引导 RNA 的转基因斑马鱼将 促进对基因功能的快速而有力的研究。我们的初步数据表明转基因 RfxCas13d 在斑马鱼中的表达具有功能性且无毒性。目标是： 1) 定义最佳系统 敲低 CRISPR-RfxCas13d 转基因斑马鱼中的 mRNA 表达。 2) 敲低 mRNA 表达 使用特定的转基因系以组织特异性的方式。该提案在概念上具有创新性，因为它基于 探索一种新技术 CRISPR-RfxCas13d，以组织特异性方式敲低 mRNA 在斑马鱼中。这种方法从未在 RNAi 不起作用的脊椎动物模型系统中使用过。 该项目的成果将是第一个通过敲入来解剖和研究基因功能的转基因系统 降低斑马鱼中 mRNA 的表达。我们的方法将帮助科学界研究基因功能 以更快和组织时间特定的方式，以及回答非常具有挑战性的问题 通过当前的方法来解决，例如非编码RNA的功能或由多种原因引起的表型 基因。此外，通过遵循我们将通过该项目进行优化的指导方针，研究人员可以产生一个 整个guideRNA集合，针对所有基因，任何人都应该可以订购来解决 他们个人的研究问题。最后，我们成功实现了CRISPR-Cas13d系统 通过注射到其他生物体中，如青鳉、鳉鱼和小鼠胚胎，我们在本提案中描述的工作 可以作为将这种有效的击倒技术转移到一系列其他物种的基础。