Repurposing systemic RNAi to simplify genome editing in nematodes

重新利用系统性 RNAi 简化线虫基因组编辑

基本信息

批准号：
10077875
负责人：
Sean Patrick Ryder
金额：
$ 25.13万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-01-01 至 2021-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10077875
关键词：
3&apos Untranslated Regions Address Adoption Adult Alleles Animal Model Animals Back Bacteria Basic Science CRISPR/Cas technology Caenorhabditis elegans Cell Line Cells Clustered Regularly Interspaced Short Palindromic Repeats Cryopreservation DNA Development Double-Stranded RNA Environment Enzymes Equipment Fluorescence Gene Deletion Gene Targeting Generations Genes Genetic Genetic Models Genetic Screening Genetic study Guide RNA Hermaphroditism Lesion Location Mediating Medical Methods Microinjections Mutagenesis Mutagens Mutation Nematoda Partner in relationship Pathway interactions Personal Communication Phase Phenotype Physiology Population Procedures Process Property RNA Interference RNA interference screen Research Somatic Mutation System Technology Testing Time Transgenic Organisms Variant disease-causing mutation equipment training feeding functional genomics gene function gene replacement gene therapy genome editing genome-wide in vivo mutant novel strategies novel therapeutics off-target mutation prevent promoter repaired reverse genetics screening skills tool transmission process whole genome zygote

项目摘要

Project Summary: CRISPR/Cas9 technology has revolutionized genome editing and holds great promise for the develop- ment of new genetic therapies. It is now possible to make precise lesions at defined locations in many model organisms and cell lines. Physical and regulatory connections identified through numerous “omics” studies can now be tested directly in vivo, establishing their relative importance to cell and animal physiology. The nema- tode Caenorhabditis elegans is an exemplary tool for gene function research. The worm is easy to culture, has a short generation time, and a large brood size. RNAi is highly efficient in this species— pathways exist to take up double stranded RNA (dsRNA) from the environment and spread it throughout the worm, enabling RNAi by soaking the worms in dsRNA, or by feeding the worms bacteria that express dsRNA, a property that has ena- bled routine genome-wide RNAi screening. Now, thanks to CRISPR / Cas9, it is possible to make both targeted gene deletions and targeted gene replacements in C. elegans a relatively straightforward way. While this approach is now widely used in many C. elegans research labs throughout the world, there remains a major limitation. The need to microinject guides creates a bottleneck that prevents high throughput mutagenesis studies. Microinjection requires both specialized equipment and training. We propose to combine the most useful features of RNAi, including deliv- ery by soaking or feeding, with CRISPR/Cas9 mediated genome editing, to eliminate this research bottleneck. Our approach—termed siCRISPR—will enable genome-wide targeted mutagenesis studies that will enable functional testing of hypotheses generated through “omics” level sequencing methods.

项目概要： CRISPR/Cas9 技术彻底改变了基因组编辑，并为未来的发展带来了巨大希望。现在可以在许多模型中的指定位置进行精确的损伤。通过大量“组学”研究确定的生物体和细胞系的物理和调控联系可以现在可以直接在体内进行测试，确定它们对细胞和动物生理学的相对重要性。秀丽隐杆线虫是基因功能研究的典范工具。该蠕虫易于培养，具有世代时间短，RNAi 规模大，在该物种中效率很高——存在可采取的途径。从环境中提取双链 RNA (dsRNA) 并将其传播到蠕虫体内，从而实现 RNAi 将蠕虫浸泡在 dsRNA 中，或者给蠕虫喂食表达 dsRNA 的细菌，这种特性具有 ena- 进行常规全基因组 RNAi 筛选。现在，借助 CRISPR/Cas9，既可以进行靶向基因删除，也可以进行靶向基因线虫中的替代物是一种相对简单的方法，虽然这种方法现在广泛应用于许多领域。世界各地的线虫研究实验室仍然存在显微注射的重大限制。指南造成了阻碍高通量诱变研究的瓶颈，这两者都需要。我们建议结合 RNAi 最有用的功能，包括提供通过浸泡或喂食，利用 CRISPR/Cas9 介导的基因组编辑，消除这一研究瓶颈。我们的方法（称为 siCRISPR）将实现全基因组靶向突变研究，从而使对通过“组学”水平测序方法产生的假设进行功能测试。