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.

项目摘要： CRISPR/CAS9技术已彻底改变了基因组编辑，并为发展带来了巨大的希望 - 新的基因疗法。现在可以在许多模型中在定义的位置进行精确病变生物和细胞系。通过许多“ OMIC”研究确定的身体和监管联系可以现在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。 NEMA- 秀丽隐杆线虫是基因功能研究的典范工具。蠕虫很容易培养，有短一代时间，大小很大。 RNAi在该物种中高效 - 存在途径将双链RNA（dsRNA）脱离环境，并在整个蠕虫中传播，从而使RNAi通过将蠕虫浸入dsRNA中，或通过喂食表达dsRNA的蠕虫细菌，该特性具有Ena- BLED常规基因组范围RNAi筛选。现在，谢谢CRISPR / CAS9，可以既有靶向基因删除又有目标基因在秀丽隐杆线虫中的替代方法相对简单。虽然现在这种方法已广泛使用秀丽隐杆线虫研究实验室在世界各地，仍有一个主要限制。需要微型注射指南会产生一种瓶颈，可防止高通量诱变研究。显微注射需要两者专业设备和培训。我们建议结合RNAi最有用的特征，包括Deliv- 通过将CRISPR/CAS9介导的基因组编辑浸泡或喂养来消除这项研究瓶颈。我们的方法 - sicrispr-将实现全基因组的靶向诱变研究，这将使通过“ OMICS”水平测序方法产生的假设的功能测试。