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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：
9916037
负责人：
Sean Patrick Ryder
金额：
$ 20.94万
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/9916037
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.

项目总结: