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Repurposing systemic RNAi to simplify genome editing in nematodes

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

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中文摘要
翻译
项目总结: CRISPR/Cas9技术使基因组编辑发生了革命性的变化,并为基因组编辑技术的发展带来了巨大的希望。 一种新的基因疗法。现在可以在许多模型中在指定位置进行精确损伤 生物体和细胞系。通过大量的“组学”研究确定的生理和监管联系可以 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型工具。蠕虫很容易培养,有 世代时间短,孵化规模大。RNAi在该物种中是高效的-存在可采取的途径 从环境中向上传播双链RNA(DsRNA),并在整个蠕虫中传播,通过以下方式实现RNAi 将蠕虫浸泡在dsRNA中,或通过喂养表达dsRNA的蠕虫细菌,这一特性具有Ena- 常规的全基因组RNAi筛查。 现在,由于CRISPR/Cas9,有可能同时进行靶向基因缺失和靶向基因 替换线虫是一种相对简单的方式。虽然这种方法现在被广泛应用于许多 线虫在世界各地的研究实验室中,仍然存在一个重大限制。微量注射的必要性 GUIDE造成了一个瓶颈,阻碍了高通量的突变研究。微量注射既需要 专门的设备和培训。我们建议结合RNAi最有用的功能,包括传递- 通过浸泡或喂养,与CRISPR/Cas9介导的基因组编辑,以消除这一研究瓶颈。 我们的方法--称为siCRISPR--将使全基因组定向突变研究成为可能 通过“组学”水平排序方法产生的假设的功能测试。
英文摘要
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.
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Post-transcriptional regulation of germline mRNAs in C. elegans
Post-transcriptional regulation of germline mRNAs in C. elegans
Repurposing systemic RNAi to simplify genome editing in nematodes
Comparative HTS to identify small molecule effectors of cell fate specification
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