Repurposing systemic RNAi to simplify genome editing in nematodes

项目概要： CRISPR/Cas9技术彻底改变了基因组编辑，并为开发新技术带来了巨大希望。 新的基因疗法。现在可以在许多模型中的定义位置进行精确的损伤 生物体和细胞系。通过大量“组学”研究确定的物理和监管联系， 现在可以直接在体内进行测试，建立它们对细胞和动物生理学的相对重要性。那条线虫 秀丽隐杆线虫是基因功能研究的典型工具。该虫易于培养， 世代时间短，育雏量大。RNAi在这个物种中是非常有效的--存在的途径 从环境中提取双链RNA（dsRNA）并将其传播到整个蠕虫中，从而实现RNA干扰 将蠕虫浸泡在dsRNA中，或通过喂养表达dsRNA的蠕虫细菌，这一特性使其具有 流血的常规的全基因组RNAi筛选。 现在，由于CRISPR / Cas9，有可能使靶向基因缺失和靶向基因 替换C. elegans优雅a relatively相对straightforward简单way.虽然这种方法现在被广泛应用于许多 C.世界各地的线虫研究实验室，仍然有一个主要的限制。微注射的必要性 向导产生了阻碍高通量诱变研究的瓶颈。显微注射需要两者 专门设备和培训。我们建议联合收割机结合RNAi最有用的特征，包括递送， 通过浸泡或喂养，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-将使全基因组靶向诱变研究， 通过“组学”水平测序方法产生的假设的功能测试。

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.