Identification of the Novel Regulatory Pathways in Skeletal Myogenesis Using a Genome-Scale Lentiviral sgRNA Library Screen

项目总结/摘要 本项目的总体目标是通过一种新的方法鉴定骨骼肌生成程序的主要激活剂。 新的功能增益（GOF）基因组规模的基因激活筛选。虽然众所周知的肌源性 调节因子（MRF，包括MYF 5、MYOD、MRF 4和肌生成素）及其上游特化 基因（PAX 3、PAX 7）已被鉴定并被广泛研究，但其上游调控因子尚不清楚。 在这种情况下，鉴定新的肌原性程序激活剂不仅对于 更好地了解肌肉的发育生物学，也用于鉴定与肌肉有关的基因 干细胞活化、再生和维持。另一方面，由于函数研究的增益已经被 由于传统cDNA过表达的缺点，这一领域的研究较少。 幸运的是，最近全基因组sgRNA激活剂文库的出现为我们提供了一个新的机会。 哺乳动物细胞中GOF基因的筛选。 我们最近已经产生了敲入肌源性谱系报告人ES/iPS细胞系，用于早期的 使用CRISPR/Cas9系统的肌源性谱系基因。这些细胞为体外研究提供了新的工具 多能干细胞向骨骼肌祖细胞的分化。因此，通过使用这些 报告ES/iPS细胞进行基因组规模的sgRNA激活剂文库筛选，我们有一个独特的机会， 激活并筛选所有已知的人类基因同种型，以确定它们诱导肌生成程序的潜力。 因此，在这个令人兴奋和探索性的R21应用中，作为第一个目标，我们计划进行sgRNA 在我们骨骼肌生成报告基因hES/iPS细胞系上筛选慢病毒文库。分离活化细胞后， 基因组DNA将通过深度测序进行分析，以鉴定整合的sgRNA，并最终鉴定其 能够激活肌原性报告基因的相关基因。这些数据有助于确定候选人 可能作为PAX 3/7或MYF 5的潜在上游激活因子的基因。 随后，在本提案的第二个目标中，我们将使用a）个体验证每个候选基因 sgRNA活化，B）cDNA过表达和c）肌原性分化实验。该验证旨在 证实基因激活是上游基因过表达的直接结果，并排除脱靶效应 的sgRNA。最后，将通过体外成肌细胞培养进一步研究cDNA过表达途径。 本发明的目的是通过细胞的分化来验证上游激活剂对骨骼肌发生起始的特异性。 因此，这种新颖的探索性全基因组筛选的结果提供了宝贵的 关于骨骼肌发生的上游激活剂的信息。这些数据可用于 骨骼肌发育研究以及潜在治疗靶点的鉴定 再生以及基于iPS细胞的治疗肌肉萎缩和疾病。

Project Summary/ Abstract The overall goal of this project is identification of master activators of skeletal myogenic program through a novel gain of function (GOF) genome-scale gene activation screening. Although well-known myogenic regulatory factors (MRFs including MYF5, MYOD, MRF4 and MYOGENIN) and their upstream specification genes (PAX3, PAX7) have been identified and studied extensively, their upstream regulators are less known. In this context, identification of novel activators of myogenic program is very important not only for better understanding of the developmental biology of muscle, also for identification of genes involved in muscle stem cell activation, regeneration and maintenance. On the other hand, since gain of function studies has been limited by traditional cDNA over-expression shortcomings, this area of research have been less explored. Fortunately, the recent advent of genome-wide sgRNA activator library provides a novel opportunity for GOF gene screen in mammalian cells. We have recently generated knock-in myogenic lineage reporter human ES/iPS cell lines for early myogenic lineage genes using CRISPR/Cas9 system. These cells provide novel tools to study in vitro differentiation of pluripotent stem cells toward skeletal myogenic progenitors. Therefore, by using these reporter ES/iPS cells for genome-scale sgRNA-activator library screen, we have a unique opportunity to activate and screen all known human gene isoforms for their potential to induce myogenic program. Therefore, in this exciting and exploratory R21 application, as the 1st aim, we plan to perform sgRNA lentiviral library screen on our skeletal myogenic reporter hES/iPS cell lines. After isolation of activated cells, genomic DNA will be analyzed by deep sequencing to identify integrated sgRNAs and eventually their associated genes which are able to activate myogenic reporters. These data leads to identification of candidate genes which might behave as potential upstream activators for PAX3/7 or MYF5. Subsequently, in the 2nd aim of this proposal, we will validate each candidate gene using a) individual sgRNA activation, b) cDNA over-expression and c) myogenic differentiation experiments. This validation is to confirm gene activation is a direct result of upstream gene over-expression and rule out the off-target effects of the sgRNAs. Finally cDNA over-expression approach will be further investigated by in vitro myogenic differentiation of the cells to validate specificity of the upstream activators for initiation of skeletal myogenesis. Therefore, the outcome of this novel and exploratory genome-wide screen provides invaluable information regarding the upstream activators of skeletal myogenesis. These data can be used for developmental studies as well as identification of potential therapeutic targets involved in skeletal muscle regeneration as well as iPS cell based therapies in muscle wasting and disorders.