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.
In vitro differentiation of human pluripotent stem cells (hES/iPS cells) toward skeletal myogenic lineage provides great insights regarding gene regulatory networks involved in this process. Here we propose to screen all known human gene isoforms (more than 23,000 isoforms) on myogenic lineage reporter hES/iPS cells using a novel genome-scale gene activation approach (sgRNA library). This provides a very unique and exceptional opportunity to discover novel gene regulatory networks important for skeletal myogenesis.
| Wu, Jianbo; Matthias, Nadine; Lo, Jonathan et al. (2018) A Myogenic Double-Reporter Human Pluripotent Stem Cell Line Allows Prospective Isolation of Skeletal Muscle Progenitors. Cell Rep 25:1966-1981.e4 |
