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

University Of Texas Hlth Sci Ctr Houston, Houston TX

Investigators

Linked publications & trials

Abstract

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.