Noncoding RNAs (ncRNAs) have emerged as key components of gene regulatory networks, yet their role in skeletal muscle diseases is still poorly understood. The maternally imprinted Dlk1‐Dio3 ncRNA locus has been shown to play an essential role in skeletal muscle development, regeneration, and metabolism. The diverse ncRNAs expressed from this locus, including over 50 microRNAs (miRNAs), several long noncoding RNAs (lncRNAs), and a cluster of C/D box snoRNAs, are thought to be transcribed as a ~200 kb polycistron initiating from the proximal promoter immediately upstream of the Gtl2 lncRNA, the most 5′ ncRNA in the locus. This project aimed to understand the importance of proper dosage of the Dlk1‐Dio3 ncRNAs in skeletal muscle differentiation. Toward this end, we utilized CRISPR‐Cas9 gene editing to generate deletions in the Gtl2 proximal promoter and the overlapping differentially methylated region (DMR) with the goal of modulating expression of the maternally expressed ncRNAs in C2C12 myoblasts, an established muscle cell line. This gene editing strategy yielded a clonal population of C2C12 myoblasts that harbored a deletion spanning a portion of the Gtl2 proximal promoter while leaving the Gtl2 promoter's TATA box intact. This CRISPR‐edited cell line showed a dramatic increase in the Dlk1‐Dio3 ncRNAs. As a result of Dlk1‐Dio3 ncRNA upregulation, this C2C12 cell line displays significantly impaired myogenic differentiation. The continued characterization of this novel C2C12 cell line will help elucidate the importance of maintaining proper dosage of the Dlk1‐Dio3 locus in skeletal muscle, which could serve as a therapeutic target for treating muscle diseases.Support or Funding InformationThis project was supported with funds from the Arnold & Mabel Beckman Foundation and the Boston University Undergraduate Research Opportunities ProgramThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.