Functional analysis of H19 long non-coding RNA by CRISPR gene editing

Authors

• B Rahat
• A Mitra
• L Brandenberger
• K Pfeiffer

Abstract

Introduction: H19-gene encodes for a non-coding RNA (ncRNA), involved in various physiological and pathological conditions. However, the functional importance of H19 is not well understood. Thus, we aim to study the functional importance of H19 and its different regions and the mechanism of its action. Experimental design: RNA-sequencing was used to analyze the effect of H19 deletion on genome-wide mRNA expression in myoblasts. We used CRISPR mutagenesis to delete different regions of H19 and plan to study their function. Non-functional region of H19 will be used to insert aptamer and pull down the proteins bound to H19. Result: Our RNA-sequencing data showed differential expression of more than 4000 genes in delta-H19 myoblasts compared to wild type myoblasts, thus, showing the role of H19 in multiple gene pathways. Using CRISPR gene editing, we have generated mutant myoblasts with deletion either in 5’- or 3’-H19, microRNA675-encoding region and let7 binding sites in exon 1 and exon 4 of H19. Quantification of H19 expression in these mutants revealed no change in H19 expression. Summary and future work: H19 regulate many important signaling pathways within myoblasts. CRISPR/Cas9 mutagenesis can be used for generating H19 mutants and these deleted regions are not important for levels of H19. Next we plan to use these mutant myoblast cell lines for functional analysis. Followed by insertion of aptamer at non-functional region of H19 to pull down protein bound to H19. Thus, our data can help to understand the role and mechanism of H19 function.

Scientific Focus Area: Molecular Biology and Biochemistry

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