Rewiring miR-22/SNAI1 via CRISPR-Based Edge Editing Destabilizes the Epithelial Phenotype

Epithelial-to-Mesenchymal Transition (EMT) is a critical biological process by which cells acquire enhanced migratory and invasive properties. A key signaling pathway involved in EMT phenotypes includes transforming growth factor ß (TGFß) and transcription factors (TFs) such as Snail, Zeb, and Twist. Additionally, microRNAs (miRNAs) – small, non-coding molecules that regulate gene expression by targeting mRNA transcripts – directly regulate genes central to the EMT process. Notably, miR-22 has been identified as a significant regulator of EMT through direct inhibition of EMT drivers like SNAI1 and indirect regulation of upstream genes. In this study, we performed CRISPR-based network rewiring by selectively removing an edge—the connection between two nodes—to investigate its impact on EMT dynamics. Specifically, we disrupted the connection between miR-22 and Snail1 without affecting other interactions involving miR-22 or Snail1 and examined the resulting effects on EMT. We demonstrate that the removal of the miR-22 target site from the SNAI1 gene renders cells more sensitive to TGFß-mediated EMT. This finding highlights the critical importance of the direct regulatory connection between miR-22 and SNAI1 in modulating EMT, distinct from miR-22's effects on other targets or indirect pathways. More generally, our results underscore the importance of CRISPR-mediated edge ablation for exploring the interactions that govern biological networks and highlight an underexplored opportunity to develop edge-based therapeutic modalities. Overall design: RNA-seq profiling of wildtype A549 cells and the miR-22/SNAI1 edge knockout derivative cultured in the precense or absence of EMT induction agent TGFß (at 10 ng/mL) for 5 days.