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

ABSTRACT: 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 β (TGFβ) 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 TGFβ-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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE300529 | GEO | 2026/02/06

REPOSITORIES: GEO

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Project description:Transforming growth factor beta (TGFβ) superfamily signaling is a prime inducer of epithelial-mesenchymal transitions (EMT) that foster cancer cell invasion and metastasis, a major cause of cancer-related deaths. Yet, TGFβ signaling is frequently inactivated in human tumor entities including colorectal cancer (CRC) and pancreatic adenocarcinoma (PAAD) with a high proportion of mutations incapacitating SMAD4, which codes for a transcription factor (TF) central to canonical TGFβ and bone morphogenetic protein (BMP) signaling. Beyond its role in initiating EMT, SMAD4 was reported to crucially contribute to subsequent gene regulatory events during EMT execution. It is therefore widely assumed that SMAD4-mutant (SMAD4mut) cancer cells are unable to undergo EMT. Here, we scrutinized this notion and probed for potential SMAD4-independent EMT execution using SMAD4mut CRC cell lines. We show that SMAD4mut cells exhibit morphological changes, become invasive, and regulate EMT marker genes upon induction of the EMT-TF SNAIL1. Furthermore, SNAIL1-induced EMT in SMAD4mut cells was found to be entirely independent of TGFβ/BMP receptor activity. Global assessment of the SNAIL1‑dependent transcriptome confirmed the manifestation of an EMT gene regulatory program in SMAD4mut cells highly related to established EMT signatures. Finally, analyses of human tumor transcriptomes showed that SMAD4 mutations are not underrepresented in mesenchymal tumor samples and that expression patterns of EMT‑associated genes are similar in SMAD4mut and SMAD4 wild-type cases. Altogether, our findings reveal considerable plasticity of gene regulatory networks operating in EMT execution and establish that EMT is not categorically precluded in SMAD4mut tumors, which is relevant for their diagnostic and therapeutic evaluation. To identify genes regulated during EMT execution in HT29 cells, two clonal HT29 cell populations (4F5 and 3C2) overexpressing Snail1-HA in a doxycycline (Dox)-inducible manner, as well as control cells were treated with Dox for different periods of time.

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

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