Article Dans Une Revue Frontiers in Genetics Année : 2023

Functional mapping of microRNA promoters with dCas9 fused to transcriptional regulators

Résumé

MicroRNAs are small non-coding RNAs that control gene expression during development, physiology, and disease. Transcription is a key factor in microRNA abundance and tissue-specific expression. Many databases predict the location of microRNA transcription start sites and promoters. However, these candidate regions require functional validation. Here, dCas9 fused to transcriptional activators or repressors - CRISPR activation (CRISPRa) and inhibition (CRISPRi)- were targeted to the candidate promoters of two intronic microRNAs, mmu-miR-335 and hsa-miR-3662, including the promoters of their respective host genes Mest and HBS1L. We report that in mouse embryonic stem cells and brain organoids, miR-335 was downregulated upon CRISPRi of its host gene Mest. Reciprocally, CRISPRa of Mest promoter upregulated miR-335. By contrast, CRISPRa of the predicted miR-335-specific promoter (located in an intron of Mest) did not affect miR-335 levels. Thus, the expression of miR-335 only depends on the promoter activity of its host gene Mest. By contrast, miR-3662 was CRISPR activatable both by the promoter of its host gene HBS1L and an intronic sequence in HEK-293T cells. Thus, CRISPRa and CRISPRi are powerful tools to evaluate the relevance of endogenous regulatory sequences involved in microRNA transcription in defined cell types.

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hal-04122098 , version 1 (08-06-2023)

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Pradeep Kumar, Mathilde Courtes, Céline Lemmers, Anne Le Digarcher, Ilda Coku, et al.. Functional mapping of microRNA promoters with dCas9 fused to transcriptional regulators. Frontiers in Genetics, 2023, 14, pp.1147222. ⟨10.3389/fgene.2023.1147222⟩. ⟨hal-04122098⟩
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