Functional mapping of microRNA promoters with dCas9 fused to transcriptional regulators

Frontiers in Genetics

Genetics

Annie Varrault¹  Runhua Liu²  Lizhong Wang²  Pradeep Kumar²  Tristan Bouschet³  Mathilde Courtes³  Ilda Coku³  Anne Le Digarcher³  Céline Lemmers⁴  Arnaud Monteil⁴  Charles Hong⁵ 

[1] Department of Genetics, University of Alabama at Birmingham, Birmingham, AL, United States;Department of Genetics, University of Alabama at Birmingham, Birmingham, AL, United States;O’Neal Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL, United States;Institut de Génomique Fonctionnelle, CNRS, INSERM, Université de Montpellier, Montpellier, France;Plateforme de Vectorologie de Montpellier (PVM), BioCampus Montpellier, CNRS, INSERM, Université de Montpellier, Montpellier, France;Vanderbilt University School of Medicine Nashville, Nashville, TN, United States;

关键词: microRNA;    CRISPRa;    CRISPRi;    promoter;    Mest;    miR-335;    miR-3662;    embryonic stem (ES) cell;   

DOI  :  10.3389/fgene.2023.1147222

 received in 2023-01-18, accepted in 2023-04-18,  发布年份 2023

来源: Frontiers

【 摘 要 】

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.

【 授权许可】

Unknown   
Copyright © 2023 Kumar, Courtes, Lemmers, Le Digarcher, Coku, Monteil, Hong, Varrault, Liu, Wang and Bouschet.