期刊
FRONTIERS IN GENETICS
卷 14, 期 -, 页码 -出版社
FRONTIERS MEDIA SA
DOI: 10.3389/fgene.2023.1147222
关键词
microRNA; CRISPRa; CRISPRi; promoter; Mest/PEG1; miR-335; miR-3662; embryonic stem (ES) cell
MicroRNAs are small non-coding RNAs that control gene expression. This study investigates the transcriptional regulation of two microRNAs, miR-335 and miR-3662, by targeting their candidate promoters using CRISPRa and CRISPRi. The results show that miR-335 expression depends on the promoter activity of its host gene, while miR-3662 can be activated by both the host gene promoter and an intronic sequence.
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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