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.

ABSTRACTBackgroundPathogenic variants in the LRRK2 gene are a common monogenic cause of Parkinson’s disease. However, only seven variants have been confirmed to be pathogenic.ObjectivesWe identified two novel LRRK2 variants (H230R and A1440P) and performed functional testing.MethodsWe transiently expressed wildtype, the two new variants, or two known pathogenic mutants (G2019S and R1441G), in HEK-293T cells, with or without LRRK2 kinase inhibitor treatment. We characterized the phosphorylation and kinase activity of the mutants by western blotting. Thermal shift assays were performed to determine the folding and stability of the LRRK2 proteins.ResultsThe two variants were found in two large families and segregate with the disease. They display altered LRRK2 phosphorylation and kinase activity.ConclusionsWe identified two novel LRRK2 variants which segregate with the disease. The results of functional testing lead us to propose these two variants as novel causative mutations for familial Parkinson’s disease.

ABSTRACTMicroRNAs are small non-coding RNAs that act as rheostats to modulate gene expression during development, physiology, and disease. Approximately half of mammalian microRNAs are intronic. It is unknown whether intronic miRNA transcription depends on their host gene or a microRNA-specific promoter. Here, we show that CRISPR inhibition of host gene Mest downregulated hosted miR-335 in mouse embryonic stem cells and brain organoids. Reciprocally, CRISPR transactivation of Mest upregulated miR-335. By contrast, activation of miR-335 predicted promoter had no effect. Thus, intronic miR-335 expression depends on the promoter activity of its host gene. This approach could serve to map microRNA promoters.