ENAGenomicsEric Lai Lab, Department of Developmental Biology, Sloan-Kettering Institutehttps://www.ebi.ac.uk/ena/browser/view/PRJNA1148192Homo sapiensThe core miRNA biogenesis enzyme Dicer sustains recurrent mutations in cancer, which specifically compromise catalysis by its RNase IIIb domain. These are proposed to act as selective hypomorphs, since Dicer-RNase IIIb cleaves the 5p arms of pre-miRNA hairpins. However, the lack of knockin models has hindered a full understanding of the consequences of Dicer hotspot mutations for miRNA biogenesis. Here, we generated Dicer-KO and Dicer-S1344L (homozygous and hemizygous) hESCs the latter is a non-catalytic mutation in RNase IIIa that impairs RNase IIIb activity. Consistent with our previous work, Dicer-KO is cell lethal in hESCs. However, in contrast to the expectation that Dicer hotspot mutations confer cell advantages, Dicer-S1344L knockin hESCs were subviable and also required a conditional genetic strategy for isolation. Small RNA sequencing using spike-in normalization reveals two broad trends: ablation of miRNA-5p species, and selective changes in miRNA-3p species. Curiously, we recognized a directional upregulation of miRNA-3p passenger strands, indicating a broad strand switch. Activity assays and Argonaute profiling of the transcriptome confirm that these confer increased repression capacity. Finally, we used in vitro assays to show 3p arm-nicked pre-miRNAs preferentially load miRNA-3p species into Argonaute, compared to corresponding miRNA/passenger duplexes. Overall, Dicer hotspot mutations have unexpected neomorphic consequences on miRNAs, including both loss- and gain-of-function. Overall design: To investigate the consequences of the Dicer cancer hotspot mutation, we generated CRISPR-Cas9 engineered Dicer-S1344L homozygous and hemizygous hESCs cells.ENAfalseHuman Dicer hotspot mutation induces both loss and gain of miRNA function [RNA-seq]Human Dicer hotspot mutation induces both loss and gain of miRNA function [RNA-seq]2025-09-242025-08-14PRJNA1148192GSE2748499606