CRISPR screen identifies microRNA-483-3p as a survival regulator

Prostate cancer remains a significant clinical challenge, with metastatic disease proving particularly difficult to treat despite recent advances in androgen-targeted therapies. While microRNAs are known to be dysregulated in prostate cancer and influence disease progression, identifying which specific microRNAs are essential for cancer cell survival has been limited by a lack of suitable screening tools.

To address this, the team developed miRKOv2 – an enhanced CRISPR-Cas knockout library specifically designed to target microRNA genes, with improved on-target efficiency and reduced off-target effects. The library was used to conduct genome-wide dropout screens in DU145 and LNCaP prostate cancer cell lines, identifying 70 candidate essential microRNAs. Among these, MIR483 had the most significant impact on cell viability.

The researchers validated these findings using both CRISPR-mediated genetic knockout (employing independent guide RNAs and a dual-knockout system) and Tough Decoy inhibitors that specifically sequester mature miR-483-3p. Both approaches reduced growth across multiple prostate cancer cell lines but not in non-malignant prostate cells, indicating cancer-specific essentiality.

Mechanistic studies using Western blotting, luciferase reporter assays, and apoptosis measurements (Annexin V staining, caspase activity, mitochondrial membrane potential) revealed that miR-483-3p suppresses apoptosis by directly targeting BCLAF1 and PUMA, whilst indirectly regulating BAK1. Tough Decoy inhibition of miR-483-3p enhanced docetaxel chemotherapy efficacy, demonstrating potential for combination therapeutic strategies in metastatic disease.

The study was led by Jonathan Tak-Sum Chow and Leonardo Salmena at the University of Toronto, Canada. The research was published in Cell Death and Disease on 24 October 2025.