CRISPR-Based Functional Screens for microRNA Biogenesis and Non-Canonical Processing Pathways

While canonical miRNA biogenesis is well-characterized, recent literature (Komatsu et al., 2023) points to numerous non-canonical sources and processing routes for functional miRNAs, including those from atypical hairpins or lncRNAs. The genome-scale, single-cell CRISPRi map described by Feng et al. (2024) demonstrates the feasibility of such screens. This idea proposes a focused CRISPR-based screen targeting all putative RNA-binding proteins, splicing factors, and uncharacterized genes, combined with single-cell small RNA-seq, to discover novel biogenesis factors or pathways. By profiling the impact of each perturbation on the miRNome and associating these changes with downstream gene expression and phenotypes, this approach could fill major gaps in our knowledge and potentially reveal new regulatory layers or therapeutic targets, especially for diseases where miRNA processing is dysregulated.

References:

1. Network Regulation of microRNA Biogenesis and Target Interaction. Shintaro Komatsu, Hiroki Kitai, Hiroshi I. Suzuki (2023). Cells.
2. A genome-scale single cell CRISPRi map of trans gene regulation across human pluripotent stem cell lines. Claudia Feng, Elin Madli Peets, Yan Zhou, Luca Crepaldi, S. Usluer, Alistair S. Dunham, Jana M. Braunger, Jing Su, Magdalena E Strauss, Daniele Muraro, Kimberly Ai Xian Cheam, M. Bonder, Edgar Garriga Nogales, Sarah Cooper, Andrew R. Bassett, Steven Leonard, Yong Gu, Bo Fussing, David Burke, Leopold Parts, O. Stegle, B. Velten (2024). bioRxiv.