Almost all human genes undergo alternative splicing to produce multiple protein isoforms potentially with distinct domain structures, localization, and functions. Despite its prevalence, the functional relevance of most isoforms remains unknown. To address this gap, we are developing RNA-targeting CRISPR technology using catalytically inactive Cas13d (dCasRx) to manipulate alternative splicing and study isoform-specific protein functions. By designing guide RNAs targeting specific splice sites or exonic regions, dCasRx can induce exon skipping by sterically hindering spliceosome assembly at the targeted exon. To further enable exon inclusion or repression in a context-dependent manner, we engineered dCasRx fusions with splicing regulators such as Quaking 5 (QKI-5) and RNA Binding Motif protein 25 (RBM25).
We demonstrate that dCasRx-QKI5 mimics the endogenous activity of QKI5 by recognizing its natural RNA motifs and modulating splicing in a position-dependent manner. Using guide RNAs targeting QKI motifs upstream of EXOC1 exon 10, dCasRx-QKI5 promotes exon skipping, while targeting downstream intronic regions with dCasRx-RBM25 promotes exon inclusion. This highlights how different splicing regulators fused to dCasRx enable precise, programmable control of exon usage.
In parallel, we used dCasRx alone to induce skipping of NFYA exon 3, resulting in the expression of the shorter NF-YAs isoform, which increases cell proliferation while reducing migration, highlighting the functional consequences of isoform switching.
This programmable system enables precise control of alternative splicing and provides a powerful strategy to dissect protein isoform functions during epithelial-mesenchymal transition (EMT). We aim to extend this approach to pooled single cell CRISPR screens to systematically identify splicing events that drive cancer cell plasticity and therapy resistance.