There is a strong clinical need for novel therapeutic options for pancreatic ductal adenocarcinoma (PDAC), where the 5-year overall survival remains poor at 15% with limited treatment options beyond chemotherapy (1, 2). Recent studies suggest alternative splicing not only drives PDAC aggressiveness but also renders it vulnerable to splicing inhibition, indicating that this may be a novel therapeutic strategy for this disease (3). Increased expression of PRMT5 is associated with a worse prognosis and interestingly, the more alternative splicing PRMT5 inhibition induces, the greater reduction in PDAC proliferation (4, 5). Whilst commercially available PRMT5 inhibitors have already shown safety in early phase trials, it remains unclear which of PRMT5’s plethora of biological effects is more important in PDAC (6-8). To understand the mechanism of action of PRMT5 inhibition in PDAC, MiaPaca2 and AsPC1 cell lines, which represent two PDAC molecular subtypes (Moffit’s Basal and Classical, respectively) (9), were treated with PRMT5 inhibitor (GSK3266595) at GI50 and GI90 doses across four time points. Changes in transcriptome and alternative splicing were then assessed. In addition, a whole genome CRISPR screen was performed to determine functionally important genes that either synergize with or are important to the response to PRMT5 inhibitor. PRMT5 inhibition resulted in a wide range of changes in RNA processing, metabolism, interferon, MYC targets, and cell cycle pathways. The pattern of transcriptome changes differed significantly between cell lines and whilst the same gene set may be enriched but it’s different genes within the set driving the enrichment. Correlating with CRISPR-identified genes of interest suggests SAGA type complexes to be important in the response of PRMT5 inhibition in addition to its effect on the splicesome. The impact of PRMT5 inhibition on alternative splicing will be updated.