Deregulated RNA splicing is emerging as a new hallmark of cancer following the identification of recurrent somatic mutations in splicing factors across different tumor types. Heterozygous hotspot mutations in SF3B1, the most frequently mutated splicing factor in cancer, induce transcriptomic changes by promoting alternative 3' splice site usage and exon skipping. These mutations are particularly frequent in hematological malignancies, which typically lack effective selective treatments. By CRISPR/Cas9, we mimicked three pathological SF3B1 mutations in the C. elegans ortholog sftb?1. We have exploited this model for two related purposes: (i) to investigate the molecular consequences of SF3B1 mutations and (ii) to identify vulnerabilities of SF3B1-mutated cells as potential therapeutic targets. We found that, unlike in mammalian cells,
sftb-1 missense mutations were tolerated in homozygosis in C. elegans. Indeed, sftb?1
(cer7[K718E]), sftb?1
(cer16[R643C]), and sftb?1
(cer17[Q552P]) worms did not display any visible phenotypes. We observed temperature-sensitive phenotypes when combining the three missense mutations in one allele (
cer39), suggesting an additive effect. A deletion allele (
cer6) resulted in early larval arrest, confirming that
sftb-1 is an essential gene. RNA-sequencing of strains harboring
cer7,
cer39, and
cer6 alleles revealed gene expression and alternative splicing (AS) changes, with exon skipping being the most deregulated type of AS event. In the search for new cancer vulnerabilities, we performed an RNAi screen of splicing genes and uncovered synthetic interactions between
sftb-1 mutations and knockdown of some U2 snRNP components. Likewise,
sftb-1 mutations interacted synthetically with
uaf-2(
cer108[S42F]) and rsp?4
(cer113[P100H]) mutations, which are equivalent to cancer-related mutations in the splicing factors U2AF1 and SRSF2, respectively. Altogether, we demonstrate that C. elegans is a valuable system for modeling cancer-related mutations in splicing factors and screening for synthetic lethal interactions.