Synthetic Lethality CRISPR Screens in Isogenic iPSCs with Splicing Factor Gene Mutations

A major recent discovery from large-scale sequencing studies was that over half of Myelodysplastic Syndromes (MDS) patients harbor mutations in splicing factor (SF) genes. SF mutations are the most common class of mutations in MDS and occur early in the course of the disease. These strongly suggest that SF mutations are key to the pathogenesis of MDS and can provide new therapeutic opportunities. However, identifying the downstream effects of SF mutations that are critical for the development of MDS presents a big challenge due to the cellular and genetic heterogeneity of primary patient samples, the unavailability of immortalized cell lines harboring SF mutations in the native genomic context and the limited conservation of alternative splicing isoforms between mice and humans. We previously showed that SF-mutant induced pluripotent stem cells (iPSCs) generated from MDS patients recapitulate key features of the disease upon differentiation into hematopoietic lineages, including cellular phenotypes (increased cell death, decreased clonogenicity and dysplastic morphology), sensitivity to splicing modulating drugs and the altered RNA binding specificity of mutant SFs (Chang et al. Stem Cell Reports, 2018). To further investigate the effects of SF mutations, we used CRISPR to introduce each of the 3 main canonical SF mutations (SRSF2 P95L, SF3B1 K700E, U2AF1 S34F) in the same normal iPSC line N-2.12 that we previously derived and extensively characterized in terms of pluripotency, genetic integrity and hematopoietic differentiation potential. The derivative iPSC lines contain the 3 SF mutations in isogenic conditions in the context of a diploid genome, in a heterozygous state, with both the normal and mutant alleles expressed at physiological and equal levels. To uncover potential new therapeutic targets and gain insights into the downstream effects of SF mutations, we set up CRISPR knockout (KO) lethality screens in hematopoietic progenitor cells (HPCs) derived from these SF-mutant iPSCs. We began with a gRNA library containing 224 gRNAs targeting 57 kinase genes (4 gRNAs per gene). The library was assembled and packaged in a lentiviral backbone also expressing GFP. Cas9 together with mCherry was expressed from a separate lentiviral vector. iPSCs were differentiated along the hematopoietic lineage, transduced on day 11, coinciding with the onset of the emergence of CD34+/CD45+ HPCs, and further cultured for up to day 27 to allow “dropout” of lethal genes, re