SRP408597 PRJNA903135 GSE218305 Targeted high throughput mutagenesis of the human spliceosome reveals its in vivo operating principles [editing] A major impediment to studying the human spliceosome in vivo has been the inability to program point mutations in endogenous genes on a large scale. CRISPR-Cas9 technology now provides such opportunities. Due to its scalability and ability to introduce point mutations, we chose CRISPR-Cas9 base editing for a forward genetic screen of the spliceosome in an eHAP haploid human cell line background. We maintained eHAP FNLS cells as haploid so that we could subsequently assign genotype-phenotype relationships. We designed a single guide RNA (sgRNA) library targeting a hand-curated list of 153 human spliceosomal proteins which engage at various steps of the splicing cycle. After mutagenesis, we interrogated the spliceosome using the potent inhibitor pladienolide B (PB), which targets U2 snRNP by binding to a pocket between the SF3B1 and PHF5A subunits of the SF3b complex, preventing stabilization of the U2-branchpoint RNA duplex. Validation and genomic sequencing revealed resistance mutations in SF3B1 and PHF5A in residues adjacent to the compound binding pocket. We mapped hypersensitive mutants to U2 snRNP components, but also to factors that act as late as the second chemical step, after SF3b has dissociated. Strikingly, we obtained resistance mutants in SUGP1, a spliceosomal G-patch protein of unknown function that lacks orthologs in yeast and is also a newly proposed tumor suppressor whose loss underpins the splicing changes induced by cancer-associated SF3B1 mutations. Overall design: We transduced eHAP FNLS with lentivirus carrying individual sgRNAs, grew cells for six days (= t0), isolated genomic DNA, amplified the edited locus and subjected the amplicons to deep sequencing. This approach revealed the consequences of base editing at the amino acid level. For sgRNAs conferring resistance to PB we also collected cells at t8 and t15 in the presence and absence of 2 nM PB treatment. GSE218305 pubmed 37402368 parent_bioproject PRJNA903130