ABSTRACT Most of understanding of how the spliceosome operates in vivo comes from studies of mutants in S. cerevisiae, yet human splicing displays major differences, including massive variation in length, much more variable intron signals, numerous additional subunits in the spliceosome, and much more regulation. While RNA-binding proteins (RBPs) that interact with the spliceosome enable many of these differences, we hypothesize that there are relevant human-specific factors and mechanisms within the spliceosome itself. However, unbiased discovery of such factors/mechanisms is extremely challenging because no forward genetic system for analyzing pre- mRNA splicing in human cells has been described. Standard CRISPR approaches are designed to produce null alleles rather than informative viable point mutations that are hypomorphic and/or disrupt specific functions. Thus, we have implemented CRISPR base editing, a method to program point mutations, in haploid human cells to mutagenize the spliceosome at thousands of residues in over 150 spliceosomal proteins. In preliminary work, we have conducted a screen using the inhibitor pladienolide B (plaB), which targets U2 snRNP protein SF3b. Following validation, sequencing of the base edits responsible for resistance revealed anticipated changes in SF3b subunits near the plaB binding pocket, validating the approach. Unexpectedly, we also identified missense mutations in SUGP1, which encodes a human-specific spliceosomal protein unknown function recently revealed to be a cancer driver gene, as well as C-terminal truncation mutations in Splicing Factor 1 (SF1), a conserved sequence-specific RNA binding protein that recognizes the intron branchpoint whose in vivo function is poorly understood. This work demonstrates large scale base editing screens in haploid human cells to investigate the function of spliceosomal proteins in vivo. Our global hypothesis is that unbiased genetics can reveal functions for human spliceosomal proteins that enable human-specific features of pre-mRNA splicing. First, we will test this hypothesis combining clonal cell lines mutant in SUGP1 or SF1 already in hand with powerful methods to investigate the phenotypic consequences of these mutations on splicing and protein interactions. This includes the global quantification of branchpoint-3’ splice site decisions made in vivo and the proximity labeling to investigate the impact of mutations on protein interactions in vivo. Analysis of these mutant molecular phenotypes will begin to inform our understanding of the functions of the core spliceosomal factors in vivo. We will also modify our genetic strategy to use bichromatic reporter-based assays to begin to identify core spliceosomal residues that promote splicing fidelity. The impact of these studies is anticipated to be high as these as this work is the first to deploy a forward genetic approach to understand the functions of essential spliceosomal components in human cells.