ABSTRACT: Histone proteins are central to regulating eukaryotic genomes, and their post-translational modifications play key roles in controlling gene expression, DNA repair and chromatin structure. Understanding the functions of individual histone modifications is a significant challenge in mammalian chromatin biology, primarily due to the multiple copies of histone genes found in mammalian genomes. In this study, we present a high-throughput CRISPR prime editing platform that allows for base-precise, combinatorial, and reversible mutagenesis of all canonical and non-canonical histone H3 genes within their native genomic context. Using this system, we systematically substituted each lysine residue in histone H3 with arginine and compared each mutation against synonymous lysine-to-lysine controls. This unbiased functional screen revealed a core set of lysines, including H3K4, H3K9, H3K14, H3K18, and H3K79, whose mutations impair cellular fitness in mouse embryonic stem cells, highlighting the critical roles of their post-translational modifications in chromatin function. In addition, our approach demonstrated that H3K56 acetylation, previously linked to genome stability in yeast and Drosophila, plays a conserved role in safeguarding the genome in mammalian cells. Extending this approach, we generated double histone H3 mutants to probe functional redundancy between several lysine residues. While individual substitutions caused minimal defects, specific combinations, most notably H3K27R+H3K36R, revealed impairments in stem cell self-renewal and distinct transcriptional consequences, uncovering regulatory crosstalk not evident from single-site perturbations. This study presents the first comprehensive functional map of histone H3 lysines in a mammalian system, establishing a broadly applicable platform for dissecting histone modifications with advanced precision.
