Uncovering Critical Lysines in Mammalian Histone H3 with High-Throughput CRISPR Prime Editing [RNA-seq]

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.