Tailoring a CRISPR/Cas-based Epigenome Editor for Programmable Chromatin Acylation and Decreased Cytotoxicity [Perturb-seq]

Precisely engineering histone acylation states can inform mechanistic epigenetics and catalyze new therapeutic epigenome editing opportunities. Here, we developed engineered lysine acyltransferases that enable the deposition of acetylation and longer-chain acylations in a programmable manner using a dead Cas9 (dCas9) fusion protein. We show that targeting an engineered lysine crotonyltransferase, developed by mutagenizing the native human p300 protein, results in relatively weak levels of endogenous enhancer activation yet retains strong potency when targeted to promoters. We further identify a single mutation within the catalytic core of human p300 that preserves enzymatic activity while promoting more target-specific acetylation and substantially reducing downstream transcriptomic perturbations. Further, this novel fusion protein exhibits low cytotoxicity but maintains high levels of H3K27ac deposition, enabling markedly improved lentiviral delivery. We leveraged this enhanced delivery and improved cytotoxicity profile to perform single-cell CRISPR activation screening. Using proteomics and a panel of engineered p300 variants, we also discover acylation-specific interactions and link the cytotoxicity of the wild-type p300 core domain to altered activities among DNA repair machinery components. These new programable epigenome editing tools and insights expand our ability to perform functional genomic screens, multiplexed cell engineering, and, more broadly, understand the mechanistic role of lysine acylation in epigenetic and cellular processes.