Tailoring a CRISPR/Cas-based Epi-editor for Decreased Cytotoxicity and Enzymatic Specificity

CRISPR-based epi-editors can robustly modulate cellular transcription and chromatin structure, but off-target activity and cytotoxicity limit their utility. Here, we engineer the acyl-CoA binding pocket of the human p300 histone acyltransferase to reduce its cytotoxicity and tune its acylation profile when fused to dCas9. We discover a single amino acid substitution (I1417N) that decreases cytotoxicity related to exogenous p300 overexpression yet preserves dCas9-p300 mediated histone acetylation and gene activation. We find that dCas9-p300 I1417N is less perturbative to the transcriptome and proteome of human cells, and that this behavior is driven by favorable stability kinetics. We also develop a crotonylation-biased dCas-p300 variant (I1395G) that selectively deposits histone crotonylation and activates transcription from endogenous promoters at levels comparable to wild-type p300. The p300 variants generated here enhance epi-editing capabilities and demonstrate that engineering of catalytic domains can be a powerful strategy for tailoring enzymatic activities and mitigating effector-driven toxicity in epi-editing.