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

Goell, J. H.; Li, J.; Mahata, B.; Ma, A. J.; Kim, S.; Shah, S.; Contreras, M.; Misra, S.; Reed, D.; Bedford, G. C.; Escobar, M.; Hilton, I. B.

doi:10.1101/2024.09.22.611000

bioRxiv · 10.1101/2024.09.22.611000

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

Goell, J. H.·Li, J.·Mahata, B.·Ma, A. J.·Kim, S.·Shah, S.·Contreras, M.·Misra, S.·Reed, D.·Bedford, G. C.·Escobar, M.·Hilton, I. B.

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Abstract

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.

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Goell, J. H., Li, J., Mahata, B., Ma, A. J., Kim, S., Shah, S., Contreras, M., Misra, S., Reed, D., Bedford, G. C., Escobar, M., Hilton, I. B.. 2024-09-22. Tailoring a CRISPR/Cas-based Epigenome Editor for Programmable Chromatin Acylation and Decreased Cytotoxicity. https://doi.org/10.1101/2024.09.22.611000

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