Escobar, M. | Search bioRxiv

bioRxiv2024

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

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.

synthetic biology↗

bioRxiv2022

Compact engineered human transactivation modules enable potent and versatile synthetic transcriptional control

Engineered transactivation domains (TADs) combined with programmable DNA binding platforms have revolutionized synthetic transcriptional control. Despite recent progress in programmable CRISPR/Cas-based transactivation (CRISPRa) technologies, the TADs used in these systems often contain poorly tolerated elements and/or are prohibitively large for many applications. Here we defined and optimized minimal TADs built from human mechanosensitive transcription factors (MTFs). We used these components to construct potent and compact multipartite transactivation modules (MSN, NMS, and eN3x9) and to build the CRISPR-dCas9 recruited enhanced activation module (CRISPR-DREAM) platform. We found that CRISPR-DREAM was specific, robust across mammalian cell types, and efficiently stimulated transcription from diverse regulatory loci. We also showed that MSN and NMS were portable across Type I, II, and V CRISPR systems, TALEs, and ZF proteins. Further, as proofs of concepts, we used dCas9-NMS to efficiently reprogram human fibroblasts into iPSCs and demonstrated that MTF TADs are efficacious and well tolerated in therapeutically important primary human cell types. Finally, we leveraged the compact and potent features of these engineered TADs to build new dual and all-in-one CRISPRa AAV systems. Altogether, these compact human TADs, fusion modules, and new delivery architectures should be valuable for synthetic transcriptional control in biomedical applications.

bioengineering↗