Chemical Control of a CRISPR-Cas9 Acetyltransferase

Abstract

Lysine acetyltransferases (KATs) play a critical role in the regulation of transcription and other genomic functions. However, a persistent challenge is the development of assays capable of defining KAT activity directly in living cells. Toward this goal, here we report the application of a previously reported dCas9-p300 fusion as a transcriptional reporter of KAT activity. First, we benchmark the activity of dCas9-p300 relative to other dCas9-based transcriptional activators and demonstrate its compatibility with second generation short guide RNA architectures. Next, we repurpose this technology to rapidly identify small molecule inhibitors of acetylation-dependent gene expression. These studies validate a recently reported p300 inhibitor chemotype and reveal a role for p300s bromodomain in dCas9-p300-mediated transcriptional activation. Comparison with other CRISPR-Cas9 transcriptional activators highlights the inherent ligand tunable nature of dCas9-p300 fusions, suggesting new opportunities for orthogonal gene expression control. Overall, our studies highlight dCas9-p300 as a powerful tool for studying gene expression mechanisms in which acetylation plays a causal role and provide a foundation for future applications requiring spatiotemporal control over acetylation at specific genomic loci.

Figure 1.

A CRISPR-Cas9 transcriptional reporter of KAT activity. (a) Scheme for induction of acetylation/gene expression by dCas9-p300, first reported by Gersbach et al. (b) Domain architecture of dCas9-p300 fusion. (c) Activation of model inducible genes IL1RN, RHOXF2, and TTN by dCas9-p300 in HEK-293 cells. D1399Y refers to a dCas9-p300 construct harboring a mutation to the KAT catalytic domain.

Figure 2.

KAT-dependent gene expression can be stimulated using diverse genomic recruitment strategies. (a) Crystal structure of Cas9-sgRNA complex (PDB 4OO8) illustrating sites for short guide RNA engineering. (b) Cartoon schematic of dCas9-dependent recruitment of MS2-p300 fusions using sgRNA 2.0. (c) Comparison of KAT-dependent expression of IL1RN using dCas9-p300 and MS2-p300 with sgRNA 2.0 architectures.

Figure 3.

Influence of small molecule stimuli on CRISPR-Cas9 acetyltransferase. (a) Single point screening data. Single point concentrations are provided in the Supporting Information. Patent KATi = NCGC00496795. (b) Structures of bromodomain-targeting small molecules identified as inhibitors of dCas9-p300-dependent gene expression. (c) Dose-dependent inhibition of KAT-dependent gene expression by NCGC00496795. (d) Dose-dependent inhibition of KAT-dependent gene expression by JQ1. (e) JQ1 alters dCas9-p300-dependent gene expression but not SAM-dependent gene expression.

Figure 4.

Impact of p300 bromodomain on KAT-dependent gene expression. (a) Dose-dependent inhibition of KAT-dependent gene expression by CBP30 and CBP112. (b) CBP30 does not affect dCas9-p300 overexpression. (c) Crystal structure of CBP bromodomain (PDB 2RNY) highlighting role of N1168 residue (N1132 in full length p300, N1516 in dCas9-p300) in acetyllysine recognition. (d) dCas9-p300 bromodomain mutant (N1168A) mutant is expressed at similar levels to wild-type dCas9-p300. Uncropped full blot data is provided in Supporting Information. (e) Mutations known to disrupt the p300 bromodomain-acetyllysine interaction reduce transcriptional activation by dCas9-p300. Significance analyzed by two-tailed Student’s t test (* = P < 0.05).