Repurposing the CRISPR‐Cas9 System for Targeted Chromatin O ‐linked β‐ N ‐acetylglucosamine Editing

Eukaryotic gene transcription is controlled by many proteins, including the basal transcription machinery, chromatin remodeling complexes, and transcription cofactors. Chromatin and genome‐mapping consortia have identified O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) as an abundant post‐translational modification involved in numerous transcriptional processes, including RNA polymerase function, chromatin dynamics, and cofactor activity. Decoding the precise function of all the intracellular O‐GlcNAc‐regulated elements identified in these studies remains challenging. Technologies to manipulate O‐GlcNAcylation at specific DNA loci for functional analysis without pleiotropic consequences were lacking, but the advent of CRISPR‐Cas9‐based technologies has enabled the precise perturbation of DNA sequences and delivery of proteins to specific cis‐acting DNA regulatory elements to elucidate their role in transcription. We reasoned that a CRISPR‐Cas9 system could be developed to study the function of O‐GlcNAcylation in transcription, which would circumvent the problems inherent in conventional gene manipulation and pharmacological approaches. We developed a programmable CRISPR‐Cas9‐based system that allows for O‐GlcNAc manipulation of transcriptional complex function at specific cis‐regulatory elements. The tools consist of nuclease‐null Cas9 (dCas9) fused to O‐GlcNAc transferase (OGT), which adds the O‐GlcNAc modification, or O‐GlcNAcase (OGA), which removes the modification. Previously, we demonstrated that O‐GlcNAc plays a role in regulating human □‐globin gene transcription. Increasing fetal hemoglobin (HbF) via activation of □‐globin chain synthesis is widely accepted as the most effective treatment for SCD and certain types of β‐thalassemias. O‐GlcNAcylation modulates the formation of a GATA‐1‐FOG‐1‐NuRD repressor complex that binds the ‐566 GATA site of the A□‐globin promoter when □‐globin gene expression is silent. OGT and OGA interact with GATA‐1 and CHD4, a component of the NuRD complex. O‐GlcNAcylation of CHD4 stimulates the formation of this repressor complex, whereas removing this PTM results in activation of A□‐globin gene expression. As proof of principle to demonstrate the utility of our system, we targeted dCas9‐OGT or dCas9‐OGA fusion proteins to DNA sequences flanking the ‐566 GATA site of the A□‐globin gene promoter. Increased □‐globin gene transcription was measured by RT‐qPCR when dCas9‐OGA was targeted to this area, whereas □‐globin gene expression decreased when dCas9‐OGT was targeted to the identical sequences. These data support our published work, confirming that O‐GlcNAcylation regulates A□‐globin gene transcription. RT‐qPCR data coupled with Cut and Run assays demonstrate the robust and highly specific nature of our targeting system, which can be employed as a generally applicable tool to study the role of O‐GlcNAcylation in transcriptional regulation.