Abstract
Purpose :
Epigenome editing utilizing the CRISPR (clustered, regularly interspaced, short palindromic repeats)-Cas9 platform has emerged as a promising alternative to cleavage-dependent gene editing, providing a novel approach for targeted control of gene expression. Previous work has shown that fusing several transcriptional regulators to dCas9 in tandem can achieve a synergistic increase in activity, although the additional payload of an epigenetic effector molecule limits AAV-based delivery strategies.
Methods :
We developed a novel 105-aa MeCp2 repressor effector comprised of the MeCp2 transcriptional repressor domain (TRD). We first designed gRNAs to target the epigenome editor systems with both S. aureus (Sa) and C. jejuni (Cj) Cas9 proteins to a HEK293 cell line harboring a proline-glutamate-serine-threonine-rich (PEST) destabilized-GFP (dGFP) sequence and measured dGFP expression by fluorescence spectroscopy. To validate our epigenetic editor using a clinically relevant target, we next employed a dual luciferase (Firefly-Renilla) assay system to screen gRNAs against neural retina leucine zipper (NRL), a transcription factor dictating a rod cell phenotype in photoreceptors. Oligonucleotides encoding the relevant sequence were cloned into a reporter construct, and then co-transfected into HEK-293T cells with plasmids expressing the dSaCas9-repressor and relevant gRNA sequence to NRL.
Results :
At 48-hour and 96-hour timepoints, we observed a ~20% reduction in dGFP expression with the dSaCas9 bipartite KRAB-MeCp2 repressor, in comparison to no detectable dGFP knockdown with a non-targeting gRNA control. Following in silico design and screening in a dual luciferase assay, significant knockdown was observed against NRL across 5 of 6 tested repressor effector combinations, with the best gRNA achieving 57.6% luciferase knockdown
Conclusions :
Our results confirm the development and in vitro validation of a novel bipartite repressor effector that can be packaged all-in-one in AAV. No discernible epigenetic repression was detected with the CjCas9 protein. To facilitate in vivo epigenetic editing, we generated AAV8 for both dSaCas9-KRAB and dSaCas9-KRAB-MeCp2 repressor systems. Future studies will evaluate the utility of Cas9-mediated repressors in retinal organoids and in an Nrl.EGFP mouse model.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.