CRISPR/dCas9-Based Tools for Epigenome Editing

Abstract

The emergence of CRISPR/Cas9 systems has transformed biotechnology, opening new opportunities for biomedical research and development of gene therapies. These tools enable diverse applications, from gene editing to modulation of the epigenome and programming complex gene regulatory networks. Epigenome editing with DNA-targeting technologies such as CRISPR/dCas9 can be used to dissect gene regulatory mechanisms and potentially treat associated disorders. Here, we both identify additional gene and epigenome editing tools and demonstrate the ability of targeted epigenetic modifications to program complex mechanisms of regulation at an endogenous imprinted locus.First, we expand the suite of CRISPR/Cas9-based tools by characterizing novel Cas9 orthologs and demonstrating their diverse applications in mammalian cells. We identified 47 Cas9 orthologs, guide RNA sequences, and putative protospacer adjacent motifs from bioinformatic analysis of the genomes of several Lactobacillales genera. We codon-optimized and assayed these Cas9 orthologs for activity in a human cell line, identifying four highly active orthologs that display potent targeted gene repression activity when their nuclease-deactivated dCas9 form is fused to a repressive KRAB domain. These four systems were also effective nucleases. In particular, we identified Streptococcus uberis Cas9 as a potent and versatile ortholog that can function as a gene activator, repressor, and nuclease with comparable performance to standard Staphylococcus aureus and Streptococcus pyogenes benchmarks, including at a v therapeutically relevant target. These additions to the CRISPR/Cas9 toolbox offer alternatives to the existing suite of targeted gene and epigenome editors and increase the number of targetable sites in the genome. Further, we sought to apply existing dCas9-based epigenome editors to reprogram a disease-relevant imprinted locus, activating epigenetically silenced genes whose expression is lost in Prader-Willi Syndrome (PWS). PWS is a human imprinting disorder caused by loss of paternally expressed genes on chromosome 15. Importantly, the relevant genes are present but epigenetically silenced on the imprinted maternal allele. We first conducted high-throughput CRISPR activation and repression screens in human induced pluripotent stem cells (iPSCs) to identify regulatory regions of the PWS locus. Then, we demonstrated that dCas9-effectors that achieve either targeted transcriptional activation or DNA demethylation can activate silenced maternal PWS genes, although these approaches work via distinct epigenetic mechanisms, at different genomic locations. Notably, transient expression of the targeted DNA demethylase can stably and heritably activate maternal PWS genes in iPSCs with a PWS deletion. This work reveals how targeted epigenetic modifications can reprogram a disease-associated imprinted locus, highlighting a potential for possible therapeutic interventions.