Casilio-ME: Enhanced CRISPR-based DNA demethylation by RNA-guided coupling methylcytosine oxidation and DNA repair pathways

Abstract

We have developed a methylation editing toolbox, Casilio-ME, that enables not only RNA-guided methylcytosine editing by targeting TET1 to genomic sites, but also by codelivering TET1 and protein factors that couple methylcytosine oxidation to DNA repair activities, and/or promote TET1 to achieve enhanced activation of methylation-silenced genes. Delivery of TET1 activity by Casilio-ME1 robustly altered the CpG methylation landscape of promoter regions and activated methylation-silenced genes. We augmented Casilio-ME1 to simultaneously deliver the TET1-catalytic domain and GADD45A (Casilio-ME2) or NEIL2 (Casilio-ME3) to streamline removal of oxidized cytosine intermediates to enhance activation of targeted genes. Using two-in-one effectors or modular effectors, Casilio-ME2 and Casilio-ME3 remarkably boosted gene activation and methylcytosine demethylation of targeted loci. We expanded the toolbox to enable a stable and expression-inducible system for broader application of the Casilio-ME platforms. This work establishes an advanced platform for editing DNA methylation to enable transformative research investigations interrogating DNA methylomes.

Targeted intragenic demethylation initiates chromatin rewiring for gene activation

“…By simultaneously engaging the 5' (promoter) and 3' (intron) regions flanking the activator core, our design introduces the requirements that enable consistent and effective demethylation spreading (Region D2, Fig. 3B) and that are missing in other CRISPR-based platforms targeting exclusively the immediate promoter (7)(8)(9)32). Intriguingly, the CRISPR-DiR induced demethylation wave propagates inward into the middle of exon 1 region, while no demethylation is observed in the surrounding regions (Region C and E, Fig.…”

“…p16, p15, MLH1, DAPK1, CEBPA, CDH1, MGMT,BRCA1) silencing is frequently associated with abnormal 5'CpG island (CGI) DNA methylation (4) and it is considered as the hallmark of most if not all cancers (5). Since 70% annotated gene promoters overlap with a CGI (6), the majority of studies have only concentrated on the correlation of CpG island promoter methylation and transcriptional repression, specifically focusing on the region just upstream of the transcription start site (TSS) (2,(7)(8)(9), but neglecting some studies showing the regulatory importance of regions downstream of TSS (10,11). Therefore, the regulatory importance and mechanism of intragenic methylation on gene expression is still not clear.…”

Nuclease-Deficient Clustered Regularly Interspaced Short Palindromic Repeat-Based Approaches for In Vitro and In Vivo Gene Activation

“…Correspondingly, several studies have used a demethylation strategy to achieve transcription activation by targeting the catalytic domain of Tet1 (ten-eleven translocation) fused to dCas9 to regions of hypermethylated genes. [36][37][38][39] In one example, targeted demethylation of the BDNF promoter IV and the MyoD distal enhancer by dCas9-Tet1 achieved robust activation of BDNF expression in postmitotic neurons and MyoD reprogramming of fibroblasts into myoblasts, respectively. 37 These studies collectively establish the utility of epigenome editing strategies to achieve targeted gene activation as an alternative to CRISPRa platforms, however, its in vivo translatability using AAV-delivery methods is hampered due to the significantly larger size of these enzymes compared with the transcription effector domains used in CRISPRa platforms.…”

CRISPR-mediated Multiplexed Live Cell Imaging of Nonrepetitive Genomic Loci

“…PUF domains contain peptide subunits that can each be programmable to recognize an RNA-base by changing amino acids contacting the RNA, thereby allowing designed PUF domains to bind different 8-mer sequences (19,20). Casilio 10 capitalizes on the versatility of PUF domains to allow simultaneous and orthogonal delivery of effector functions to distinct genome loci, as well as multimerization of effector molecules, for efficient epigenetic editing (18,21). In the case of genomic labeling, we demonstrated not only greatly enhanced SNR but also simultaneous dual color labeling of telomeric and centromeric repeats (18).…”