Abstract
Here we develop a methylation editing toolbox, Casilio-ME, that enables not only RNA-guided methylcytosine editing by targeting TET1 to genomic sites, but also by co-delivering 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 alters the CpG methylation landscape of promoter regions and activates methylation-silenced genes. We augment 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 boost gene activation and methylcytosine demethylation of targeted loci. We expand the toolbox to enable a stable and expression-inducible system for broader application of the Casilio-ME platforms. This work establishes a platform for editing DNA methylation to enable research investigations interrogating DNA methylomes.
Highlights
We develop a methylation editing toolbox, Casilio-Methylation Editing (ME), that enables RNA-guided methylcytosine editing by targeting TET1 to genomic sites, and by co-delivering TET1 and protein factors that couple methylcytosine oxidation to DNA repair activities, and/or promote TET1 to achieve enhanced activation of methylation-silenced genes
We show that Casilio-ME-mediated delivery of TET1 activity to gene promoters induces robust cytosine demethylation within the targeted CpG island (CGI) and activation of gene expression
Casilio-ME1 is a three-component DNA Methylation Editing platform built on Casilio which uses nuclease-deficient Cas[9], an effector module made of Pumilio/FBF (PUF) domain linked to an effector protein, and a modified single guide RNA (sgRNA) containing PUF-binding sites (PBS) (Fig. 1a)[30]
Summary
We develop a methylation editing toolbox, Casilio-ME, that enables RNA-guided methylcytosine editing by targeting TET1 to genomic sites, and by co-delivering TET1 and protein factors that couple methylcytosine oxidation to DNA repair activities, and/or promote TET1 to achieve enhanced activation of methylation-silenced genes. We develop an advanced DNA methylation editing technology which allows targeted bridging of TET1 activity to BER machinery to efficiently alter the epigenetic state of CpG targets and activate methylation-silenced genes. Casilio-DNA Methylation Editing (ME) platforms enable targeted delivery of the TET1 effector alone (Casilio-ME1) or in association with GADD45A (Casilio-ME2) or NEIL2 (Casilio-ME3) to achieve enhanced 5mC demethylation and gene activation. The ability of Casilio-ME to mediate co-delivery of TET1 activity along with other protein factors, which enhance turnover of oxidized cytosine intermediates, paves the way for new areas of research to efficiently address the cause–effect relationships of DNA methylation in normal and pathological processes
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
