Enzyme-free targeted DNA demethylation using CRISPR–dCas9-based steric hindrance to identify DNA methylation marks causal to altered gene expression

Sapozhnikov, Daniel M.; Szyf, Moshe

doi:10.1038/s41596-022-00741-3

Cited by 6 publications

(5 citation statements)

References 116 publications

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“…In addition, TET1 is the ten-eleven translocation methylcytosine dioxygenase 1, and its catalytic domain is used for CRISPR approaches [33]. As a control, we used dCas alone, without effector, that was reported to induce moderate demethylation that was replication-dependent [34]. For epigenetic editing, the cells were transfected in 6-well dishes at an initial density of approximately 80% with a total of 4 µg plasmid DNA.…”

Section: Methodsmentioning

confidence: 99%

Biomarker RIPK3 Is Silenced by Hypermethylation in Melanoma and Epigenetic Editing Reestablishes Its Tumor Suppressor Function

Arroyo Villora,

Castellanos Silva,

Zenz

et al. 2024

Genes

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For several decades, cancers have demonstrably been one of the most frequent causes of death worldwide. In addition to genetic causes, cancer can also be caused by epigenetic gene modifications. Frequently, tumor suppressor genes are epigenetically inactivated due to hypermethylation of their CpG islands, actively contributing to tumorigenesis. Since CpG islands are usually localized near promoters, hypermethylation of the promoter can have a major impact on gene expression. In this study, the potential tumor suppressor gene Receptor Interacting Serine/Threonine Protein Kinase 3 (RIPK3) was examined for an epigenetic regulation and its gene inactivation in melanomas. A hypermethylation of the RIPK3 CpG island was detected by bisulfite pyrosequencing and was accompanied by a correlated loss of its expression. In addition, an increasing RIPK3 methylation rate was observed with increasing tumor stage of melanomas. For further epigenetic characterization of RIPK3, epigenetic modulation was performed using a modified CRISPR/dCas9 (CRISPRa activation) system targeting its DNA hypermethylation. We observed a reduced fitness of melanoma cells by (re-)expression and demethylation of the RIPK3 gene using the epigenetic editing-based method. The tumor suppressive function of RIPK3 was evident by phenotypic determination using fluorescence microscopy, flow cytometry and wound healing assay. Our data highlight the function of RIPK3 as an epigenetically regulated tumor suppressor in melanoma, allowing it to be classified as a biomarker.

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Section: Methodsmentioning

confidence: 99%

Biomarker RIPK3 Is Silenced by Hypermethylation in Melanoma and Epigenetic Editing Reestablishes Its Tumor Suppressor Function

Arroyo Villora,

Castellanos Silva,

Zenz

et al. 2024

Genes

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“…Plasmids. All S. pyogenes gRNA expression plasmids were generated by mutagenesis of pLenti-gRNA-puro (Addgene plasmid #180426) with the Q5® Site-Directed Mutagenesis Kit as previously described 40 using primers listed in Supplementary Table 1. A modified species-specific pLenti-gRNA-puro plasmid for S. aureus gRNAs was constructed as described previously for S. pyogenes 18 by ordering modified gene fragments (gBlocks, Integrated DNA Technologies) encoding the U6 promoter, gRNA sequence, and species-specific scaffold (Supplementary Table 1).…”

Section: Methodsmentioning

confidence: 99%

The PROTECTOR strategy employs dCas orthologs to sterically shield off-target sites from CRISPR/Cas activity

Sapozhnikov

Szyf

2023

Sci Rep

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Off-target mutagenesis of CRISPR/Cas systems must be solved to facilitate safe gene therapy. Here, we report a novel approach, termed "PROTECTOR", to shield known off-target sites by directing the binding of an orthologous nuclease-dead Cas protein to the off-target site to sterically interfere with Cas activity. We show that this method reduces off-target mutation rates of two well-studied guide RNAs without compromising on-target activity and that it can be used in combination with high-fidelity Cas enzymes to further reduce off-target editing. This expands the suite of off-target mitigation strategies and offers an ability to protect off-target sites even when their sequences are fully identical to target sites.

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“…A tool for site-specific epigenetic editing typically must consist of two components: an enzymatic component with epigenetic-modifying activity (with the exception of targeted DNA methylation editing [34,35], discussed below in the section titled "Steric inhibition for DNA demethylation") and a targeting component-a domain that can bind a specific DNA sequence so that the epigenetic activity can be targeted to specific genes or genomic locations. At present, targeting is achieved by one of three categories of targeting domains: zinc-fingers (ZFs), transcription activator-like effectors (TALEs), or CRISPR/dCas9.…”

Section: Targeting Architecture Of Epigenetic Editorsmentioning

confidence: 99%

Increasing Specificity of Targeted DNA Methylation Editing by Non-Enzymatic CRISPR/dCas9-Based Steric Hindrance

Sapozhnikov

Szyf

2023

Biomedicines

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As advances in genome engineering inch the technology towards wider clinical use—slowed by technical and ethical hurdles—a newer offshoot, termed “epigenome engineering”, offers the ability to correct disease-causing changes in the DNA without changing its sequence and, thus, without some of the unfavorable correlates of doing so. In this review, we note some of the shortcomings of epigenetic editing technology—specifically the risks involved in the introduction of epigenetic enzymes—and highlight an alternative epigenetic editing strategy using physical occlusion to modify epigenetic marks at target sites without a requirement for any epigenetic enzyme. This may prove to be a safer alternative for more specific epigenetic editing.

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Enzyme-free targeted DNA demethylation using CRISPR–dCas9-based steric hindrance to identify DNA methylation marks causal to altered gene expression

Cited by 6 publications

References 116 publications

Biomarker RIPK3 Is Silenced by Hypermethylation in Melanoma and Epigenetic Editing Reestablishes Its Tumor Suppressor Function

Biomarker RIPK3 Is Silenced by Hypermethylation in Melanoma and Epigenetic Editing Reestablishes Its Tumor Suppressor Function

The PROTECTOR strategy employs dCas orthologs to sterically shield off-target sites from CRISPR/Cas activity

Increasing Specificity of Targeted DNA Methylation Editing by Non-Enzymatic CRISPR/dCas9-Based Steric Hindrance

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