RESUMEN
The discovery and adaptation of CRISPR/Cas systema for epigenome editing has allowed for a straightforward design of targeting modules that can direct epigenome editors to virtually any genomic site. This advancement in DNA-targeting technology brings allele-specific epigenome editing into reach, a "super-specific" variation of epigenome editing whose goal is an alteration of chromatin marks at only one selected allele of the genomic target locus. This technology could be useful for the treatment of diseases caused by a mutant allele with a dominant effect, because allele-specific epigenome editing allows the specific silencing of the mutated allele leaving the healthy counterpart expressed. Moreover, it may allow the direct correction of aberrant imprints in imprinting disorders where editing of DNA methylation is required exclusively in a single allele. Here, we describe a basic protocol for the design and application of allele-specific epigenome editing systems using allele-specific DNA methylation at the NARF gene in HEK293 cells as an example. An sgRNA/dCas9 unit is used for allele-specific binding to the target locus containing a SNP in the seed region of the sgRNA or the PAM region. The dCas9 protein is connected to a SunTag allowing to recruit up to 10 DNMT3A/3L units fused to a single-chain Fv fragment, which specifically binds to the SunTag peptide sequence. The plasmids expressing dCas9-10x SunTag, scFv-DNMT3A/3L, and sgRNA, each of them co-expressing a fluorophore, are introduced into cells by co-transfection. Cells containing all three plasmids are enriched by FACS, cultivated, and later the genomic DNA and RNA can be retrieved for DNA methylation and gene expression analysis.
Asunto(s)
Alelos , Sistemas CRISPR-Cas , Metilación de ADN , Epigenoma , Edición Génica , Humanos , Edición Génica/métodos , Células HEK293 , ARN Guía de Sistemas CRISPR-Cas/genética , Epigenómica/métodos , Epigénesis GenéticaRESUMEN
DNA methylation is critically involved in the regulation of chromatin states and cell-type-specific gene expression. The exclusive expression of imprinted genes from either the maternal or the paternal allele is regulated by allele-specific DNA methylation at imprinting control regions (ICRs). Aberrant DNA hyper- or hypomethylation at the ICR1 of the H19/IGF2 imprinting locus is characteristic for the imprinting disorders Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS), respectively. In this paper, we performed epigenome editing to induce targeted DNA demethylation at ICR1 in HEK293 cells using dCas9-SunTag and the catalytic domain of TET1. 5-methylcytosine (5mC) levels at the target locus were reduced up to 90% and, 27 days after transient transfection, >60% demethylation was still observed. Consistent with the stable demethylation of CTCF-binding sites within the ICR1, the occupancy of the DNA methylation-sensitive insulator CTCF protein increased by >2-fold throughout the 27 days. Additionally, the H19 expression was increased by 2-fold stably, while IGF2 was repressed though only transiently. Our data illustrate the ability of epigenome editing to implement long-term changes in DNA methylation at imprinting control regions after a single transient treatment, potentially paving the way for therapeutic epigenome editing approaches in the treatment of imprinting disorders.
Asunto(s)
Desmetilación del ADN , Trastornos de Impronta , Humanos , Dominio Catalítico , Epigenoma , Células HEK293 , Alelos , Oxigenasas de Función Mixta/genética , Proteínas Proto-Oncogénicas , Factor II del Crecimiento Similar a la Insulina/genéticaRESUMEN
BACKGROUND: Activation of dominant oncogenes by small or structural genomic alterations is a common driver mechanism in many cancers. Silencing of such dominantly activated oncogenic alleles, thus, is a promising strategy to treat cancer. Recently, allele-specific epigenome editing (ASEE) has been described as a means to reduce transcription of genes in an allele-specific manner. In cancer, specificity to an oncogenic allele can be reached by either targeting directly a pathogenic single-nucleotide variant or a polymorphic single-nucleotide variant linked to the oncogenic allele. To investigate the potential of ASEE in cancer, we here explored this approach by targeting variants at the TERT promoter region. The TERT promoter region has been described as one of the most frequently mutated non-coding cancer drivers. RESULTS: Sequencing of the TERT promoter in cancer cell lines showed 53% (41/77) to contain at least one heterozygous sequence variant allowing allele distinction. We chose the hepatoblastoma cell line Hep-G2 and the lung cancer cell line A-549 for this proof-of-principle study, as they contained two different kinds of variants, namely the activating mutation C228T in the TERT core promoter and the common SNP rs2853669 in the THOR region, respectively. These variants were targeted in an allele-specific manner using sgRNA-guided dCas9-DNMT3A-3L complexes. In both cell lines, we successfully introduced DNA methylation specifically to the on-target allele of the TERT promoter with limited background methylation on the off-target allele or an off-target locus (VEGFA), respectively. We observed a maximum CpG methylation gain of 39% and 76% on the target allele when targeting the activating mutation and the common SNP, respectively. The epigenome editing translated into reduced TERT RNA expression in Hep-G2. CONCLUSIONS: We applied an ASEE-mediated approach to silence TERT allele specifically. Our results show that the concept of dominant oncogene inactivation by allele-specific epigenome editing can be successfully translated into cancer models. This new strategy may have important advantages in comparison with existing therapeutic approaches, e.g., targeting telomerase, especially with regard to reducing adverse side effects.
Asunto(s)
Neoplasias Pulmonares , Telomerasa , Humanos , Alelos , Metilación de ADN , Epigenoma , ARN Guía de Sistemas CRISPR-Cas , Regiones Promotoras Genéticas , Nucleótidos , Mutación , Telomerasa/genéticaRESUMEN
BACKGROUND: Epigenome editing refers to the targeted reprogramming of genomic loci using an EpiEditor which may consist of an sgRNA/dCas9 complex that recruits DNMT3A/3L to the target locus. Methylation of the locus can lead to a modulation of gene expression. Allele-specific DNA methylation (ASM) refers to the targeted methylation delivery only to one allele of a locus. In the context of diseases caused by a dominant mutation, the selective DNA methylation of the mutant allele could be used to repress its expression but retain the functionality of the normal gene. RESULTS: To set up allele-specific targeted DNA methylation, target regions were selected from hypomethylated CGIs bearing a heterozygous SNP in their promoters in the HEK293 cell line. We aimed at delivering maximum DNA methylation with highest allelic specificity in the targeted regions. Placing SNPs in the PAM or seed regions of the sgRNA, we designed 24 different sgRNAs targeting single alleles in 14 different gene loci. We achieved efficient ASM in multiple cases, such as ISG15, MSH6, GPD1L, MRPL52, PDE8A, NARF, DAP3, and GSPT1, which in best cases led to five to tenfold stronger average DNA methylation at the on-target allele and absolute differences in the DNA methylation gain at on- and off-target alleles of > 50%. In general, loci with the allele discriminatory SNP positioned in the PAM region showed higher success rate of ASM and better specificity. Highest DNA methylation was observed on day 3 after transfection followed by a gradual decline. In selected cases, ASM was stable up to 11 days in HEK293 cells and it led up to a 3.6-fold change in allelic expression ratios. CONCLUSIONS: We successfully delivered ASM at multiple genomic loci with high specificity, efficiency and stability. This form of super-specific epigenome editing could find applications in the treatment of diseases caused by dominant mutations, because it allows silencing of the mutant allele without repression of the expression of the normal allele thereby minimizing potential side-effects of the treatment.
Asunto(s)
Metilación de ADN , ARN Guía de Sistemas CRISPR-Cas , Humanos , Epigénesis Genética , Alelos , Células HEK293 , Epigenoma , Sistemas CRISPR-Cas , Edición GénicaRESUMEN
Epigenome editing is a powerful approach for the establishment of a chromatin environment with desired properties at a selected genomic locus, which is used to influence the transcription of target genes and to study properties and functions of gene regulatory elements. Targeted DNA methylation is one of the most often used types of epigenome editing, which typically aims for gene silencing by methylation of gene promoters. Here, we describe the design principles of EpiEditors for targeted DNA methylation and provide step-by-step guidelines for the realization of this approach. We focus on the dCas9 protein as the state-of-the-art DNA targeting module fused to 10×SunTag as the most frequently used system for editing enhancement. Further, we discuss different flavors of DNA methyltransferase modules used for this purpose including the most specific variants developed recently. Finally, we explain the principles of gRNA selection, outline the setup of the cell culture experiments, and briefly introduce the available options for the downstream DNA methylation data analysis.
Asunto(s)
Metilación de ADN , ARN Guía de Sistemas CRISPR-Cas , Sistemas CRISPR-Cas/genética , Cromatina , ADN/metabolismo , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilasas de Modificación del ADN/genética , Epigénesis Genética , Edición Génica , Metiltransferasas/metabolismo , ARN Guía de Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-Cas/metabolismo , Factores de Transcripción/metabolismoRESUMEN
In eukaryotes, cytosine methylation regulates diverse biological processes such as gene expression, development and maintenance of genomic integrity. However, cytosine methylation and its functions in pathogenic apicomplexan protozoans remain enigmatic. To address this, here we investigated the presence of cytosine methylation in the nucleic acids of the protozoan Plasmodium falciparum. Interestingly, P. falciparum has TRDMT1, a conserved homologue of DNA methyltransferase DNMT2. However, we found that TRDMT1 did not methylate DNA, in vitro. We demonstrate that TRDMT1 methylates cytosine in the endogenous aspartic acid tRNA of P. falciparum. Through RNA bisulfite sequencing, we mapped the position of 5-methyl cytosine in aspartic acid tRNA and found methylation only at C38 position. P. falciparum proteome has significantly higher aspartic acid content and a higher proportion of proteins with poly aspartic acid repeats than other apicomplexan pathogenic protozoans. Proteins with such repeats are functionally important, with significant roles in host-pathogen interactions. Therefore, TRDMT1 mediated C38 methylation of aspartic acid tRNA might play a critical role by translational regulation of important proteins and modulate the pathogenicity of the malarial parasite.