Development of super-specific epigenome editing by targeted allele-specific DNA methylation
Abstract 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...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2023-10-01
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| Series: | Epigenetics & Chromatin |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s13072-023-00515-5 |
| _version_ | 1827708226862317568 |
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| author | Nivethika Rajaram Alexandra G. Kouroukli Susanne Bens Pavel Bashtrykov Albert Jeltsch |
| author_facet | Nivethika Rajaram Alexandra G. Kouroukli Susanne Bens Pavel Bashtrykov Albert Jeltsch |
| author_sort | Nivethika Rajaram |
| collection | DOAJ |
| description | Abstract 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. |
| first_indexed | 2024-03-10T17:00:42Z |
| format | Article |
| id | doaj.art-41d1de7855f14d409bafb99c8a958394 |
| institution | Directory Open Access Journal |
| issn | 1756-8935 |
| language | English |
| last_indexed | 2024-03-10T17:00:42Z |
| publishDate | 2023-10-01 |
| publisher | BMC |
| record_format | Article |
| series | Epigenetics & Chromatin |
| spelling | doaj.art-41d1de7855f14d409bafb99c8a9583942023-11-20T10:57:30ZengBMCEpigenetics & Chromatin1756-89352023-10-0116111810.1186/s13072-023-00515-5Development of super-specific epigenome editing by targeted allele-specific DNA methylationNivethika Rajaram0Alexandra G. Kouroukli1Susanne Bens2Pavel Bashtrykov3Albert Jeltsch4Institute of Biochemistry and Technical Biochemistry, Department of Biochemistry, University of StuttgartInstitute of Human Genetics, University of Ulm and Ulm University Medical CenterInstitute of Human Genetics, University of Ulm and Ulm University Medical CenterInstitute of Biochemistry and Technical Biochemistry, Department of Biochemistry, University of StuttgartInstitute of Biochemistry and Technical Biochemistry, Department of Biochemistry, University of StuttgartAbstract 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.https://doi.org/10.1186/s13072-023-00515-5Epigenome editingTargeted DNA methylationdCas9DNMT3A/3LAllele discriminationSpecificity |
| spellingShingle | Nivethika Rajaram Alexandra G. Kouroukli Susanne Bens Pavel Bashtrykov Albert Jeltsch Development of super-specific epigenome editing by targeted allele-specific DNA methylation Epigenetics & Chromatin Epigenome editing Targeted DNA methylation dCas9 DNMT3A/3L Allele discrimination Specificity |
| title | Development of super-specific epigenome editing by targeted allele-specific DNA methylation |
| title_full | Development of super-specific epigenome editing by targeted allele-specific DNA methylation |
| title_fullStr | Development of super-specific epigenome editing by targeted allele-specific DNA methylation |
| title_full_unstemmed | Development of super-specific epigenome editing by targeted allele-specific DNA methylation |
| title_short | Development of super-specific epigenome editing by targeted allele-specific DNA methylation |
| title_sort | development of super specific epigenome editing by targeted allele specific dna methylation |
| topic | Epigenome editing Targeted DNA methylation dCas9 DNMT3A/3L Allele discrimination Specificity |
| url | https://doi.org/10.1186/s13072-023-00515-5 |
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