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Targeted DNA Methylation and Inactivation of Endogenous Genes Using CAS9-DNMT3a Fusion Protein; An Update
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A7082 - Targeted DNA Methylation and Inactivation of Endogenous Genes Using CAS9-DNMT3a Fusion Protein; An Update
Author Block: S. D. Spivack1, M. Shi1, A. Yan2, Y. Peter3, M. Levy2, W. Han4; 1Pulmonary Medicine, Albert Einstein College of Med, Bronx, NY, United States, 2Biochemistry, Albert Einstein College of Med, Bronx, NY, United States, 3Biology, Touro College, Queens, NY, United States, 4Immunotoxicology, Wadsworth Center, NYSDOH, Albany, NY, United States.
Background: Developing technologies for precise manipulation of individual DNA methylation loci is an attractive challenge in cancer and lung biology, because aberrant expression via hyper- or hypo-methylation is so common. Projects such as ENCODE and the Roadmap Epigenetics Project, have identified thousands of epigenetic marks from human genome. However, the functional evaluation of these marks has been largely limited to determining their associations with gene expression. Technologies for targeting manipulation of epigenetic marks would allow direct experimental testing of the impact of DNA methylation at specific residues, mechanisms of gene regulation, and potential use as interventions, for example to silence constitutively active oncogenes. Methods: Here, we developed a CRISPR Cas9-based tool for specific DNA methylation in which the catalytic domain of DNMT3a (DNMT3a-CD) is fused to the carboxy-terminus of Cas9 D10A-H840A mutant (dCas9). Both construct promoter strength and transfection strategies were optimized. Results: We demonstrated targeted and consistent CpG methylation in 30~100bp regions downstream of the PAM (binding) site of the gRNA guided fusion protein in HEK293 cells. The multiple guide RNAs could target the dCas9-DNMT3A to multiple sites consistently. DNA methylation activity was specific for the targeted region and was partally heritable across cell divisions. We also found directed promoter DNA methylation of DAL1, GATA5 and C-MYC could decrease the corresponding mRNA expression by up to 80%; different DNA methylation positions had consequently different effects on gene expression. We have recently accomplished KRAS promoter methylation, and are now evaluating resulting expression and cellular phenotypes. Conclusion: If this new technology can be directed to lung cells for the purpose of reprogramming otherwise unregulated epithelial and other accessible cells, one can envision targeted preventive and therapeutic uses.
Author Block: S. D. Spivack1, M. Shi1, A. Yan2, Y. Peter3, M. Levy2, W. Han4; 1Pulmonary Medicine, Albert Einstein College of Med, Bronx, NY, United States, 2Biochemistry, Albert Einstein College of Med, Bronx, NY, United States, 3Biology, Touro College, Queens, NY, United States, 4Immunotoxicology, Wadsworth Center, NYSDOH, Albany, NY, United States.
Background: Developing technologies for precise manipulation of individual DNA methylation loci is an attractive challenge in cancer and lung biology, because aberrant expression via hyper- or hypo-methylation is so common. Projects such as ENCODE and the Roadmap Epigenetics Project, have identified thousands of epigenetic marks from human genome. However, the functional evaluation of these marks has been largely limited to determining their associations with gene expression. Technologies for targeting manipulation of epigenetic marks would allow direct experimental testing of the impact of DNA methylation at specific residues, mechanisms of gene regulation, and potential use as interventions, for example to silence constitutively active oncogenes. Methods: Here, we developed a CRISPR Cas9-based tool for specific DNA methylation in which the catalytic domain of DNMT3a (DNMT3a-CD) is fused to the carboxy-terminus of Cas9 D10A-H840A mutant (dCas9). Both construct promoter strength and transfection strategies were optimized. Results: We demonstrated targeted and consistent CpG methylation in 30~100bp regions downstream of the PAM (binding) site of the gRNA guided fusion protein in HEK293 cells. The multiple guide RNAs could target the dCas9-DNMT3A to multiple sites consistently. DNA methylation activity was specific for the targeted region and was partally heritable across cell divisions. We also found directed promoter DNA methylation of DAL1, GATA5 and C-MYC could decrease the corresponding mRNA expression by up to 80%; different DNA methylation positions had consequently different effects on gene expression. We have recently accomplished KRAS promoter methylation, and are now evaluating resulting expression and cellular phenotypes. Conclusion: If this new technology can be directed to lung cells for the purpose of reprogramming otherwise unregulated epithelial and other accessible cells, one can envision targeted preventive and therapeutic uses.
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