Abstract 5380: Inactivation of endogenous genes in cancer cells using targeted promoter DNA methylation via CRISPR-DNMT3a fusion protein

Abstract

Abstract 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 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 now performed C-MYC methylation-mediated knockdown in A549 lung cancer cells and are evaluating resulting cell cycle and apoptosis phenotypes. Conclusion: We believe this new targeted epigenetic technology can be directed to cancer cells for the purpose of reprogramming lung cancer, and unregulated stromal and microenvironment cells, for preventive and therapeutic uses. Note: This abstract was not presented at the meeting. Citation Format: Simon D. Spivack. Inactivation of endogenous genes in cancer cells using targeted promoter DNA methylation via CRISPR-DNMT3a fusion protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5380. doi:10.1158/1538-7445.AM2017-5380