Author of

• 14490

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  P2.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 234)

  • Event: WCLC 2015
  • Type: Poster
  • Track: Biology, Pathology, and Molecular Testing
  • Presentations: 1
  • Moderators:
  • Coordinates: 9/08/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)

  • 14544

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    P2.04-054 - Targeting DNA Methylation in Chromatin (ID 3177)

    09:30 - 09:30  |  Author(s): A. Yan
    • Abstract

    Background:
    DNA methylation is heritable during mitosis, and has been reported to serve as a strong molecular mark for gene silencing memory. Therefore, to more permanently down-regulate a gene’s expression than by siRNA or other means, target-directed DNA methyltransferases are desirable. Recently, the first gene-specific targeted DNA methylation using a zinc finger protein fused to the catalytic domain of DNMT3a (DNMT3a-CD) was reported in a chromatin context for the tumor suppressor gene MASPIN, and the oncogene SOX2, and showed definite but modest activity, but require that a new protein be re-engineered for every new target site. Cas9 directed constructs can potentially be retargeted by simply changing the identity of the guide RNA (gRNA) sequence.
    
    Methods:
    We synthesized a Cas9x-DNMT3a-P2A-EGFP fusion in which the catalytic domain of DNMT3a (DNMT3a-CD) is fused to the carboxy terminus of Cas9 D10A-H840A mutant (Cas9x) along with a fluorescent reporter (EGFP) for targeting to the SOX2 promoter. The constructs was transfected into 293T cells and the transfected cells were sorted by flow cytometry. DNA methylation was analyzed by bisulfite sequencing and SOX2 expression was determined by real-time RT-PCR.
    
    Results:
    We sorted transfected 293T cells with flow cytometry and found ~50% of the GFP positive cells were methylated with an average methylation level of ~17% (~4 of 21 CpG sites with the target region, but vaired from clone-to-clone); the Cas9x-only expressing vector (as control) showed no methylation. SOX2 mRNA expression was reduced 31% compared to the Cas9x control. Cell adhesion was disrupted, as was growth in culture, compared to empty vector Cas9x controls. Replication studies in A549 lung cancer and other cells are ongoing, as are optimization refinements. Figure 1 Figure. The methylation state of SOX2 promoter targeted by Cas9x-DNMT3a-CD. The Cas9x-DNMT3a-2A-GFP expression vector was transfected into 293Tcells with LipoFectamin 2000. The sgRNA-targeted site is depicted in yellow. GFP expressing cells were sorted with flow cytometry. Genomic DNA was extracted. After bisulfite treatment, the 400 bp promoter region was amplified and cloned into sequencing vector. Four out of eight colonies (50%) showed some degree of targeted methylation (red marks) adjacent to the Cas9x binding site.
    
    
    
    Conclusion:
    These results suggest that one can use de-activated Cas9 to direct methyltransferases to specific sites within the genome and regulate gene expression. This has not previously been reported, and may represent a significant advance in the ability to methylate and regulate specific target sites in the cancer genome on a heritable basis.