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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session D

Poster I4 - Programmable DNA methylation and tunable transcriptional silencing using engineered CRISPR/Cas9-based DNA methyltransferase variants

Friday, October 25, 2024
3:30 PM - 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction:  DNA methylation can naturally regulate transcription in cells by depositing methyl marks at CpG islands. However, abnormal DNA methylation patterns have been linked to a wide range of both inherited and de novo diseases. Interestingly, aberrant DNA methylation has also been linked to immune cell dysfunction upon chronic immunological stimulation, ultimately leading to “epigenetic scarring” and dampened immunity. Despite this importance, it remains unclear how DNA methylation drives immune cell dysfunction.
The goal of this project is to engineer programmable dCas9-based DNA methyltransferases (DNMTs) to quantify the role of DNA methylation in human cells. In the longer term these technologies could be used to better understand and control primary human immune cell functions. To this end, we designed three different rationally engineered DNMT3A-3L variants using point mutations intended to minimize off-target activity, improve protein stability, and increase methyltransferase activity. We directly fused those variants to dCas9 and targeted them to a synthetic reporter locus to evaluate their methyltransferase and gene silencing activity in HEK293T cells. Their efficacy was compared to that of wild-type (WT) DNMT3A-3L, and the data was then used to characterize the most effective tool between our engineered variants and WT DNMT3A-3L. The most effective engineered methyltransferase was then targeted to IFNGR1, an immune regulatory gene, in HEK293T cells.

Materials and

Methods:  After deciding on the point mutations to introduce, the DNMT3A-3L variants were designed in silico and cloned into a plasmid downstream from an EFS promoter. There were 6 methyltransferase plasmids: one negative control including dCas9 not fused to DNMT, one control with dCas9 fused to inactive DNMT, WT DNMT, and the three rationally engineered variants. A stable HEK39T cell line expressing eGFP was then established, and that line was co-transfected with a dCas9-DNMT encoding plasmid and gRNA targeted to the eGFP synthetic reporter locus. Two days post-transfection, FACS was used to sort cells that displayed mCherry fluorescence. The level of eGFP fluorescence and silencing was used to assess the gene silencing efficacy of the engineered constructs. Bisulfite conversion was used to determine the methyltransferase activity of each construct.
The construct with the greatest silencing activity was then targeted to IFNGR1, a immune regulatory gene, in WT HEK293T cells. qPCR was used to quantify IFNGR1 expression in cells with the most effective engineered methyltransferase v. WT DNMT3A-3L.

Results, Conclusions, and Discussions: Our data indicated that engineered variant V2 was the most effective engineered variant at both methylating and silencing target genes. V2 more effectively deposits methyl marks at synthetic loci than wild type DNMT3A-3L, which was demonstrated through lower eGFP fluorescence and increased methylation, as indicated by bisulfite sequencing. Notably, dCas9 fused to inactive DNMT3A-3L did not produce significant eGFP silencing, indicating that the methyltransferase activity, and not steric hindrance, is the cause of gene silencing.
Our data also indicates that, under transient transfection conditions, the most methylation is deposited 6 days after initial transfection, and all methyl marks disappear 12 days post-transfection. This led us to conclude that there is endogenous machinery that removes methyl marks soon after they are deposited by exogenous machinery, preventing the endurance of methyl markers in the absence of the targeted methyltransferase.
Engineered variant V2 also silenced IFNGR1 more effectively than WT DNMT3A-3L in HEK293T cells. This could allow for epigenetic immune engineering and the development of an in vitro immune exhaustion model that replicates patient symptoms— allowing for the development of better treatments. We plan to further investigate this potential by testing the epigenetic engineering potential of V2 in primary human T cells, and how its methyltransferase and gene silencing activity impact T-cell activation.

Acknowledgements (Optional):  I would like to thank Dr. Isaac Hilton for all his mentorship and support of this project. I would also like to thank my bench mentor, Dr. Rosa Guerra-Resendez, for her hard work and enthusiasm in teaching me both lab and life skills. Finally, thank you to all of my coauthors, the members of the Hilton lab, and the bioengineering community at Rice for their scientific and personal support.