Future Directions
Expanding Our Epigenetic Toolkit
Our work with targeted epigenetic modifications represents just the beginning of what's possible with programmable DNA methylation systems. From optimizing mammalian cell lines for biopharmaceutical production to creating novel memory circuits and antiviral defense systems, we are exploring diverse applications that could revolutionize biotechnology and agriculture.
Part 1: CHO Cells
The CHO-K1 System Challenge
Chinese Hamster Ovary (CHO) cells, especially the K1 variant, are the workhorses of the biopharmaceutical industry. They are used to produce the majority of therapeutic antibodies and complex biologics because they are robust, easy to adapt to suspension culture, and capable of adding human-like glycosylation patterns to proteins. The cell line we are working with is based on a specially engineered version of these CHO-K1 cells, where the dihydrofolate reductase (DHFR) gene has been knocked out. Without DHFR, cells cannot make nucleotides efficiently and will die unless they receive a functional copy of the gene. This feature turns DHFR into a powerful "selection marker" for ensuring that only the cells carrying our gene cassette survive.
RMCE and MTX Selection
To insert therapeutic genes into the genome in a controlled way, our system uses Recombinase-Mediated Cassette Exchange (RMCE). RMCE ensures that the gene of interest, for example, an antibody like dupilumab, is integrated into a specific, pre-characterized genomic site, rather than randomly. This avoids position effects and makes expression more predictable. Importantly, the cassette also includes DHFR alongside the therapeutic gene, linking the two.
The system also leverages methotrexate (MTX) selection. MTX inhibits DHFR, creating pressure on the cell to survive by amplifying the chromosomal region that contains the DHFR gene. Because our therapeutic gene sits right next to DHFR, it also gets amplified. This leads to higher protein expression which is useful in industrial applications.
The Efficiency Problem
However, this approach has a drawback. Alongside our gene of interest, DHFR itself becomes highly overexpressed. This creates unnecessary metabolic load - the cells divert energy and resources into producing vast amounts of DHFR protein, which they don't need in such abundance. This can stress the cells, reduce folding and secretion efficiency of the therapeutic protein, and in some cases lower the stability of the production system. In other words, the system is not perfectly efficient because a significant part of the cell's production capacity is wasted on DHFR.
Our Epigenetic Solution
To solve this we propose a tunable epigenetic silencing system to selectively reduce DHFR expression without compromising the expression of the gene of interest. Using a CRISPR-based epigenetic tool (dCas9 fused to a DNA methyltransferase, under control of a Tet-On promoter), we can direct methylation to specific sites in the CMV core promoter driving DHFR, and dynamically reduce its expression. Because the system is inducible, we can finely control the timing and extent of silencing with doxycycline, avoiding premature shutdown of DHFR during selection but reducing unnecessary overexpression afterward. The result is a more efficient, balanced production system, where the therapeutic gene stays amplified and active, but the metabolic burden from excess DHFR is relieved.
Experimental Design and Workflow
Validation Strategy
To validate our epigenetic programmable control system in mammalian cells, we are working with CHO-K1 Dupilumab-producing cells, a DHFR knockout cell line that uses the DHFR–MTX amplification system for high-level recombinant protein production. Our goal is to establish whether using a Tet-On inducible dCas9–DNMT3A system can selectively downregulate DHFR expression post-amplification by inducing methyl marks at certain sites in the CMV core promoter that drives expression of the DHFR gene, thereby reducing cellular stress while maintaining high levels of therapeutic protein output.
1. Establishing and Maintaining Cell Cultures
We began by subculturing the CHO-K1 Dupilumab cells under standard suspension culture conditions. Maintaining optimal cell viability and morphology is crucial to ensure consistent experimental results during downstream transfection and gene expression assays.
2. Baseline Methylation Profiling
Before any intervention, we aim to characterize the methylation status of the CMV core promoter driving DHFR expression. Using bisulfite treatment followed by PCR amplification and Sanger sequencing, we will determine whether the target CpG sites are initially unmethylated. Housekeeping genes will serve as internal controls to confirm the accuracy and efficiency of the bisulfite conversion process. This step establishes the baseline for subsequent methylation changes induced by our system.
3. Transfection Strategy
Following baseline analysis, cells will be transfected under four different conditions to decouple the effects of each component:
- Untransfected control
- Tet-On-dCas9-DNMT3A plasmid only
- sgRNA vector only
- Co-transfection of Tet-On-dCas9-DNMT3A and sgRNA vectors
The Tet-On construct allows doxycycline-dependent expression of the dCas9–DNMT3A fusion protein, enabling tunable activation of the methylation system.
4. Verification of Transfection
Successful transfection will be confirmed through Fluorescence-Activated Cell Sorting (FACS) based on the reporter markers in the transfected plasmids. This ensures the selection of successfully transformed cells.
5. Post-Transfection Methylation Analysis
Once transfection is verified, bisulfite PCR and sequencing will be used again to detect newly acquired methylation marks at the CMV promoter of the DHFR gene. Comparing methylation levels before and after doxycycline induction will provide direct evidence of targeted, titratable methylation.
6. Gene Expression Quantification
To evaluate the functional outcome of methylation, quantitative RT-PCR (qRT-PCR) will be performed to measure DHFR mRNA expression levels across the experimental conditions. We expect the system to show reduced DHFR expression only in the induced co-transfected cells, confirming selective transcriptional silencing.
7. Assessing Protein Production Efficiency
Finally, we will assess whether lowering DHFR expression improves the cell's resource allocation toward recombinant protein synthesis. The expression and secretion of Dupilumab will be quantified using protein assays such as ELISA. Consistent or enhanced Dupilumab production, coupled with reduced DHFR levels, would indicate a more metabolically efficient production system.
Current Progress
So far, we have subcultured the CHO-K1 Dupilumab cells and received primers for bisulfite sequencing. The Tet-On-dCas9-DNMT3A plasmid has been ordered, while the sgRNA vector has been designed. These tools will form the backbone of our experimental validation pipeline as we proceed to the transfection and analysis phases.
Part 2: Epigenetic Memory System
Concept and Design
An epigenetic memory system allows cells to retain information about past stimuli without altering their DNA sequence. Unlike permanent genetic mutations, which irreversibly change the genetic code, epigenetic modifications act as reversible molecular "bookmarks" that can be passed on through successive divisions. Using this principle, a synthetic genetic circuit can be engineered to establish stable, heritable memory with the dCas9–Dam methylation toolkit.
Three-Module Architecture
The design consists of three functional modules:
- Trigger module: A stimulus-sensitive, tightly regulated inducible promoter driving expression of a dCas9–Dam fusion and its guide RNA. This ensures that methylation is initiated only when an external signal is present.
- Memory module: A custom promoter targeted by the guide RNA from the trigger module, controlling both a reporter gene and another copy of dCas9–Dam with its guide RNA directed back to the same promoter. This creates the basis for a positive feedback loop.
- Zinc finger repressor: A methylation-sensitive zinc finger protein engineered to bind the unmethylated promoter and strongly repress transcription in the absence of methylation.
Mechanism of Action
In the uninduced state, the zinc finger protein remains bound to the promoter, keeping the memory module silent. When a stimulus activates the trigger module, dCas9–Dam methylates the promoter, displacing the zinc finger and allowing transcription of both the reporter and the additional dCas9–Dam. This is the critical point: as soon as the memory module begins expressing dCas9–Dam, the promoter is further methylated by its own product. The result is a self-reinforcing positive feedback loop that locks the system into an "ON" state. Once established, the methylation pattern and continued dCas9–Dam expression ensure that the memory is stably propagated across generations, even after the original signal disappears.
Advantages Over Previous Systems
Earlier work has demonstrated the feasibility of such designs using non-specific methyltransferases. However, a dCas9–based fusion offers higher precision, minimizes off-target methylation, and lowers the threshold of stimulus required to trigger the system, greatly improving both robustness and efficiency.
This system could potentially be used in large scale industrial protein production, where induction of regulated expression systems is expensive and sometimes difficult to control. In the systems developed here, inducers like IPTG would be required only transiently to activate the expression of the gene of interest.
Applications in Biomanufacturing
Gene Regulation in Biomanufacturing
In the microbial production of various compounds, by-products produced in excess often reduce the efficiency of the whole system. The genes responsible for the production of these by-products cannot always be knocked out as they could be essential for the survival of the chassis. In this case, epigenetic silencing can be used to reduce the activity of these genes to a bare minimum.
GABA Production Example
An example of such a system would be microbes producing GABA (Gamma-Aminobutyric Acid), a non-protein amino acid with immense applications in neuropharmacology and other industries owing to its functions as a neurotransmitter. They produce byproducts like acetate as well which lead to increased downstreaming costs. Knocking out (removing) acetate-producing genes isn't an option, as acetate is essential for microbial survival. Instead of removing the gene, epigenetic silencing can be used to reduce acetate production to a minimal level while still keeping the cell alive, improving the efficiency of GABA production.
Mimicking DNA Methylation Patterns
Researchers have developed a systematic approach to overcome restriction-modification barriers in difficult-to-transform bacterial strains by mimicking their native methylation patterns. Cell free systems are used with the right methyltransferases to methylate the DNA to be transformed, before transforming it into the host cells. This process can be optimized and more streamlined if targeted methylation is used, which is where our system would come in.
This systematic approach overcomes restriction–modification barriers in otherwise difficult-to-transform bacterial strains by replicating their native methylation patterns. In this strategy, cell-free systems are employed with the appropriate methyltransferases to methylate the DNA prior to transformation into host cells. This process can be further optimized and streamlined through the use of targeted methylation, which is precisely where our system would provide an advantage.
Secondary Metabolite Production
DNA methylation patterns affect the expression of biosynthetic gene clusters responsible for the production of certain secondary metabolites, and modulating these patterns can activate "silent" pathways to produce novel compounds or increase yields of existing products.
DNA hypermethylation has been known to silence biosynthetic pathways that produce valuable secondary metabolites in fungi. These pathways can be re-activated by making use of targeted de-methylation using a dCAS9 based fusion protein utilising de-methylating enzymes like TET.
Plant Viral Defense Systems
dCas9-Based Epigenetic Silencing for Plant Viral Defense
The dCas9 system can be engineered to silence virulence factors in plant pathogens through targeted epigenetic modification.
Biological Precedent: The Natural RdDM System
Transgenic deployment of dCas9-based antiviral systems is biologically validated by existing plant defense mechanisms, particularly the AGO4/DCL3-mediated RNA-directed DNA methylation (RdDM) pathway. This endogenous system combats geminiviruses which cause billions of dollars in global crop losses annually by methylating viral minichromosomes and establishing transcriptional gene silencing. The natural precedent demonstrates that epigenetic silencing of viral DNA is feasible for plant protection.
Overcoming Natural Defense Limitations
Many viral pathogens have evolved sophisticated suppressor proteins that disable plant RdDM machinery. The dCas9 system could circumvent these vulnerabilities by functioning independently of the multi-step siRNA biogenesis and AGO4-dependent targeting pathways that viruses commonly disrupt.
Technical Advantages of Synthetic Approach
The dCas9 platform could offer several key improvements over natural systems: direct targeting via programmable guide RNAs eliminates dependence on complex RNA processing machinery; rapid response enables immediate binding and silencing upon guide RNA expression; multiple viral genes can be targeted simultaneously and adaptability permits rapid retargeting against emerging viral strains by simply modifying guide RNA sequences. This synthetic biology approach essentially provides plants with a more robust, flexible, and suppressor-resistant antiviral system that complements existing natural defenses.
Future Works
Memory System Implementation
Our Vision
We plan to construct the above mentioned memory system in a prokaryotic host as a future project.
DNA Parts to be Designed
Trigger Module: Inducible promoter (for example, pLac or pTet) driving expression upon stimulus upstream of a dCas9-Dam gene and sgRNA expression cassette targeting the memory module promoter.
Memory Module: Custom promoter engineered with sgRNA binding sites and Zinc Finger binding sites coinciding with each other, upstream of the eGFP reporter gene and another dCAS9-Dam gene along with its sGRNA cassette.
Zinc Finger Repressor Module: Zinc finger(ZnF) protein gene designed to bind the unmethylated promoter, present downstream of a promoter that has ZnF binding site of its own, thus providing autoregulation for the ZnF.
System Components Table
| Module | Part | Function |
|---|---|---|
| Trigger module | Inducible promoter (pLac/pTet) | Activates the system in response to external stimulus |
| dCas9–Dam fusion gene | Methylates target promoter with high precision | |
| sgRNA cassette | Directs dCas9–Dam to the custom promoter | |
| Memory module | Custom synthetic promoter | Acts as the memory element, target of dCas9–Dam |
| Reporter gene (GFP) | Provides a measurable readout of the memory state | |
| Self-amplifying dCas9–Dam cassette | Creates positive feedback to maintain promoter methylation | |
| Zinc finger repressor | Zinc finger protein gene | Binds and represses unmethylated promoter |
| Auto regulatory promoter | Ensures constant zinc finger repressor expression |