Plant DNMT3 Fusion Proteins for Targeted CHH and CpG Methylation
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Solution Overview
Problem
The role of plant DNA methyltransferase 3 (DNMT3) orthologs in DNA methylation has remained elusive, particularly in angiosperms, and their potential applications in non-plant systems like mammals have not been explored.
Innovation Solution
Development of a fusion protein comprising a DNA targeting moiety linked to a catalytic domain of a plant DNMT3 protein, specifically utilizing a DNA endonuclease protein like Cas9, optimized for expression in non-gymnosperm and non-bryophyte organisms, which enhances methylation at CHH and CpG sites.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If plant DNMT3 is used in angiosperms, then de novo methylation can be achieved, but the function remains elusive and uninvestigated
Solution Approach 1:
The patent uses a fusion protein approach where plant DNMT3 is combined with a DNA targeting moiety (such as dCas9) to create an intermediary system that can deliver methylation activity to specific genomic locations. This mediator system enables functional investigation of plant DNMT3 in angiosperms where it was previously uninvestigated, resolving the contradiction between having the enzyme and lacking functional knowledge.
2Adaptability or versatility
If DNMT3 is expressed in mammalian cells, then methylation activity can be demonstrated, but the enzyme is plant-specific and may not function naturally in mammals
Solution Approach 1:
The patent creates a universal methylation system by fusing plant DNMT3 with a DNA targeting moiety that can function in both plant and mammalian cells. The catalytic domain of plant DNMT3 retains its methyltransferase activity when expressed in mammalian cells, demonstrating cross-species functionality while maintaining reliable enzymatic function through the conserved catalytic domain.
3Measurement precision
If fusion protein with DNA targeting moiety is created, then precise methylation at specific sites is achieved, but the device complexity increases
Solution Approach 1:
The patent segments the methylation system into two functional parts: a DNA targeting moiety (such as dCas9) that provides site-specificity and a catalytic domain of plant DNMT3 that provides methylation activity. This segmentation allows each component to perform its specific function independently while working together to achieve precise methylation at desired genomic sites.
4Productivity
If plant DNMT3 is used instead of human DNMT3, then higher methylation efficiency at CHH and CpG sites is achieved, but the enzyme is optimized for plant genomes
Solution Approach 1:
The patent changes the parameters of the methylation system by using plant DNMT3 instead of human DNMT3, taking advantage of the higher methylation efficiency of plant DNMT3 at CHH and CpG sites. The fusion protein approach allows this parameter change while maintaining genome compatibility through the DNA targeting moiety that can guide the enzyme to specific locations in both plant and mammalian genomes.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The fusion protein effectively increases DNA methylation at CHH and CpG sites in mammalian and plant cells, demonstrating higher methylation efficiency compared to human DNMT3, and regulates gene expression.
Implementation Method 1
DNA methylation, the addition of a methyl group to a cytosine base, is a prominent epigenetic modification in many eukaryotes. It is catalyzed by distinct DNA methyltransferase (DNMT) families of proteins that share a conserved methyl-transferase domain (MTD).
Implementation Method 2
The fusion protein effectively increases DNA methylation at CHH and CpG sites in mammalian and plant cells, demonstrating higher methylation efficiency compared to human DNMT3
Data Source
AI summary
An isolated polynucleotide encoding a fusion protein which comprises a DNA targeting moiety linked to a catalytic domain of a plant DNA methyltransferase 3 (DNMT3) protein is disclosed. Uses thereof are also disclosed.