Targeted DNA Methylation via CRISPR-PUF Hybrid System
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Solution Overview
Problem
Current methods for modulating DNA methylation status in target cells result in non-specific global changes, making it challenging to achieve targeted conversion of 5-methylcytosine to unmethylated cytosine, which is essential for understanding cytosine methylation biology and developing therapies for related diseases.
Innovation Solution
A demethylation or methylation complex comprising a ribonucleoprotein complex with a nuclease-deficient RNA-guided DNA endonuclease enzyme and a polynucleotide with a DNA-targeting sequence, combined with a PUF domain conjugate, allows for targeted delivery of demethylation or methylation activity to specific genomic loci using a three-component hybrid system that includes CRISPR/Cas9 and Pumilio proteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If DNA demethylase or methyltransferase is introduced to modulate methylation status, then methylation modulation capability is improved, but specificity deteriorates resulting in non-specific global changes
Solution Approach 1:
The system segments the methylation modulation function into two independent components: (1) a targeting module using CRISPR/dCas9-sgRNA complex that directs localization to specific genomic loci, and (2) a effector module using PUF domain fused to demethylase or methyltransferase that performs the actual methylation modification. This segmentation allows the system to achieve both versatility in methylation modulation and precision in target localization, resolving the contradiction between capability and specificity.
Solution Approach 2:
The patent introduces an intermediary PUF domain that acts as a bridge between the CRISPR targeting system and the methylation effector enzymes. The PUF domain binds to the sgRNA and recruits the fused demethylase or methyltransferase to the target site, enabling precise spatial control of methylation activity while maintaining the versatility of the effector enzymes. This intermediary mechanism resolves the contradiction by decoupling targeting from enzymatic activity.
2Adaptability or versatility
If multiple effector domains are fused to dCas9, then functional versatility is improved, but device complexity increases due to large protein size
Solution Approach 1:
The system divides the effector functionality into separate modules: the CRISPR/dCas9-sgRNA complex handles targeting, while the PUF domain fused to the effector enzyme (demethylase or methyltransferase) handles the biochemical function. This segmentation avoids the need to fuse multiple large effector domains to dCas9, thereby reducing protein size and complexity while maintaining functional versatility through modular assembly of different PUF-effector combinations.
Solution Approach 2:
The PUF domain serves as a universal adaptor that can be fused to different effector enzymes (demethylases, methyltransferases) to achieve multiple functions. Rather than creating separate multi-functional fusions with dCas9, the system uses the universal PUF domain to recruit different effectors, simplifying the overall protein architecture while maintaining versatility.
Data Source
AI summary
Provided herein are, inter alia, compositions and methods for demethylating and methylating a target DNA sequences in a mammalian cell. The compositions and methods are, inter alia, useful for modulating the expression of a target gene, or to create a gene regulatory network.