dCas9-TET1 Fusion Protein for Site-Specific DNA Demethylation

Resolve Bottlenecks,
Find Innovative SolutionsGenerate Solutions

Solution Overview

Problem

Current methods for regulating DNA methylation, such as using 5-azacytosine for demethylation, are not site-specific and raise safety concerns, while previous genome editing technologies like TALEN are time-consuming and have low demethylation efficiency.

Innovation Solution

A DNA methylation editing kit and method utilizing a CRISPR/Cas system with a fusion protein of inactivated Cas9, a tag peptide array, and guide RNA to specifically target and regulate methylation or demethylation at particular sites using a catalytic domain of TET1 or DNA methyltransferase 3 beta.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If 5-azacytosine is used for demethylation treatment, then demethylation effect is achieved, but site-specificity is lost and safety concerns arise due to whole-genome effects

Engineering Contradiction:

Improvesite-specificityVSAvoidsafety concerns

Core Design Contradiction:

Measurement precisionVSObject-affected harmful factors

Solution Approach 1:

The patent applies local quality by using CRISPR/Cas9 system with guide RNA to target specific DNA sequences, enabling methylation regulation at precise locations rather than genome-wide. The dCas9-TET1 fusion protein delivers demethylation activity only to the targeted site, achieving site-specific epigenetic editing without affecting other genes.

Inventive Principle:

Principle #3Local quality

Solution Approach 2:

The patent uses dCas9 as an intermediary protein that binds to target DNA sequences via guide RNA and recruits TET1 enzyme to perform demethylation. This intermediary mechanism enables precise delivery of demethylation activity to specific sites without direct chemical modification of the entire genome.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Measurement precision

If TALEN fusion protein is used for demethylation, then site-specific demethylation is achieved, but the process becomes time-consuming and demethylation efficiency is low

Engineering Contradiction:

Improvesite-specificityVSAvoiddemethylation efficiency

Core Design Contradiction:

Measurement precisionVSProductivity

Solution Approach 1:

The patent replaces the TALEN-based mechanical genome editing system with the CRISPR/Cas9 system, which uses RNA-guided DNA binding instead of protein-DNA recognition. This substitution enables faster and more efficient targeting and demethylation at specific sites.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Solution Approach 2:

The patent changes the fundamental mechanism from TALEN protein fusion to CRISPR RNA-guided endonuclease system, altering the key parameters of target recognition and binding speed, thereby improving demethylation efficiency and reducing processing time.

Inventive Principle:

Principle #35Parameter changes

3Productivity

If CRISPR/Cas system is used for methylation regulation, then speed and efficiency are improved, but the complexity of the system increases

Engineering Contradiction:

Improveediting speedVSAvoidsystem complexity

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The patent merges the dCas9 protein with TET1 enzyme into a single fusion protein, combining the targeting function with the demethylation function in one component. This integration simplifies the overall system by reducing the number of separate elements that need to be coordinated.

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The dCas9-TET1 fusion protein serves multiple functions: it acts as a targeting vehicle via guide RNA binding, a delivery mechanism for TET1 enzyme, and the demethylation catalyst itself. This multi-functionality reduces system complexity despite using CRISPR/Cas technology.

Inventive Principle:

Principle #6Universality (Multi-functionality)

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

Enables precise regulation of DNA methylation, achieving high efficiency in demethylation and methylation at specific sites with reduced off-target effects and improved speed compared to previous technologies.

Implementation Method 1

a guide RNA(s) (gRNA(s)) comprising a sequence complementary to a DNA sequence within 1 kb of a desired site of methylation or demethylation

Methodology Applied

Scientific EffectBase pairing complementarity:

Implementation Method 2

a fusion protein of inactivated CRISPR-associated endonuclease Cas9 (dCas9) having no nuclease activity and a tag peptide array

Methodology Applied

Scientific EffectSequence-specific recognition:

Implementation Method 3

the demethylase is a catalytic domain (TET1CD) of ten-eleven translocation 1

Methodology Applied

Scientific EffectOxidative demethylation:

Implementation Method 4

the methylase is DNA methyltransferase 3 beta (DNMT3B)

Methodology Applied

Scientific EffectMethyl group transfer:

Data Source

PatentUS11591623B2DNA methylation editing kit and DNA methylation editing method

Publication Date: 2023.02.28 GUNMA UNIVERSITY

AI summary

A DNA methylation editing kit comprises: (1) a fusion protein of inactivated CRISPR-associated endonuclease Cas9 (dCas9) having no nuclease activity and a tag peptide array in which plural tag peptides are linked by linkers, or an RNA or DNA coding therefor; (2) a fusion protein(s) of a tag peptide-binding portion and a methylase or demethylase, or an RNA(s) or DNA(s) coding therefor; and (3) a guide RNA(s) (gRNA(s)) comprising a sequence complementary to a DNA sequence within 1 kb of a desired site of methylation or demethylation, or a DNA(s) expressing the gRNA(s).