Modified sgRNA Scaffold for Targeted Gene Demethylation
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for gene demethylation and activation are non-specific, leading to unintended effects and high toxicity, and existing gene editing technologies like Crispr/Cas9 do not effectively address gene deactivation by methylation.
Innovation Solution
Development of oligonucleotide constructs with a targeting portion and a modified single guide RNA (sgRNA) scaffold that inhibits DNA methyltransferase 1 (DNMT1) activity using deactivated Cas9 (dCas9), specifically targeting methylated genomic regions to achieve targeted demethylation and activation of genes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If broad demethylating agents (azacitidine, decitabine) are used to treat hypermethylation-associated diseases, then demethylation effect is achieved, but specificity for genetic loci is lost and high toxicity occurs
Solution Approach 1:
The invention segments the demethylation effect to target specific genetic loci rather than applying broad demethylation across the genome. The CRISPR-dCas9 system with DiR components is directed to specific genomic regions through guide RNA, achieving localized demethylation at hypermethylated genes while leaving other regions unaffected, thereby reducing systemic toxicity.
Solution Approach 2:
The invention applies demethylation activity locally at specific hypermethylated genomic loci rather than uniformly across the genome. The CRISPR-dCas9-DiR complex is recruited to specific locations through sequence-specific guide RNA binding, creating localized demethylation zones that restore gene expression at target sites without affecting other genomic regions, thus reducing off-target effects and toxicity.
2Quantity of substance
If traditional non-specific demethylating agents are used, then demethylation is achieved, but unintended or undesirable effects are created
Solution Approach 1:
The invention divides the demethylation process into targeted segments using CRISPR guide RNA to direct the dCas9-DiR complex to specific hypermethylated loci. This segmentation ensures that demethylation occurs only at intended target sites with specific sequences, eliminating the non-specific demethylation and unintended effects characteristic of traditional agents.
Solution Approach 2:
The invention introduces guide RNA as an intermediary that mediates between the CRISPR-dCas9-DiR system and the target genomic DNA. The guide RNA provides sequence-specific recognition and binding to hypermethylated regions, acting as a mediator that directs demethylation activity precisely to intended targets while preventing off-target demethylation and undesirable effects.
3Measurement precision
If CRispr/Cas9 is used as a gene editing system, then sequence-specific targeting is achieved, but gene deactivation by methylation is not addressed
Solution Approach 1:
The invention creates a multi-functional CRISPR system where dCas9 serves multiple purposes: it maintains the sequence-specific targeting capability of traditional CRISPR/Cas9 while additionally recruiting DiR components to perform demethylation. This universal platform combines precise targeting with epigenetic modification functionality, allowing the same system to both locate and actively modify hypermethylated regions.
Solution Approach 2:
The invention merges the CRISPR-dCas9 targeting system with the DiR demethylation component into a single functional complex. The dCas9 provides precise genomic localization through guide RNA binding, while the attached DiR components perform the actual demethylation chemistry at the targeted site, combining targeting precision with demethylation capability in one integrated system.
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
This approach provides a more natural and specific demethylation effect, leading to increased gene expression and is maintained over extended periods, with improved safety and efficacy compared to traditional methods.
Implementation Method 1
a targeting portion having sequence complementarity and binding affinity with a region of genomic DNA within a gene, near a gene, or both
Data Source
AI summary
Provided herein are methods and agents for gene specific demethylation and/or activation. Oligonucleotide constructs are provided, the oligonucleotide constructs including: [1] a targeting portion having sequence complementarity and binding affinity with a region of genomic DNA within a gene, near a gene, or both; and [2] a single guide RNA (sgRNA) scaffold portion, wherein a tetra-loop portion of the sgRNA is modified and includes an R2 stem loop of DNMT1-interacting RNA (DiR), and wherein a stem loop 2 portion of the sgRNA is modified and includes an R5 step loop of DiR. The oligonucleotide constructs may be used, together with deactivated (dead) Cas9 (dCas9) for providing gene specific demethylation and/or activation of gene(s) of interest in a cell or subject in need thereof.