CRISPR Guide RNA Targeting CTCF Boundaries
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Solution Overview
Problem
Current methods lack understanding and effective regulation of gene expression in insulated neighborhoods of the genome, particularly in embryonic stem cells, where cohesin-associated chromosome structures play a crucial role in maintaining pluripotency and cell identity, but the integrity of these structures is not well understood.
Innovation Solution
The use of gene modulatory molecules, such as small molecules, proteins, peptides, and nucleic acids, to alter the expression of genes within insulated neighborhoods by targeting CTCF boundaries, specifically using CRISPR technology to modify CTCF sites within topologically active domains and super-enhancer domains.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If current methods are used to regulate gene expression, then general gene targets can be accessed, but precise regulation within insulated neighborhoods and CTCF boundaries cannot be achieved
Solution Approach 1:
The patent applies local quality by designing guide RNA molecules with specific sequences that target only particular CTCF boundary regions within insulated neighborhoods. This allows the CRISPR-Cas9 system to exert precise local effects on gene expression at specific genomic locations without affecting other regions, thereby achieving precise regulation within insulated neighborhoods while maintaining the ability to target different locations through sequence-specific guide RNAs.
Solution Approach 2:
The patent segments the genome into functional insulated neighborhoods bounded by CTCF sites, and further segments these into addressable regions using guide RNA molecules. Each guide RNA targets a specific segment (CTCF boundary region), allowing independent regulation of individual insulated neighborhoods or sub-regions within them, enabling precise control of gene expression at multiple discrete locations simultaneously.
2Manufacturing precision
If CRISPR technology is used to modify CTCF sites, then precise regulation of gene expression within insulated neighborhoods is achieved, but complexity of the method increases
Solution Approach 1:
The patent introduces guide RNA molecules as intermediary components that mediate between the CRISPR-Cas9 system and the target CTCF boundary regions. The guide RNA serves as a programmable intermediary that recognizes specific DNA sequences through base pairing, directing the Cas9 enzyme to precise locations. This intermediary layer simplifies the overall system by providing sequence-specific targeting capability without requiring complex protein-protein interactions or multiple components at each target site.
Data Source
AI summary
Provided herein are improved compositions and methods for the directed control of gene expression.