Modular CRISPR Epigenetic Editing With Linker-Spaced Effector Arrays

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Solution Overview

Problem

Current epigenetic editing tools struggle to precisely and systematically manipulate chromatin modifications at specific loci, limiting the understanding and therapeutic applications of chromatin function in biological processes and diseases.

Innovation Solution

A catalytically inactive site-specific nuclease, such as dCas9, linked to an array of effector domains with chromatin-modifying activities, separated by linkers to avoid interference, allows for precise and combinatorial epigenetic editing, including histone methylation, acetylation, and demethylation, facilitated by a modular CRISPR-based toolkit.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If multiple effector domains are fused directly to the nuclease, then the chromatin modifying activities are enhanced, but the specific chromatin modifying activities are substantially interfered with due to steric hindrance

Engineering Contradiction:

Improvechromatin modifying activityVSAvoidspecific chromatin modifying activity

Core Design Contradiction:

ProductivityVSManufacturing precision

Solution Approach 1:

The effector domains are segmented and arranged in an array format rather than being directly fused to the nuclease. This segmentation allows each effector domain to maintain its specific chromatin modifying activity while collectively enhancing the overall productivity of the complex.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

Linker sequences serve as intermediaries between the nuclease and the effector domains. These linkers provide sufficient distance and flexibility to prevent steric hindrance, allowing the effector domains to function independently while being positioned effectively by the nuclease.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Device complexity

If effector domains are positioned close to the nuclease, then the complex size is reduced, but the chromatin modifying activities are interfered with

Engineering Contradiction:

Improvecomplex structureVSAvoidchromatin modifying activity

Core Design Contradiction:

Device complexityVSProductivity

Solution Approach 1:

The linker sequences provide dynamic flexibility, allowing the effector domains to move and adjust their positions relative to the nuclease. This dynamic arrangement ensures that the domains can access their substrates effectively while maintaining a compact overall structure when not in use.

Inventive Principle:

Principle #15Dynamics

Data Source

PatentUS20250339557A1Crispr-based modular tool for the specific introduction of epigenetic modifications at target loci

Publication Date: 2025.11.06 EURO LAB FUER MOLEKULARBIOLOGIE EMBL

AI summary

The present invention relates to a complex comprising i) a catalytically inactive site-specific nuclease linked to ii) an array of between two and ten, preferably three to seven effector domains each having a specific chromatin modifying activity, such as, for example, a specific DNA methylation activity, a histone methylation activity, a specific histone acetylation or ubiquitination activity, and/or a specific chromatin demethylation/deacetylation activity, wherein the effector domains are each separated by a linker providing sufficient distance between the domains and the nuclease in order not to substantially interfere with their specific chromatin modifying activities, and the binding of the site-specific nuclease, as well as respective methods involving the complex and use of the complex.