MRE11-Mediated Off-Target Detection in CRISPR Therapies

Resolve Bottlenecks,
Find Innovative SolutionsGenerate Solutions

Solution Overview

Problem

Current methods for detecting off-target genome editing, particularly with CRISPR-Cas systems, suffer from low sensitivity and are limited to purified DNA or restricted cellular systems, making it difficult to accurately identify off-target mutagenesis in vivo, which is a concern for therapeutic applications.

Innovation Solution

A method involving the use of a gene-editing complex with a guide RNA and an inhibitor of DNA repair, such as DNA-PKcs inhibitors, to modulate MRE11 localization, allowing for the detection of off-target sites through chromatin immunoprecipitation with sequencing (ChIP-seq) and quantitative PCR, enhancing the sensitivity of off-target detection in both in vitro and in vivo settings.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If current off-target discovery methods are used, then detection can be performed on purified DNA or restricted cellular systems, but sensitivity is low and in vivo detection is not possible

Engineering Contradiction:

Improveoff-target detection sensitivityVSAvoidapplicability to in vivo systems

Core Design Contradiction:

Measurement precisionVSAdaptability or versatility

Solution Approach 1:

The patent introduces MRE11 protein as an intermediary marker that binds to off-target cleavage sites. By using MRE11 as a mediator, the method enables detection of off-target events in complex in vivo systems where direct detection would be impossible. The MRE11 binding serves as a detectable signal that bridges the gap between the actual off-target cleavage event and the sequencing detection system.

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent changes the detection parameter from direct sequencing of off-target mutations to sequencing of MRE11-bound DNA fragments. This parameter change enables detection in in vivo systems because MRE11 provides a consistent, detectable signal at off-target sites that can be isolated and sequenced, overcoming the limitations of direct mutation detection in complex genomic backgrounds.

Inventive Principle:

Principle #35Parameter changes

2Measurement precision

If DNA repair is not inhibited, then normal cellular processes continue, but MRE11 localization at target sequences is reduced, lowering detection sensitivity

Engineering Contradiction:

ImproveMRE11 enrichment at target sitesVSAvoidDNA repair activity

Core Design Contradiction:

Measurement precisionVSObject-generated harmful factors

Solution Approach 1:

The patent converts the potentially harmful effect of DNA repair (which removes MRE11 from target sites) into a beneficial detection signal. By inhibiting DNA repair, MRE11 accumulates at off-target sites, creating a stronger signal for detection. The harmful DNA repair activity is transformed into a useful marker enrichment that enhances off-target detection sensitivity.

Inventive Principle:

Principle #22Blessing in disguise (Convert harm into benefit)

Solution Approach 2:

The patent applies preliminary anti-action by inhibiting DNA repair pathways before MRE11 can be removed from target sites. This preventive measure ensures that MRE11 remains bound to off-target cleavage sites long enough to be detected and sequenced, counteracting the natural DNA repair processes that would otherwise eliminate the detection signal.

Inventive Principle:

Principle #9Preliminary anti-action

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

The approach significantly increases the sensitivity of off-target detection, up to over five-fold, enabling more accurate identification of off-target genome editing in cell lines, induced pluripotent stem cells, and mice, making it the most sensitive method to date for in vivo off-target discovery.

Implementation Method 1

contacting a cell with a gene-editing complex and at least one guide RNA (gRNA) that targets a nucleic acid sequence of interest

Methodology Applied

Scientific EffectEndonuclease cleavage: Enzyme

Implementation Method 2

administering to a cell an inhibitor of DNA repair, wherein the inhibitor modulates meiotic recombination 11 (MRE11) localization at the target nucleic acid sequence

Methodology Applied

Scientific EffectDNA-PKcs inhibition: Enzyme

Implementation Method 3

detecting MRE11 at on- and off-target nucleic acid sequences

Methodology Applied

Scientific EffectProtein-DNA binding:

Implementation Method 4

the MRE11 is detected by chromatin immunoprecipitation with sequencing (ChIP-seq)

Methodology Applied

Scientific EffectChromatin immunoprecipitation:

Data Source

PatentUS20250002991A1Methods and compositions for genome editing

Publication Date: 2025.01.02 JOHNS HOPKINS UNIVERSITY

AI summary

Compositions and methods for discovery of off-target CRISPR-Cas genome editing in patients, patient-derived cells and animal models are provided.