Cas9-HUH Donor DNA Tethering for HDR Efficiency

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Solution Overview

Problem

The efficiency of gene editing using CRISPR/Cas9 systems is limited by the inefficient delivery and integration of donor DNA during the Homology Directed Repair (HDR) process, which is outcompeted by the more efficient Non-homologous End Joining (NHEJ) pathway.

Innovation Solution

Fusing a Cas9 endonuclease with an HUH tag allows for the covalent tethering of a donor DNA molecule to the Cas9-HUH/gRNA complex, creating a single complex that includes all components required for DNA cleavage and HDR, thereby enhancing the delivery and integration of donor DNA.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If donor DNA is transfected or electroporated into cells along with Cas9 and gRNA, then gene editing can be performed, but the delivery and integration of donor DNA is inefficient and outcompeted by NHEJ pathway

Engineering Contradiction:

ImproveHDR efficiencyVSAvoidgene editing frequency

Core Design Contradiction:

ReliabilityVSProductivity

Solution Approach 1:

The patent merges the Cas9 endonuclease, gRNA, and donor DNA into a single integrated complex. The donor DNA is covalently tethered to the Cas9-gRNA complex through a protein tag system, ensuring that all components required for HDR are delivered together to the target site, thereby improving both reliability and productivity of gene editing.

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The patent uses a protein tag as an intermediary to covalently link the donor DNA to the Cas9-gRNA complex. This intermediary ensures stable association of donor DNA with the editing complex, preventing loss during delivery and enhancing HDR efficiency by maintaining close proximity of donor DNA to the cleavage site.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Adaptability or versatility

If multiple separate components (Cas9, gRNA, donor DNA) are delivered to cells, then flexibility in design is maintained, but delivery efficiency and integration of donor DNA decreases

Engineering Contradiction:

Improvedesign flexibilityVSAvoiddonor DNA integration efficiency

Core Design Contradiction:

Adaptability or versatilityVSReliability

Solution Approach 1:

The patent combines multiple separate components (Cas9, gRNA, and donor DNA) into a single integrated complex through covalent tethering. This merging maintains design flexibility while dramatically improving donor DNA integration efficiency by ensuring all components are delivered together and remain associated during nuclear entry and repair.

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The patent uses modular protein tags that can be attached to either Cas9 or donor DNA, allowing flexible design choices. The segmentation of the tagging system enables researchers to choose different tag configurations while maintaining the core functionality of integrated complex formation.

Inventive Principle:

Principle #1Segmentation

Data Source

PatentUS20250051743A1Materials and methods for increasing gene editing frequency

Publication Date: 2025.02.13 REGENTS OF THE UNIVERSITY OF MINNESOTA

AI summary

Materials and methods for gene editing using improved targeted endonucleases and endonuclease systems (e.g., Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) endonuclease systems) are provided herein.