Split Cas9 Fusion Proteins for High-Specificity Genome Editing

Solution Overview

Problem

Existing site-specific endonucleases and recombinases exhibit significant off-target activity and limited specificity, posing challenges for clinical and research applications, leading to cellular toxicity and undesired genomic alterations.

Innovation Solution

Engineering Cas9 variants with improved specificity by splitting nuclease activities between two or more proteins, utilizing fusion proteins with nuclease-inactivated Cas9 domains and FokI DNA cleavage domains, and employing RNA-guided recombinases for targeted genomic manipulations.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If site-specific endonucleases are used for targeted genome manipulation, then genomic editing capability is improved, but off-target activity increases leading to cellular toxicity and undesired genomic alterations

Engineering Contradiction:

Improvespecificity of DNA cleavageVSAvoidoff-target activity

Core Design Contradiction:

ReliabilityVSObject-generated harmful factors

Solution Approach 1:

The Cas9 protein is divided into two separate proteins (Cas9α and Cas9β), each containing a catalytically inactive nuclease domain and a guide RNA binding domain. These two proteins must simultaneously bind to adjacent sites on the target DNA to form a functional complex, thereby requiring precise target recognition and reducing off-target effects.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

A bridging molecule or interface is introduced between the two Cas9 halves to mediate their interaction. This intermediary ensures that both Cas9 proteins are correctly positioned and oriented at the target site, enhancing specificity through cooperative binding while preventing individual Cas9 proteins from acting alone at off-target sites.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Reliability

If nuclease activities are split between two or more proteins to improve specificity, then off-target effects are reduced, but device complexity increases

Engineering Contradiction:

Improvespecificity of DNA cleavageVSAvoidprotein structure complexity

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The Cas9 protein is divided into two separate proteins (Cas9α and Cas9β), each containing a catalytically inactive nuclease domain and a guide RNA binding domain. These two proteins must simultaneously bind to adjacent sites on the target DNA to form a functional complex, thereby requiring precise target recognition and reducing off-target effects.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

Two separate Cas9 proteins (Cas9α and Cas9β) are combined to form a functional dimeric complex at the target DNA site. The merging of these two proteins restores full nuclease activity only when both are correctly positioned at the target site, thereby achieving high specificity while distributing the complexity across two simpler individual proteins.

Inventive Principle:

Principle #5Merging (Combining)

3Reliability

If multiple sequences are bound at the target site to increase specificity, then off-target effects are decreased, but binding complexity increases

Engineering Contradiction:

Improvespecificity of DNA cleavageVSAvoidbinding mechanism complexity

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The Cas9 protein is divided into two separate proteins (Cas9α and Cas9β), each containing a catalytically inactive nuclease domain and a guide RNA binding domain. These two proteins must simultaneously bind to adjacent sites on the target DNA to form a functional complex, thereby requiring precise target recognition and reducing off-target effects.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The system incorporates a feedback mechanism where the binding of the first Cas9 protein facilitates or is required for the binding of the second Cas9 protein, and vice versa. This cooperative binding creates a positive feedback loop that enhances specificity, as both proteins must correctly recognize their respective target sites to stabilize the complex and enable cleavage activity.

Inventive Principle:

Principle #23Feedback

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

Enhances the specificity of DNA cleavage and recombination processes, reducing off-target effects and enabling precise genomic modifications.

Implementation Method 1

bind guide RNAs (gRNAs) that direct cleavage of specific target sites

Methodology Applied

Scientific EffectBase pairing:

Implementation Method 2

Cas9 endonuclease engineered to have improved specificity

Methodology Applied

Scientific EffectEnzymatic cleavage: Enzyme

Data Source

PatentUS12559737B2Cas9 variants and uses thereof

Publication Date: 2026.02.24 PRESIDENT & FELLOWS OF HARVARD COLLEGE

AI summary

Some aspects of this disclosure provide compositions, methods, and kits for improving the specificity of RNA-programmable endonucleases, such as Cas9. Also provided are variants of Cas9, e.g., Cas9 dimers and fusion proteins, engineered to have improved specificity for cleaving nucleic acid targets. Also provided are compositions, methods, and kits for site-specific nucleic acid modification using Cas9 fusion proteins (e.g., nuclease-inactivated Cas9 fused to a nuclease catalytic domain or a recombinase catalytic domain). Such Cas9 variants are useful in clinical and research settings involving site-specific modification of DNA, for example, genomic modifications.