Novel Cas9 Protein Guide RNA Targeting

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Solution Overview

Problem

Current methods for genome editing and gene regulation using synthetic transcription factors are laborious, require specialized expertise, and often result in variable expression levels and transient gene activation, limiting their therapeutic potential.

Innovation Solution

Development of novel Cas9 proteins and fusion proteins that can specifically target and edit genes, utilizing guide RNAs to direct the Cas9 proteins to precise genomic locations, thereby enabling efficient and precise genome editing and regulation.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If synthetic transcription factors are engineered to control gene expression, then gene regulation capability is improved, but the process becomes laborious and requires specialized expertise

Engineering Contradiction:

Improvegene regulation capabilityVSAvoidengineering process complexity

Core Design Contradiction:

Adaptability or versatilityVSEase of manufacture

Solution Approach 1:

The patent uses guide RNA molecules as programmable templates that direct Cas9 to specific genomic sequences. Instead of engineering complex protein-DNA interaction domains like zinc fingers or TALEs, the system copies the simplicity of RNA base-pairing to achieve programmable targeting. The guide RNA sequence is designed to match the target DNA sequence, enabling non-experts to program gene targeting by simple sequence selection rather than complex protein engineering.

Inventive Principle:

Principle #26Copying

Solution Approach 2:

The patent replaces the mechanical protein-DNA recognition system (zinc fingers, TALEs) with a biochemical RNA-DNA hybridization system. The guide RNA forms an RNA-DNA hybrid with the target sequence through complementary base pairing, substituting the complex mechanical protein-DNA interface with a simpler, more predictable RNA-DNA interaction that follows universal base-pairing rules.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

2Ease of operation

If plasmid DNA delivery methods are used to deliver transcription factors to cells, then delivery is achieved, but expression levels become highly variable and gene activation is transient

Engineering Contradiction:

Improvedelivery capabilityVSAvoidexpression level consistency

Core Design Contradiction:

Ease of operationVSReliability

Solution Approach 1:

The patent merges the Cas9 nuclease and guide RNA into a single functional complex that can be delivered together, ensuring stoichiometric equivalence and consistent expression. This unified delivery approach eliminates the variability caused by separate plasmid transfection of multiple components, as the Cas9 and gRNA are co-delivered in a fixed ratio, ensuring reliable and consistent gene editing activity across cell populations.

Inventive Principle:

Principle #5Merging (Combining)

3Ease of manufacture

If transient gene expression is achieved through plasmid transfection, then delivery is simplified, but therapeutic effects are insufficient due to temporary activation

Engineering Contradiction:

Improvedelivery simplicityVSAvoidgene activation duration

Core Design Contradiction:

Ease of manufactureVSDuration of action of moving object

Solution Approach 1:

The patent extracts the essential functional components (Cas9 and guide RNA) from complex plasmid delivery systems and delivers them in a streamlined format. This extraction allows for more controlled and sustained expression compared to transient plasmid transfection, as the delivered components can be integrated into the cell's existing machinery for longer-lasting activity without the limitations of plasmid copy number variability and degradation.

Inventive Principle:

Principle #2Taking out (Extraction)

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 novel Cas9 proteins and fusion proteins achieve efficient and precise genome editing and regulation, overcoming the limitations of existing technologies by providing stable and controlled gene expression, which is crucial for therapeutic applications.

Implementation Method 1

utilizing guide RNAs to direct the Cas9 proteins to precise genomic locations

Methodology Applied

Scientific EffectComplementary base pairing:

Implementation Method 2

Site-specific nucleases can be used to introduce site-specific double strand breaks at targeted genomic loci. This DNA cleavage stimulates the natural DNA-repair machinery

Methodology Applied

Scientific EffectDNA cleavage:

Implementation Method 3

In the absence of a donor template, the break will be repaired by non-homologous end joining (NHEJ), an error-prone repair pathway that leads to small insertions or deletions of DNA

Methodology Applied

Scientific EffectNon-homologous end joining:

Implementation Method 4

If a donor template is provided along with the nucleases, then the cellular machinery will repair the break by homologous recombination

Methodology Applied

Scientific EffectHomologous recombination:

Data Source

PatentUS20250171754A1Crispr-cas9 compositions and methods with a novel cas9 protein for genome editing and gene regulation

Publication Date: 2025.05.29 NORTH CAROLINA STATE UNIV

AI summary

Disclosed herein is a novel Cas9 protein. Further described herein are fusion proteins, compositions, and methods comprising the same. The novel Cas9 protein may be used, for example, in compositions and methods for modulating expression of a gene, for correcting a mutant gene, and for treating a disease.