Modular Genome Editing Complex for AAV-Size DNA Targeting

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Solution Overview

Problem

Existing RNA-guided CRISPR-Cas systems, such as CRISPR-Cas9, are limited by size and immunogenicity, which restricts efficient AAV packaging and therapeutic application in humans, and alternative systems like Fok1 nuclease domains have doubts over efficiency.

Innovation Solution

A totally synthetic modular system for gene modification using a nucleoprotein complex with a targeting nucleic acid and a modular polypeptide component, including a DNA-binding domain (DBD) of no more than 70-75 amino acids, linked to an effector component for site-specific DNA modification, allowing smaller size and various delivery means.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If CRISPR-Cas9 system is used for genome editing, then DNA targeting ability is achieved, but system size becomes too large for efficient AAV packaging

Engineering Contradiction:

ImproveDNA targeting abilityVSAvoidsystem size

Core Design Contradiction:

ReliabilityVSLength of moving object

Solution Approach 1:

The system is divided into separate functional modules: a targeting nucleic acid (guide RNA) and a modular polypeptide component. The polypeptide further contains separate modules for DNA binding, recognition, and effector functions. This segmentation allows each component to be optimized independently and reduces the overall size for AAV packaging.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The invention extracts and removes the large Cas9 protein from the system, replacing it with a smaller modular polypeptide component that performs similar functions. This extraction of the bulky element directly reduces system size while maintaining DNA targeting capability.

Inventive Principle:

Principle #2Taking out (Extraction)

2Reliability

If CRISPR-Cas9 system is used for genome editing, then DNA modification is achieved, but immunogenicity arises in human population

Engineering Contradiction:

ImproveDNA modification capabilityVSAvoidimmunogenicity

Core Design Contradiction:

ReliabilityVSObject-affected harmful factors

Solution Approach 1:

The invention uses a synthetic, non-natural polypeptide component that does not exist in the human genome, reducing immunogenicity. The system can be designed to be transient and degradable, minimizing long-term immunological responses.

Inventive Principle:

Principle #27Cheap short-living objects (Disposable)

Solution Approach 2:

The system changes the biochemical parameters by using a synthetic polypeptide with modified amino acid sequences that differ from natural Cas9 proteins. This parameter change in protein sequence reduces recognition by the human immune system while maintaining functional capability.

Inventive Principle:

Principle #35Parameter changes

3Adaptability or versatility

If Fok1 nuclease domain with SCNA is used for genome editing, then alternative targeting is achieved, but efficiency remains doubtful

Engineering Contradiction:

Improvetargeting flexibilityVSAvoidediting efficiency

Core Design Contradiction:

Adaptability or versatilityVSReliability

Solution Approach 1:

The modular polypeptide component is designed with universal functionality that can be applied to various target sequences. The separate modules for DNA binding and recognition can be configured to work with different targeting nucleic acids, providing both flexibility and efficiency.

Inventive Principle:

Principle #6Universality (Multi-functionality)

Solution Approach 2:

The invention introduces a recognition module as an intermediary between the targeting nucleic acid and the effector component. This intermediary enhances the interaction specificity and efficiency by providing a dedicated binding interface that facilitates accurate target recognition and effector activation.

Inventive Principle:

Principle #24Intermediary (Mediator)

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 system achieves efficient site-specific DNA modification with smaller size, compatible with AAV vector delivery, and avoids immunogenicity, enhancing genome editing efficiency and versatility.

Implementation Method 1

a targeting nucleic acid possessing both DNA targeting ability and ability to bind a recognition module of a modular polypeptide

Methodology Applied

Scientific EffectMolecular recognition and binding:

Implementation Method 2

a short peptide DNA-binding component, devoid of enzyme activity and which recognises a pre-determined sequence in the target

Methodology Applied

Scientific EffectSequence-specific DNA binding:

Implementation Method 3

an effector component for use in modifying the target... an artificial nickase comprising a multimer of linked, self-assembling short peptides

Methodology Applied

Scientific EffectEnzymatic DNA modification: Enzyme

Data Source

PatentUS20260009008A1Synthetic genome editing system

Publication Date: 2026.01.08 PENCIL BIOSCIENCES LTD

AI summary

The present invention provides a synthetic, modular system for DNA modification as may be employed for genome editing comprising a targeting nucleic acid possessing both DNA targeting ability and ability to bind a recognition module of a modular polypeptide component, where the modular polypeptide component also includes an effector component and a short peptide DNA-binding sequence (DBD) which binds a pre-determined sequence (PBS) in the target. The DBD may preferably be no more than a 15 mer and serves to destabilize the structure of a targeted dsDNA upon binding thereby facilitating the desired DNA modification. The effector component may, for example, be an artificial nickase comprising linked self-assembling peptides thereby providing a complete genome-editing system of advantageous small size for vector delivery to cells.