Genome Editing With RT-Endonuclease Fusion for Site-Specific Insertion

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Solution Overview

Problem

Existing methods for integrating nucleic acid sequences into a genome lack site specificity and efficiency, particularly for longer sequences, and often require multiple steps or rely on host repair pathways.

Innovation Solution

A system comprising a polypeptide with a reverse transcriptase (RT) domain, DNA-binding domain (DBD), and endonuclease domain, combined with a template RNA or DNA, to specifically target and modify genomic sequences, enabling insertion, deletion, or alteration of nucleic acid sequences with high precision and efficiency.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If existing integration methods are used without specialized proteins, then the process is simple, but the integration occurs at low frequency and with little site specificity

Engineering Contradiction:

Improvesite specificityVSAvoidprocess complexity

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The patent combines multiple functional domains into a single specialized protein: a DNA-binding domain for target recognition, an endonuclease domain for DNA cleavage, and a reverse transcriptase domain for template-dependent DNA synthesis. This merging of functions enables site-specific integration while maintaining process efficiency.

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The patent introduces a template RNA or DNA molecule as an intermediary that carries the sequence to be integrated and guides the specialized protein to the target site through homology-based pairing. This intermediary mediates between the protein complex and the genomic DNA, enabling precise integration.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Manufacturing precision

If CRISPR/Cas9 is used for genome editing, then small edits are achieved, but integration of longer sequences is less effective

Engineering Contradiction:

Improveediting precisionVSAvoidsequence integration capacity

Core Design Contradiction:

Manufacturing precisionVSQuantity of substance

Solution Approach 1:

The patent changes the fundamental mechanism from non-homologous end joining (CRISPR/Cas9) to homology-directed repair using reverse transcriptase. This parameter change enables the integration of longer sequences by using template RNA/DNA with homology arms that can be extended in length, while maintaining precision through homology-based targeting.

Inventive Principle:

Principle #35Parameter changes

3Manufacturing precision

If Cre/loxP system is used, then sequence insertion is achieved, but a first step of inserting loxP site is required before inserting the sequence of interest

Engineering Contradiction:

Improveinsertion precisionVSAvoidnumber of steps

Core Design Contradiction:

Manufacturing precisionVSLoss of time

Solution Approach 1:

The patent performs preliminary action by incorporating the target site recognition capability directly into the specialized protein through the DNA-binding domain. This eliminates the need for preliminary insertion of recognition sites like loxP, as the protein directly recognizes and binds to the target sequence through homology-based pairing with the template RNA/DNA.

Inventive Principle:

Principle #10Preliminary action

4Productivity

If existing methods are used, then host repair pathways are relied upon, but this reduces efficiency and control

Engineering Contradiction:

Improveintegration efficiencyVSAvoiddependence on host pathways

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The patent implements self-service by providing all necessary functions within the introduced specialized protein and template molecule. The reverse transcriptase domain performs template-dependent DNA synthesis autonomously, and the endonuclease domain creates the necessary breaks for integration without requiring host repair pathways, thereby increasing efficiency and control.

Inventive Principle:

Principle #25Self-service

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 precise and efficient insertion, deletion, or alteration of nucleic acid sequences in the genome, overcoming the limitations of existing technologies by providing site-specific integration and reduced reliance on host repair pathways.

Implementation Method 1

a polypeptide or a nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain

Methodology Applied

Scientific EffectReverse transcription:

Implementation Method 2

an endonuclease domain, e.g., a nickase domain

Methodology Applied

Scientific EffectEndonuclease cleavage:

Implementation Method 3

a DNA-binding domain (DBD)

Methodology Applied

Scientific EffectSequence-specific DNA binding:

Data Source

PatentUS20250346927A1Methods and compositions for modulating a genome

Publication Date: 2025.11.13 FLAGSHIP PIONEERING INNOVATIONS VI LLC

AI summary

Methods and compositions for modulating a target genome are disclosed. This disclosure relates to novel compositions, systems and methods for altering a genome at one or more locations in a host cell, tissue or subject, in vivo or in vitro. In particular, the invention features compositions, systems and methods for inserting, altering, or deleting sequences of interest in a host genome.