Retroviral Integrase-Cas9 Fusion for Targeted Genome Editing

Solution Overview

Problem

Current genome editing technologies, such as CRISPR-Cas9 and lentiviral vectors, face inefficiencies and safety concerns due to limited ability to deliver large DNA sequences and random integration, which hampers the treatment of monogenetic diseases and can cause off-target mutations.

Innovation Solution

A fusion protein combining retroviral integrase and CRISPR-associated Cas protein with a nuclear localization signal is developed, allowing for targeted and specific integration of large DNA sequences into the genome without the need for homology arms, using a system that includes guide RNAs and donor templates with U3 and U5 sequences.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If CRISPR-Cas9 mediated HDR is used for targeted delivery of large DNA sequences, then genome editing capability is improved, but delivery efficiency deteriorates and requires significant flanking homology regions

Engineering Contradiction:

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The patent merges CRISPR-Cas9 with lentiviral integrase into a fusion protein, combining the targeted DNA binding capability of Cas9 with the efficient integration capability of integrase. This allows the system to deliver and integrate large DNA sequences without requiring homology arms, thereby improving delivery efficiency while maintaining genome editing capability

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The lentiviral integrase acts as an intermediary that facilitates the integration of donor DNA sequences into the host genome at the Cas9-targeted location. The integrase mediates the insertion process by recognizing specific sequences in the donor DNA and catalyzing their integration into the genome, bypassing the need for homology-directed repair

Inventive Principle:

Principle #24Intermediary (Mediator)

2Quantity of substance

If lentiviral vectors are used for delivering large genes, then delivery capacity is improved, but integration specificity deteriorates due to random integration

Engineering Contradiction:

Core Design Contradiction:

Quantity of substanceVSManufacturing precision

Solution Approach 1:

The patent combines the high delivery capacity of lentiviral vectors with the targeted binding capability of CRISPR-Cas9. The fusion protein maintains the ability to deliver large DNA sequences while the Cas9 component provides sequence-specific targeting, thereby achieving both high delivery capacity and precise integration specificity

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The CRISPR-Cas9 complex acts as an intermediary that directs the lentiviral integrase to specific genomic locations. The guide RNA directs Cas9 to the target sequence, and the fusion integrase随之 integrates the donor DNA at that specific location, converting random integration into targeted integration while maintaining delivery capacity

Inventive Principle:

Principle #24Intermediary (Mediator)

3Productivity

If CRISPR-Cas9 induces double-strand DNA cleavage, then genome editing efficiency is improved, but off-target effects and p53 pathway activation worsen

Engineering Contradiction:

Core Design Contradiction:

ProductivityVSObject-generated harmful factors

Solution Approach 1:

The patent extracts the DNA cleavage function from the integration function by using a catalytically inactive Cas9 (dCas9) or Cas9 with reduced cleavage activity. The essential genome editing capability is maintained through the integration of donor DNA sequences without requiring extensive double-strand breaks, thereby reducing off-target effects and p53 pathway activation while preserving editing efficiency

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

This approach enables efficient and targeted genome editing with reduced off-target effects, allowing for the permanent correction of genetic material and the delivery of large gene sequences that exceed the limits of traditional vectors, enhancing the precision and safety of gene therapy.

Implementation Method 1

The fusion of viral Integrase (IN) with CRISPR-Cas9 allows for the integration of donor DNA sequences at targeted locations in the host genome

Methodology Applied

Scientific EffectEnzymatic catalysis: Enzyme

Implementation Method 2

A fusion protein combining retroviral integrase and CRISPR-associated Cas protein with a nuclear localization signal is developed

Methodology Applied

Scientific EffectNuclear localization signal-mediated transport:

Data Source

PatentUS20210363509A1Genome Editing by Directed Non-Homologous DNA Insertion Using a Retroviral Integrase-Cas9 Fusion Protein

Publication Date: 2021.11.25

AI summary

The present invention provides proteins, nucleic acids, systems and methods for editing genomic material.