Cas9 Trex2 Coexpression for Precise Genome Editing

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Solution Overview

Problem

Current DNA double-strand break (DSB) repair pathways in cells are prone to mistakes, leading to mutations and chromosomal rearrangements, which can result in malignancy or other pathologies, and there is a need for systems that can enhance the frequency of desired repair outcomes.

Innovation Solution

A composition and system comprising a CAS9 endonuclease, a guide RNA sequence complementary to a target genomic nucleotide sequence, and a Trex2 exonuclease, which are coexpressed in cells to induce targeted mutagenesis by creating specific chromosomal breaks and promoting precise repair outcomes.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If DSB repair pathways are used to repair DNA damage, then DNA breaks are repaired, but mutations and chromosomal rearrangements occur due to repair mistakes

Engineering Contradiction:

ImproveDNA repair accuracyVSAvoidmutations and chromosomal rearrangements

Core Design Contradiction:

ReliabilityVSObject-generated harmful factors

Solution Approach 1:

The patent introduces a homology-directed repair template as an intermediary molecule that mediates the repair process. This template provides the correct sequence information that the cellular repair machinery uses to accurately repair DSBs, thereby reducing mutagenic outcomes while maintaining repair efficiency

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent modifies the cellular repair environment by introducing exogenous DNA templates with specific homology regions. This changes the parameters of the repair process by providing alternative repair substrates that guide the repair machinery toward more accurate outcomes

Inventive Principle:

Principle #35Parameter changes

2Reliability

If HDR pathway is used for precise repair, then repair accuracy is improved, but repair efficiency is reduced compared to EJ pathways

Engineering Contradiction:

Improverepair precisionVSAvoidrepair speed

Core Design Contradiction:

ReliabilityVSProductivity

Solution Approach 1:

The patent performs preliminary action by introducing homology-directed repair templates before the DSB repair process occurs. These pre-introduced templates are already in place and ready to be used by the repair machinery, eliminating the need for de novo template synthesis and accelerating the HDR process

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The patent uses copied sequences from the desired final state as repair templates. By providing copies of the correct sequence flanking the DSB site, the repair machinery can directly copy this information to restore the original sequence, speeding up the repair process while maintaining accuracy

Inventive Principle:

Principle #26Copying

3Productivity

If EJ pathways are used for rapid repair, then repair speed is improved, but mutagenic outcomes increase

Engineering Contradiction:

Improverepair speedVSAvoiddeletion and insertion mutations

Core Design Contradiction:

ProductivityVSObject-generated harmful factors

Solution Approach 1:

The patent introduces homology templates as intermediary molecules that mediate between the DSB and the repair outcome. Even when EJ pathways are active, these templates provide a template for more accurate repair, reducing the mutagenic effects of rapid repair mechanisms

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent converts the naturally occurring but mutagenic EJ process into a more accurate repair mechanism by providing homology templates. The same rapid repair machinery that normally causes mutations is redirected to use template-guided repair, turning a harmful process into a beneficial one

Inventive Principle:

Principle #22Blessing in disguise (Convert harm into benefit)

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 effectively enhances the frequency of desired mutagenic outcomes, such as insertion or deletion of nucleotides, reducing the occurrence of rearrangements and allowing for efficient targeted genome modification and therapeutic interventions.

Implementation Method 1

a CAS9 endonuclease... which are coexpressed in cells to induce targeted mutagenesis by creating specific chromosomal breaks

Methodology Applied

Scientific EffectEndonuclease cleavage: Enzyme

Implementation Method 2

a Trex2 exonuclease... which are coexpressed in cells to induce targeted mutagenesis by creating specific chromosomal breaks and promoting precise repair outcomes

Methodology Applied

Scientific EffectExonuclease digestion: Enzyme

Implementation Method 3

a guide RNA sequence complementary to a target genomic nucleotide sequence in a cell

Methodology Applied

Scientific EffectNucleic acid hybridization:

Data Source

PatentUS11427819B2Coexpression of CAS9 and TREX2 for targeted mutagenesis

Publication Date: 2022.08.30 CITY OF HOPE

AI summary

A composition used in targeted mutagenesis is provided, which includes a first expression cassette comprising a nucleotide sequence which encodes a CAS9 endonuclease; a second expression cassette comprising a nucleotide sequence which encodes a guide RNA sequence, wherein the guide RNA sequence is complementary to a target genome nucleotide sequence in a cell; and a third expression cassette comprising a nucleotide sequence which encodes a Trex2 exonuclease (Trex2) gene. The first, second, and third expression cassettes may be a part or a portion of one or more expression vectors.