FusionCRISPR Nucleic Acid Editing for Efficient Homologous Recombination

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Solution Overview

Problem

Current CRISPR/Cas systems face challenges in efficiently editing genomes of organisms with inefficient non-homologous end joining (NHEJ) systems, such as Clostridium cellulolyticum and Escherichia coli, where double-strand breaks often result in cell death, and in diploid or polyploid cells, achieving precise gene knockouts is complex and costly.

Innovation Solution

The FusionCRISPR technology covalently links a guide nucleic acid to a donor nucleic acid, providing a fusion nucleic acid molecule that synchronizes with the CRISPR components to facilitate homologous recombination repair, allowing for efficient gene editing by introducing a donor nucleic acid promptly after a double-strand break, thereby enhancing repair efficacy and predictability.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If CRISPR/Cas9 is used to create double-strand breaks in organisms with inefficient NHEJ systems (e.g., Clostridium cellulolyticum, Escherichia coli), then gene disruption is achieved, but cell death occurs due to inability to repair breaks

Engineering Contradiction:

Improvegene disruption success rateVSAvoidcell death

Core Design Contradiction:

ReliabilityVSObject-affected harmful factors

Solution Approach 1:

The patent combines the CRISPR/Cas9 nuclease system with a donor DNA template into a single integrated construct. The donor template contains homology arms that enable homologous recombination repair, merging the gene disruption function with the repair function into one unified system, thereby achieving reliable gene knockout without cell death in organisms with inefficient NHEJ

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The donor DNA template is designed and prepared in advance with appropriate homology arms flanking the target sequence. This preliminary preparation ensures that when the double-strand break occurs, the repair machinery immediately has the correct template available, preventing cell death and ensuring successful gene disruption in organisms that rely on homologous recombination

Inventive Principle:

Principle #10Preliminary action

2Productivity

If separate CRISPR components and donor nucleic acids are introduced into cells, then gene editing can occur, but delivery efficiency and synchronization are reduced

Engineering Contradiction:

Improvegene editing efficiencyVSAvoiddelivery system complexity

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The patent merges the guide RNA, Cas9 nuclease, and donor DNA template into a single integrated construct or co-delivers them as a coordinated system. This ensures all components arrive at the target location simultaneously and in the correct stoichiometric ratios, dramatically improving gene editing efficiency while simplifying the delivery process compared to introducing multiple separate components

Inventive Principle:

Principle #5Merging (Combining)

3Reliability

If multiple separate nucleic acid donors are used for homologous recombination, then repair can occur, but timing and localization are uncoordinated, reducing repair efficacy

Engineering Contradiction:

Improvehomologous recombination efficiencyVSAvoiddelay in donor availability

Core Design Contradiction:

ReliabilityVSLoss of time

Solution Approach 1:

The donor nucleic acid is designed with homology arms that match the sequences flanking the target site, and is prepared in advance as part of the integrated CRISPR system. This preliminary preparation ensures the donor is immediately available at the correct location when the double-strand break occurs, eliminating delays and maximizing homologous recombination efficiency

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The integrated construct acts as an intermediary that brings the donor nucleic acid into close proximity with the target site through the guide RNA-Cas9 complex. This intermediary function ensures the donor is delivered precisely to the break location at the right time, coordinating timing and localization to enhance repair efficacy

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

FusionCRISPR simplifies gene editing in various microbial species, including those with inefficient NHEJ systems, and in diploid or polyploid cells, by ensuring precise and predictable outcomes, reducing off-target effects and costs associated with high-throughput knockouts and gene corrections.

Implementation Method 1

a guide nucleic acid (gNA) molecule that directs a site specific nucleic acid modifying polypeptide to a target sequence within a target DNA molecule

Methodology Applied

Scientific EffectBase pairing:

Implementation Method 2

the nuclease cleaves the target region

Methodology Applied

Scientific EffectNuclease cleavage:

Implementation Method 3

providing a template for repair allowed for editing the genome with nearly any desired sequence at nearly any site

Methodology Applied

Scientific EffectHomologous recombination:

Data Source

PatentUS20230183686A1Methods for modification of target nucleic acids

Publication Date: 2023.06.15 BASF PLANT SCI GMBH

AI summary

Methods for modification of target nucleic acids. The method involves a construct in which guide RNA is covalently linked to donor RNA (fusion NA) to be introduced into the target nucleic acid by homologous recombination and is based on the introduction of a nuclease, e.g. CRISPR or TALEN, into the cell containing the target nucleic acid. The fusion NA may be introduced as a DNA vector.