Plant Gene Editing HDR Efficiency via Replicon and Inhibitors

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Solution Overview

Problem

The efficiency of homologous recombination-based gene editing in plants is low, limiting its industrial applicability and requiring optimization of temperature, photoperiod conditions, and the use of multiple replicons to enhance HDR pathway activity and inhibit NHEJ pathway, while ensuring stable expression of nucleases and high activity of CRISPR/Cpf1 enzymes.

Innovation Solution

Optimizing temperature and photoperiod conditions during plant tissue culture, using a multiple replicon system with HDR and NHEJ pathway regulation, and employing CRISPR/Cpf1-based gene scissors with enhanced stability and activity, along with chemical inhibitors like SCR7 pyrazine to increase HDR efficiency.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Manufacturing precision

If CRISPR/Cas9 system is used to induce DSB for gene editing, then random Indel mutations are generated, but accurate HDR-based gene editing efficiency remains low at about 1/100 of NHEJ

Engineering Contradiction:

ImproveHDR editing precisionVSAvoidHDR efficiency

Core Design Contradiction:

Manufacturing precisionVSProductivity

Solution Approach 1:

The patent optimizes temperature conditions (25-30°C) and photoperiod conditions (16/8 light/dark cycle) during tissue culture to enhance HDR efficiency. These parameter changes create optimal environmental conditions that shift the balance from NHEJ to HDR pathway, achieving both high precision and improved productivity in gene editing

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent introduces a viral replicon system that serves as an intermediary to amplify and deliver HDR templates to target sites. This replicon system overcomes the low efficiency of direct HDR by providing abundant template DNA, thereby improving HDR productivity while maintaining editing precision

Inventive Principle:

Principle #24Intermediary (Mediator)

2Productivity

If NHEJ pathway is the predominant DNA repair pathway in somatic cells, then DNA repair occurs rapidly, but HDR efficiency remains low compared to NHEJ

Engineering Contradiction:

ImproveHDR efficiencyVSAvoidDNA repair pathway reliability

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The patent applies chemical inhibitors (SCR7, CuSO4) that preliminarily block the NHEJ pathway before HDR can occur. By inhibiting NHEJ components (ligase IV, ATM kinase) in advance, the cell is forced to use HDR pathway instead, thereby improving HDR efficiency while maintaining reliable DNA repair through the enhanced HDR mechanism

Inventive Principle:

Principle #9Preliminary anti-action

3Ease of manufacture

If conventional DSB-based plant HDR technique is used, then some HDR events can be obtained, but the technique is far from commercialization due to low efficiency

Engineering Contradiction:

ImproveCommercial applicabilityVSAvoidHDR efficiency

Core Design Contradiction:

Ease of manufactureVSProductivity

Solution Approach 1:

The patent establishes optimized tissue culture parameters including temperature (25-30°C), photoperiod (16/8 light/dark), and hormonal conditions that dramatically improve HDR efficiency. These parameter optimizations make the technique commercially viable by achieving sufficient productivity while maintaining ease of operation in plant tissue culture systems

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent employs a viral replicon system as an intermediary mechanism to amplify HDR templates and enhance delivery to target sites. This intermediary system bridges the gap between conventional low-efficiency HDR and commercial applicability by providing robust template amplification and delivery

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

Significantly increases HDR efficiency in plants, allowing for accurate gene editing and potential industrial applications, such as pyramiding genes in GM crops and in planta plant engineering, with improved stability and activity of CRISPR/Cpf1 enzymes.

Implementation Method 1

When a DSB is created in a gene at specific site of a genome according to application of the CRISPR/Cas9 system

Methodology Applied

Scientific EffectDouble-strand break (DSB):

Implementation Method 2

HR easily occurs in meiosis of germ line cells, yielding haploids, but it hardly occurs in mitosis of somatic cells

Methodology Applied

Scientific EffectHomologous recombination (HR):

Implementation Method 3

HDR is a repair pathway which occurs, along with non-homologous end joining (NHEJ), in case of having DNA damage

Methodology Applied

Scientific EffectNon-homologous end joining (NHEJ):

Implementation Method 4

after the injection to tobacco using Agrobacterium, a circular replicon was yielded based on rolling circle replication

Methodology Applied

Scientific EffectRolling circle replication:

Data Source

PatentUS11542530B2Method for increasing efficiency of homologous recombination-based gene editing in plant

Publication Date: 2023.01.03 INDUSTRYACADEMIC COOPERATION FOUNDATION GYEONGSANG NATIONAL UNIVERSITY

AI summary

A method for increasing the efficiency of homologous recombination-based gene editing in a plant according to an embodiment of the present invention includes optimizing temperature and photoperiod conditions during tissue culture of plant cells, expressing factors required for homology-directed DNA repair (HDR) and factors for increasing the HDR efficiency by using a multiple replicon, or regulating the HDR pathway or non-homologous end joining (NHEJ) pathway.