Gene-Edited iPSC Subcloning for PRRSV Resistance in Pigs

Resolve Bottlenecks,
Find Innovative SolutionsGenerate Solutions

Solution Overview

Problem

Current methods for breeding gene-edited non-human animals lack precision and efficiency, particularly in large domestic species, and there is a need for more effective techniques to introduce desired genetic edits and traits, such as resistance to porcine reproductive and respiratory syndrome virus (PRRSV).

Innovation Solution

A method involving simultaneous reprogramming and gene-editing of somatic cells to produce induced pluripotent stem cells (iPSCs), followed by subcloning and genotyping, which are then used in somatic nuclear transfer (SCNT) to generate gene-edited non-human embryos and animals, specifically using CRISPR/Cas9 technology to edit the CD163 gene for PRRSV resistance in pigs.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Manufacturing precision

If traditional gene editing methods are used in non-human animals, then genetic modifications can be achieved, but precision and efficiency are insufficient

Engineering Contradiction:

Improvegene editing precisionVSAvoidbreeding efficiency

Core Design Contradiction:

Manufacturing precisionVSProductivity

Solution Approach 1:

The patent performs gene editing and reprogramming on somatic cells before nuclear transfer, allowing genetic modifications to be established in vitro with high precision before being transferred to the embryo. This preliminary action ensures accurate genome editing while improving breeding efficiency by selecting only successfully edited cells for nuclear transfer.

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The patent segments the breeding process into distinct stages: somatic cell gene editing, reprogramming to iPSCs, in vitro subcloning and genotyping, and finally nuclear transfer. This segmentation allows each step to be optimized independently, achieving both high precision in gene editing and high efficiency in the overall breeding process.

Inventive Principle:

Principle #1Segmentation

2Ease of manufacture

If gene editing is performed without selection, then the process is simpler, but the accuracy of desired genetic traits is reduced

Engineering Contradiction:

Improveprocess simplicityVSAvoidgenotype accuracy

Core Design Contradiction:

Ease of manufactureVSMeasurement precision

Solution Approach 1:

The patent implements feedback control through in vitro subcloning and genotyping of iPSC candidates before nuclear transfer. This feedback mechanism identifies and selects only those cells with the desired genetic modifications, ensuring high genotype accuracy while maintaining process simplicity by using established cell culture and genotyping techniques.

Inventive Principle:

Principle #23Feedback

3Manufacturing precision

If multiple breeding steps are used to ensure genetic precision, then accuracy improves, but time and cost increase

Engineering Contradiction:

Improvegenome editing accuracyVSAvoidbreeding time

Core Design Contradiction:

Manufacturing precisionVSLoss of time

Solution Approach 1:

The patent performs all gene editing verification and selection steps in vitro before nuclear transfer, including subcloning and genotyping of iPSC candidates. This preliminary action ensures genome editing accuracy is established before the animal breeding process begins, reducing the time required for subsequent breeding steps.

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The patent uses in vitro subcloning to create multiple copies of successfully edited iPSC candidates, allowing selection of the best clones for nuclear transfer. This copying approach ensures genetic precision is maintained while enabling parallel processing of multiple candidates, reducing overall breeding time.

Inventive Principle:

Principle #26Copying

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 the precise generation of gene-edited non-human animals with desired traits, such as resistance to PRRSV, by ensuring accurate genome editing and efficient breeding, reducing the time and cost associated with traditional methods.

Implementation Method 1

CRISPR/Cas9 technology to edit the CD163 gene for PRRSV resistance in pigs

Methodology Applied

Scientific EffectCRISPR/Cas9 gene editing:

Implementation Method 2

transferring the gene-edited iPSC subclone into an enucleated oocyte to generate a reconstituted embryo

Methodology Applied

Scientific EffectSomatic cell nuclear transfer:

Implementation Method 3

simultaneous reprograming and gene-editing carried out in somatic cells to produce gene-edited induced pluripotency stem cells

Methodology Applied

Scientific EffectCell reprogramming:

Data Source

PatentUS20230292720A1Precise breeding methods for gene-edited non-human animals

Publication Date: 2023.09.21 AGRICULTURAL TECHNOLOGY RESEARCH INSTITUTE

AI summary

The present invention relates to a breeding method for generating gene-edited non-human animals. In particular, the method of the present invention features simultaneous reprograming and gene-editing carried out in somatic cells and subsequent subcloning and genotyping conducted at the in vitro cell stage to obtain precisely gene-edited induced pluripotency stem cells (iPSCs) which are then used in somatic nuclear transfer (SCNT) to generate a precisely gene-edited non-human animal embryo and a resultant gene-edited non-human animal.