CRISPR-Cas3 Poultry Genome Editing via Segmented Delivery
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Solution Overview
Problem
Efficient genome editing in poultry using Class 1 CRISPR-Cas systems, such as CRISPR-Cas3, is unclear and less developed compared to Class 2 systems like CRISPR-Cas9, particularly for targeting sequences in poultry cells, due to the complexity of multiple molecules involved and limited accuracy in eukaryotic cells.
Innovation Solution
A method involving the CRISPR-Cas3 system is developed to knock-in a gene encoding a protein of interest into poultry cells by introducing the CRISPR-Cas3 system, including the CRISPR-Cas3 protein, cascade complex, and crRNA, targeting specific egg white protein genes like ovalbumin, ovomucoid, ovotransferrin, ovoinhibitor, or lysozyme, with optimized PAM and target sequences for precise DNA editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If CRISPR-Cas3 system is used for genome editing in poultry, then target sequence recognition accuracy is improved, but system complexity increases due to multiple molecules involved
Solution Approach 1:
The CRISPR-Cas3 system is divided into separate functional components: Cas3 protein, Cascade complex, and crRNA. Each component is delivered separately into poultry cells, allowing independent optimization and control of each element's function and reducing the complexity of managing a single multifunctional complex.
Solution Approach 2:
The Cascade complex serves as an intermediary that bridges crRNA and Cas3. It binds crRNA and uses it to recognize target DNA sequences, then recruits Cas3 for cleavage. This intermediary structure organizes the multiple molecules into a coordinated system, improving target recognition accuracy while managing system complexity through structured interaction.
2Ease of operation
If CRISPR-Cas9 system is used for genome editing in poultry, then ease of operation is improved, but target sequence recognition accuracy deteriorates compared to CRISPR-Cas3
Solution Approach 1:
The invention changes the key parameter of target sequence length recognition from 18-24 bases (CRISPR-Cas9) to 32-37 bases (CRISPR-Cas3). This parameter change in the crRNA design enables more accurate target recognition while maintaining operational simplicity through standardized delivery methods similar to Cas9 systems.
3Measurement precision
If Class 1 CRISPR-Cas systems are used in poultry, then target sequence accuracy is improved, but manufacturing efficiency deteriorates due to lack of developed protocols
Solution Approach 1:
The invention performs preliminary actions by pre-optimizing the CRISPR-Cas3 components for poultry cells before actual genome editing. This includes pre-designing crRNA sequences with 32-37 base targets, pre-testing Cascade complex formulations, and pre-establishing delivery protocols. These preliminary optimizations enable efficient and accurate genome editing in poultry without requiring extensive trial-and-error during the actual editing process.
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 allows for precise and efficient genome editing in poultry cells, achieving targeted gene knock-in and high expression of the protein of interest in egg white, with reduced off-target effects and stable expression in subsequent generations.
Implementation Method 1
In the CRISPR-Cas3 system, Cas3 (a protein with nuclease and helicase activities), cascade and crRNA cooperate to cleave DNA
Implementation Method 2
crRNAs have been reported to recognize target sequences of 32-37 bases
Data Source
AI summary
According to the present invention, a poultry cell knocked-in at an egg white protein gene, a knock-in method, a method for producing a knocked-in poultry cell and an egg or a poultry containing the knocked-in poultry cell may be provided.