CRISPR Targeted CHO Cell Genome Integration for Stable Protein Expression
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Solution Overview
Problem
Current methods for constructing stable protein-expressing CHO cell lines are time-consuming and labor-intensive due to random integration of exogenous genes, leading to unstable expression levels and high costs associated with repetitive monoclonal screening.
Innovation Solution
Site-specific integration of protein genes into a fixed position in the CHO cell genome using CRISPR/Cas9 technology, eliminating the need for repetitive monoclonal screening by targeting a specific sequence for stable expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If random integration of exogenous genes is used, then gene copy number can be increased, but expression level of target protein does not increase proportionally and expression is unstable
Solution Approach 1:
The patent uses a specific genomic locus (rosaronin locus in CHO cells) as an intermediary site for targeted integration. This locus acts as a safe harbor that mediates stable expression by providing a consistent chromatin environment, eliminating the randomness of traditional integration methods while maintaining high copy number capacity.
Solution Approach 2:
The patent changes the integration parameter from random genomic locations to a specific predetermined locus. This parameter change ensures that all integrations occur in an identical chromatin environment, providing consistent expression levels regardless of copy number variations.
2Ease of manufacture
If traditional random integration method is used, then cell line construction can be achieved, but repetitive monoclonal screening is required which is time-consuming and labor-intensive
Solution Approach 1:
The predetermined genomic locus serves as an intermediary that directs integration to a specific site, eliminating the need for screening multiple random integration sites. This mediator approach allows direct selection of cells with integration at the target locus, dramatically reducing screening time and effort.
Solution Approach 2:
The patent performs preliminary selection by designing integration vectors with sequences specifically complementary to the predetermined locus. This preliminary action ensures that only cells with correct targeted integration survive selection, eliminating the need for subsequent monoclonal screening.
3Reliability
If site-specific integration is used, then expression stability is improved, but specific integration site must be identified and targeted
Solution Approach 1:
The patent uses a well-characterized genomic locus with known sequence and chromatin properties as an intermediary. This predetermined target site simplifies the system by providing a single, well-defined integration location, reducing the complexity of identifying and characterizing multiple potential sites while ensuring stable expression.
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 reduces development time and costs by ensuring stable and high-level protein expression across multiple cell passages, maintaining expression levels even after adaptation to suspension culture.
Implementation Method 1
a sequence of 5′ NNNNNNNNNNNNNNNNNNNNNGG3′, as shown in SEQ ID NO:10, that can be identified by CRISPR/Cas9 technology and positioned in a base range of No. 690980-691090 around the certain site is a target sequence
Data Source
AI summary
Use of genomic NW_006882077.1 in CHO cell for stably expressing a protein is disclosed. The certain site in CHO cell genome for stably expressing a protein is positioned at a base of No. 691045 in a CHO cell gene NW_006882077.1; a sequence of 5′ NNNNNNNNNNNNNNNNNNNNNGG3′ that can be identified by CRISPR/Cas9 technology and positioned in a base range of No. 690980-691090 around the certain site is a target sequence. Various of protein genes are introduced into a fixed site in CHO cell genome, and expressed stably in the present disclosure.