CRISPR/Cas9 Site-Specific Integration in CHO Cells
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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 repetitive monoclonal screening, which increases costs and development time.
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, thereby stabilizing protein expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If random integration method is used to construct stable protein-expressing CHO cell lines, then exogenous genes can be integrated into the cell genome, but the expression levels become unstable and repetitive monoclonal screening is required, increasing time and labor costs
Solution Approach 1:
The patent applies preliminary action by pre-selecting a specific integration site (ROS1 gene locus) in the CHO cell genome that is known to support stable and high-level protein expression. By targeting this predetermined optimal location before integration, the method eliminates the need for subsequent monoclonal screening to identify stable expressors, as all integrations at this site inherently provide stable expression.
Solution Approach 2:
The patent uses a specific integration vector designed to target the ROS1 locus as an intermediary mechanism. This vector includes homology arms that specifically recognize and integrate into the ROS1 gene sequence, acting as a mediator between the exogenous gene and the genome. This targeted integration approach replaces the random integration process and eliminates the need for screening intermediaries (monoclonal selection).
2Quantity of substance
If transgene copy number is increased in CHO cells, then more protein can be expressed, but the expression level does not correlate well with copy number and remains unstable
Solution Approach 1:
The patent applies local quality by focusing on the quality of the integration site rather than the quantity of transgene copies. The ROS1 locus has specific chromatin characteristics and regulatory elements that create a favorable local environment for stable, high-level expression. This approach prioritizes the quality of the genomic neighborhood over the number of gene copies, achieving stable expression without relying on high copy numbers.
Solution Approach 2:
The patent uses a single-copy integration strategy at the ROS1 locus, replacing the traditional multi-copy random integration approach. By integrating one well-chosen copy at a predetermined optimal site, the method achieves stable expression equivalent to or better than multiple random copies, eliminating the correlation problem between copy number and expression stability.
3Productivity
If site-specific integration technology is used to target a specific sequence, then monoclonal screening can be eliminated, but the technology requires precise targeting capability
Solution Approach 1:
The patent applies parameter changes by modifying the integration vector design parameters to include homology arms matching the ROS1 locus sequence. This specific sequence parameter (the homology arms) enables precise targeting without requiring complex additional systems. The approach changes the parameter of vector sequence composition to achieve specificity, rather than increasing overall system complexity.
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 method reduces development time and costs by ensuring stable protein expression across multiple cell passages without the need for repetitive screening, achieving consistent high-expression levels in CHO cell lines.
Implementation Method 1
a sequence of 5′ NNNNNNNNNNNNNNNNNNNNNGG3′, as shown in SEQ ID NO: 15, and that can be identified by CRISPR/Cas9 technology and positioned in a base range of No. 1235284-1235429 around the certain site is a target sequence
Data Source
AI summary
Use of genomic NW_006880285.1 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. 1235357 in a CHO cell gene NW_006880285.1; a sequence of 5′ NNNNNNNNNNNNNNNNNNNNNGG3′ that can be identified by CRISPR/Cas9 technology and positioned in a base range of No. 1235284-1235429 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.