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CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy
Published on: March 12, 2018
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Efficient site-specific integration in CHO-K1 cells using CRISPR/Cas9-modified donors
Mohammad Hassan Kheirandish1,2, Behnaz Rahmani2,3, Hossein Zarei Jaliani4
1Medical Biotechnology Department, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Molecular Biology Reports
|May 27, 2023
Summary
This study enhances CRISPR-mediated gene integration in CHO cells using novel methods, improving efficiency and productivity for recombinant protein development. These strategies accelerate the generation of stable, high-expressing cell lines.
Area of Science:
- Biotechnology
- Molecular Biology
- Cell Line Engineering
Background:
- Conventional recombinant CHO (rCHO) cell line development relies on random integration, leading to lengthy clone selection processes.
- CRISPR/Cas9 offers site-specific integration for homogenous clones and faster selection, but requires efficient integration rates and stable expression sites.
- Challenges remain in optimizing CRISPR-mediated integration efficiency and identifying suitable genomic loci for sustained transgene expression in rCHO cells.
Purpose of the Study:
- To improve the integration rate of a GFP reporter into the Chromosome 3 (Chr3) pseudo-attP site in CHO-K1 cells using CRISPR/Cas9.
- To evaluate the efficacy of PCR-based donor linearization and mSA-biotin tethering for enhancing site-specific integration.
- To assess the productivity of a recombinant cell line generated by targeting the Chr3 pseudo-attP site with an expression cassette.
Main Methods:
- Employed CRISPR/Cas9 for site-specific gene targeting to the Chr3 pseudo-attP site in CHO-K1 cells.
- Utilized two strategies to enhance knock-in efficiency: PCR-based donor linearization and mSA-biotin tethering to increase local donor concentration.
- Quantified integration efficiency and assessed single-copy integration using quantitative PCR.
Main Results:
- The donor linearization and tethering methods improved knock-in efficiency by 1.6- and 2.4-fold, respectively, compared to conventional CRISPR targeting.
- Quantitative PCR confirmed that 84% and 73% of on-target clones achieved single-copy integration with the respective methods.
- Targeting an hrsACE2 expression cassette to the Chr3 pseudo-attP site via tethering resulted in a 2-fold increase in productivity compared to random integration cell lines.
Conclusions:
- Developed and validated reliable strategies to enhance CRISPR-mediated gene integration efficiency in CHO cells.
- Identified the Chr3 pseudo-attP site as a promising locus for sustained transgene expression in rCHO cell line development.
- The findings provide a pathway to accelerate the development of high-performance rCHO cell lines for biopharmaceutical production.
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