Details

Autor(en) / Beteiligte

• Kheirandish, Mohammad Hassan
• Rahmani, Behnaz
• Zarei Jaliani, Hossein
• Barkhordari, Farzaneh
• Mazlomi, Mohammad Ali
• Davami, Fatemeh

Titel

Efficient site-specific integration in CHO-K1 cells using CRISPR/Cas9-modified donors

Ist Teil von

• Molecular biology reports, 2023-07, Vol.50 (7), p.5889-5899

Ort / Verlag

Dordrecht: Springer Netherlands

Erscheinungsjahr

2023

Quelle

Alma/SFX Local Collection

Beschreibungen/Notizen

• Background Conventional methods applied to develop recombinant CHO (rCHO) cell line as a predominant host for mammalian protein expression are limited to random integration approaches, which can prolong the process of getting the desired clones for months. CRISPR/Cas9 could be an alternative by mediating site-specific integration into transcriptionally active hot spots, promoting homogenous clones, and shortening the clonal selection process. However, applying this approach for the rCHO cell line development depends on an acceptable integration rate and robust sites for the sustained expression. Methods and results In this study, we aimed at improving the rate of GFP reporter integration to the Chromosome 3 (Chr3) pseudo-attP site of the CHO-K1 genome via two strategies; these include the PCR-based donor linearization and increasing local concentration of donor in the vicinity of DSB site by applying the monomeric streptavidin (mSA)-biotin tethering approach. According to the results, compared to the conventional CRISPR-mediated targeting, donor linearization and tethering methods exhibited 1.6- and 2.4-fold improvement in knock-in efficiency; among on-target clones, 84% and 73% were determined to be single copy by the quantitative PCR, respectively. Finally, to evaluate the expression level of the targeted integration, the expression cassette of hrsACE2 as a secretory protein was targeted to the Chr3 pseudo-attP site by applying the established tethering method. The generated cell pool reached 2-fold productivity, as compared to the random integration cell line. Conclusion Our study suggested reliable strategies for enhancing the CRISPR-mediated integration, introducing Chr3 pseudo-attP site as a potential candidate for the sustained transgene expression, which might be applied to promote the rCHO cell line development.