Rapid characterization of the CHO platform cell line and identification of pseudo attP sites for PhiC31 integrase

Damavandi, Narges; Raigani, Mozhgan; Joudaki, Atefeh; Davami, Fatemeh; Zeinali, Sirous

doi:10.1016/j.pep.2017.08.002

Cited by 7 publications

(5 citation statements)

References 21 publications

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“…89 In another study, pseudosites in CHO cells were also identified and utilized for industrial production of therapeutic proteins such as monoclonal antibodies (mAbs). 95 On the basis of a systematic comparison among 15 Ser recombinases, Bxb1 was identified as the most efficient integrase in both human and mouse cells. 96 Furthermore, mammalian-codon optimized Bxb1 integrase could yield a 3fold improvement in integration efficiency compared to the wild type Bxb1, with ∼10% integration efficiency of a ∼ 12 kb donor plasmid without drug enrichment.…”

Section: Applications Of Native Ssrs In Mammalian Cellsmentioning

confidence: 99%

“…One example demonstrated the stable genetic correction of an inherited human skin disease through the integration of a ∼9 kb COL7A1 cDNA by the integrase, with 16 out of 30 clones after drug selection showing integration of the donor at one pseudosite on chromosome 8 . In another study, pseudosites in CHO cells were also identified and utilized for industrial production of therapeutic proteins such as monoclonal antibodies (mAbs) . On the basis of a systematic comparison among 15 Ser recombinases, Bxb1 was identified as the most efficient integrase in both human and mouse cells .…”

Section: More Than Just Crispr: Site-specific Recombinasesmentioning

confidence: 99%

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Expanding the Potential of Mammalian Genome Engineering via Targeted DNA Integration

et al. 2021

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Inserting custom designed DNA sequences into the mammalian genome plays an essential role in synthetic biology. In particular, the ability to introduce foreign DNA in a site-specific manner offers numerous advantages over random DNA integration. In this review, we focus on two mechanistically distinct systems that have been widely adopted for targeted DNA insertion in mammalian cells, the CRISPR/Cas9 system and site-specific recombinases. The CRISPR/Cas9 system has revolutionized the genome engineering field thanks to its high programmability and ease of use. However, due to its dependence on linearized DNA donor and endogenous cellular pathways to repair the induced double-strand break, CRISPR/Cas9-mediated DNA insertion still faces limitations such as small insert size, and undesired editing outcomes via error-prone repair pathways. In contrast, site-specific recombinases, in particular the Serine integrases, demonstrate large-cargo capability and no dependence on cellular repair pathways for DNA integration. Here we first describe recent advances in improving the overall efficacy of CRISPR/Cas9-based methods for DNA insertion. Moreover, we highlight the advantages of site-specific recombinases over CRISPR/Cas9 in the context of targeted DNA integration, with a special focus on the recent development of programmable recombinases. We conclude by discussing the importance of protein engineering to further expand the current toolkit for targeted DNA insertion in mammalian cells.

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Section: Applications Of Native Ssrs In Mammalian Cellsmentioning

confidence: 99%

Section: More Than Just Crispr: Site-specific Recombinasesmentioning

confidence: 99%

Expanding the Potential of Mammalian Genome Engineering via Targeted DNA Integration

et al. 2021

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“…These are low protein expression, unstable cell lines, and high costs [ 2 ]. These challenges can be addressed by using PhiC31 site-specific integrase, which recognizes pseudo-attP sites in mammalian cells and facilitates site-specific recombination, thereby resulting in stable, long-term, and high-level transgene expression [ 3 , 4 ]. Compared with random integration, PhiC31 phage-mediated integration achieves a five- to ten-fold higher efficiency in HEK293 and 3T3 cells [ 5 ].…”

Section: Introductionmentioning

confidence: 99%

Study on chromatin regulation patterns of expression vectors in the PhiC31 integration site

Liu,

Chen,

Yin

et al. 2024

Epigenetics

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The PhiC31 integration system allows for targeted and efficient transgene integration and expression by recognizing pseudo attP sites in mammalian cells and integrating the exogenous genes into the open chromatin regions of active chromatin. In order to investigate the regulatory patterns of efficient gene expression in the open chromatin region of PhiC31 integration, this study utilized Ubiquitous Chromatin Opening Element (UCOE) and activating RNA (saRNA) to modulate the chromatin structure in the promoter region of the PhiC31 integration vector. The study analysed the effects of DNA methylation and nucleosome occupancy changes in the integrated promoter on gene expression levels. The results showed that for the OCT4 promoter with moderate CG density, DNA methylation had a smaller impact on expression compared to changes in nucleosome positioning near the transcription start site, which was crucial for enhancing downstream gene expression. On the other hand, for the SOX2 promoter with high CG density, increased methylation in the CpG island upstream of the transcription start site played a key role in affecting high expression, but the positioning and clustering of nucleosomes also had an important influence. In conclusion, analysing the DNA methylation patterns, nucleosome positioning, and quantity distribution of different promoters can determine whether the PhiC31 integration site possesses the potential to further enhance expression or overcome transgene silencing effects by utilizing chromatin regulatory elements.

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“…Bi et al introduced phiC31 recombinase target sites termed pseudo-attP as the potential safe harbor loci for robust recombinant expression [ 15 ]. We recently characterized novel pseudo-attP sites in CHO-K1 cells [ 16 ]; however, their functionality to express a recombinant secretory protein has not been examined.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2023

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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. Supplementary information The online version contains supplementary material available at 10.1007/s11033-023-08529-8.

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Rapid characterization of the CHO platform cell line and identification of pseudo attP sites for PhiC31 integrase

Cited by 7 publications

References 21 publications

Expanding the Potential of Mammalian Genome Engineering via Targeted DNA Integration

Expanding the Potential of Mammalian Genome Engineering via Targeted DNA Integration

Study on chromatin regulation patterns of expression vectors in the PhiC31 integration site

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

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