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Abstract：
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.
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.
CONCLUSIONS: 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.
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.
CONCLUSIONS: 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.
摘要：
背景：用于开发重组CHO（rCHO）细胞系作为哺乳动物蛋白质表达的主要宿主的常规方法仅限于随机整合方法，这可以延长几个月获得所需克隆的过程。CRISPR/Cas9可以通过介导位点特异性整合到转录活性热点来替代，促进同质克隆，缩短克隆选择过程。然而,将这种方法应用于rCHO细胞系开发取决于可接受的整合速率和持续表达的稳健位点。
结果：在这项研究中，我们旨在通过两种策略提高GFP报告分子整合到CHO-K1基因组3号染色体(Chr3)伪attP位点的速率;这些策略包括基于PCR的供体线性化和通过应用单体链霉亲和素(mSA)-生物素系链方法增加DSB位点附近供体的局部浓度。根据结果,与传统的CRISPR介导的靶向相比，供体线性化和系链方法在敲入效率方面表现出1.6和2.4倍的改善；在中靶克隆中，通过定量PCR确定84%和73%为单拷贝，分别。最后,为了评估目标整合的表达水平，通过应用已建立的连接方法，将作为分泌蛋白的hrsACE2的表达盒靶向Chr3假attP位点。生成的细胞池达到2倍的生产率，与随机整合细胞系相比。
结论：我们的研究提出了增强CRISPR介导的整合的可靠策略，引入Chr3伪ATTP位点作为持续转基因表达的潜在候选者，可用于促进rCHO细胞系的发展。
结果：在这项研究中，我们旨在通过两种策略提高GFP报告分子整合到CHO-K1基因组3号染色体(Chr3)伪attP位点的速率;这些策略包括基于PCR的供体线性化和通过应用单体链霉亲和素(mSA)-生物素系链方法增加DSB位点附近供体的局部浓度。根据结果,与传统的CRISPR介导的靶向相比，供体线性化和系链方法在敲入效率方面表现出1.6和2.4倍的改善；在中靶克隆中，通过定量PCR确定84%和73%为单拷贝，分别。最后,为了评估目标整合的表达水平，通过应用已建立的连接方法，将作为分泌蛋白的hrsACE2的表达盒靶向Chr3假attP位点。生成的细胞池达到2倍的生产率，与随机整合细胞系相比。
结论：我们的研究提出了增强CRISPR介导的整合的可靠策略，引入Chr3伪ATTP位点作为持续转基因表达的潜在候选者，可用于促进rCHO细胞系的发展。
