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.
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。