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首页> 外文期刊>Biotechnology and bioprocess engineering >Efficient CRISPR/Cas9-mediated multiplex genome editing in CHO cells via high-level sgRNA-Cas9 complex
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Efficient CRISPR/Cas9-mediated multiplex genome editing in CHO cells via high-level sgRNA-Cas9 complex

机译：通过高级sgRNA-Cas9复合体在CHO细胞中进行高效的CRISPR / Cas9介导的多重基因组编辑

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摘要

Increasing demand for recombinant therapeutic proteins has warranted the need for an efficient host cell to produce high-quality proteins, with a high yield. Chinese hamster ovary (CHO) cells appear to meet this demand, and their genetic tailoring will facilitate improvements in their productivity for recombinant proteins. Recent advances in programmable RNA-guided Cas9 nuclease (RGN) have facilitated CHO cell engineering via site-specific genome editing. One critical determinant for increasing genomeediting efficiency is attaining a balanced expression level of Cas9 nuclease and guide RNAs in the nucleus. Here, we achieved high-level expression of Cas9 nuclease and single guide RNA (sgRNA), enhancing expression levels approximately three-fold over the conventional methodology by using an iterative transfection approach. We demonstrated that high abundance of sgRNA and Cas9 nuclease induced a two-fold increase in the site-specific mutation rate on average for both single and multiple genetic targets. Sequencing results confirmed frame-shift mutations at targeted genomic loci created by error-prone NHEJassociated mutations. Moreover, we controlled the amount of sgRNA-Cas9 complex formation in vitro and delivered the complex directly to cells, resulting in the maximization of mutation frequency by the high-level of sgRNA-Cas9 complex. Importantly, mutation rates of putative off-target sites remained minimal in spite of the improved genome-editing efficiency. These results provide an efficient strategy for editing the CHO genome with the reduction of the time-consuming screening efforts aimed at isolating clones with desirable properties.
机译：对重组治疗性蛋白质的需求不断增加，因此需要一种有效的宿主细胞来高产量地生产高质量蛋白质。中国仓鼠卵巢（CHO）细胞似乎可以满足这一需求，其基因剪裁将有助于提高重组蛋白的生产率。可编程RNA引导的Cas9核酸酶（RGN）的最新进展通过位点特异性基因组编辑促进了CHO细胞的工程设计。提高基因组编辑效率的一个关键决定因素是在细胞核中实现Cas9核酸酶和指导RNA的平衡表达水平。在这里，我们实现了Cas9核酸酶和单向导RNA（sgRNA）的高水平表达，通过使用迭代转染方法，表达水平比常规方法提高了约三倍。我们证明，对于单个和多个遗传靶标，高丰度的sgRNA和Cas9核酸酶平均会导致位点特异性突变率平均提高两倍。测序结果证实了由容易出错的NHEJ相关突变产生的靶向基因组位点的移码突变。此外，我们在体外控制了sgRNA-Cas9复合物的形成量，并将该复合物直接递送至细胞，从而通过高水平的sgRNA-Cas9复合物使突变频率最大化。重要的是，尽管提高了基因组编辑效率，但假定的脱靶位点的突变率仍保持最小。这些结果提供了编辑CHO基因组的有效策略，减少了旨在分离具有所需特性的克隆的费时筛选工作。

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  • 来源
    《Biotechnology and bioprocess engineering》 |2015年第5期|825-833|共9页
  • 作者

    Shin Jongoh; Lee Namil; Song Yoseb; Park Jinhyung; Kang Taek Jin; Kim Sun Chang; Lee Gyun Min; Cho Byung-Kwan;

  • 作者单位

    Korea Adv Inst Sci & Technol, KI BioCentury, Taejon 305701, South Korea|Korea Adv Inst Sci & Technol, Dept Biol Sci, Taejon 305701, South Korea;

    Korea Adv Inst Sci & Technol, KI BioCentury, Taejon 305701, South Korea|Korea Adv Inst Sci & Technol, Dept Biol Sci, Taejon 305701, South Korea;

    Korea Adv Inst Sci & Technol, KI BioCentury, Taejon 305701, South Korea|Korea Adv Inst Sci & Technol, Dept Biol Sci, Taejon 305701, South Korea;

    Korea Adv Inst Sci & Technol, Dept Biol Sci, Taejon 305701, South Korea;

    Dongguk Univ Seoul, Dept Chem & Biochem Engn, Seoul 100715, South Korea;

    Korea Adv Inst Sci & Technol, Dept Biol Sci, Taejon 305701, South Korea|Korea Adv Inst Sci & Technol, KI BioCentury, Taejon 305701, South Korea|Intelligent Synthet Biol Ctr, Taejon 305701, South Korea;

    Korea Adv Inst Sci & Technol, Dept Biol Sci, Taejon 305701, South Korea;

    Korea Adv Inst Sci & Technol, Dept Biol Sci, Taejon 305701, South Korea|Korea Adv Inst Sci & Technol, KI BioCentury, Taejon 305701, South Korea|Intelligent Synthet Biol Ctr, Taejon 305701, South Korea;

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  • 正文语种 eng
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  • 关键词

    genome editing; CRISPR/Cas9; Chinese hamster ovary (CHO) cells; iterative transfection;

    机译：基因组编辑CRISPR / Cas9中国仓鼠卵巢细胞迭代转染;

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