While plant cells in suspension are becoming a popular platform for expressing biotherapeutic proteins, the need to pre-engineer these cells to better comply with their role as host cell lines is emerging. Heterologous DNA and selectable markers are used for transformation and genome editing designated to produce improved host cell lines for overexpression of recombinant proteins. The removal of these heterologous DNA and selectable markers, no longer needed, can be beneficial since they limit additional gene stacking in subsequent transformations and may pose excessive metabolic burden on the cell machinery. In this study we developed an innovative stepwise methodology in which the CRISPR-Cas9 is used sequentially to target genome editing, followed by its own excision. The first step included a stable insertion of a CRISPR-Cas9 cassette, targeted to knockout the β(1,2)-xylosyltranferase (XylT) and the α(1,3)-fucosyltransferase (FucT) genes in Nicotiana tabacum L. cv Bright Yellow 2 (BY2) cell suspension. The second step included the excision of the inserted cassette of 14.3 kbp by induction of specific sgRNA designed to target the T-DNA boundaries. The genome editing step and the transgene removal step are achieved in one transformation run. This mechanism enables CRISPR genome editing and subsequently eliminating the introduced transgenes thus freeing the cells from foreign DNA no longer needed.

尽管悬浮中的植物细胞正在成为表达生物治疗蛋白的流行平台，但对这些细胞进行预工程改造以更好地发挥其作为宿主细胞系的作用的需求正在出现。异源DNA和选择标记用于转化和基因组编辑，其指定用于产生用于重组蛋白过表达的改良宿主细胞系。这些不再需要的异源DNA和选择标记的去除可能是有益的，因为它们限制了后续转化中额外的基因堆积，并可能给细胞机制带来过多的代谢负担。在这项研究中，我们开发了一种创新的逐步方法，其中，CRISPR-Cas9被顺序用于靶向基因组编辑，然后进行自身切除。第一步包括稳定插入CRISPR-Cas9盒，烟草L. cv亮黄色2（BY2）细胞悬液。第二步包括通过诱导旨在靶向T-DNA边界的特异性sgRNA切除14.3 kbp的插入盒。基因组编辑步骤和转基因去除步骤可以一次转化完成。这种机制能够进行CRISPR基因组编辑，并随后消除引入的转基因，从而使细胞脱离不再需要的外源DNA。