Abstract
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.
Highlights
Using plant cell suspensions for the production of biotherapeutic proteins is considered a promising direction to overcome some of the limitations of using whole plants as the production platform (Tekoah et al, 2015; Zagorskayaa and Deinekoa, 2017)
In a previous publication (Hanania et al, 2017), we reported the construction of a vector for stable transformation, which was designed to drive the constitutive expression of Cas9 together with five sets of sgRNAs targeted to the N. tabacum genome, which are known to contain two XylT genes (Ntab-XylT A and B) and five FucT genes (Ntab-FucT A, B, C, D, and E)
To enable its own self-excision and the subsequent selection of “transgene-free” cell lines thereafter, the previously described vector (Hanania et al, 2017) was further supplemented with the following components: (1) three repeats of 23 nucleotides each, designated as “ZZZ,” which were framed on its two edges; (2) an additional g-RNA-Z driven by the heat inducible HSP18.2 promoter (Takahashi and Komeda, 1989); and (3) a cytosine deaminase negative selectable marker driven by a 35S promoter (Figure 1 and Supplementary Table 1)
Summary
Using plant cell suspensions for the production of biotherapeutic proteins is considered a promising direction to overcome some of the limitations of using whole plants as the production platform (Tekoah et al, 2015; Zagorskayaa and Deinekoa, 2017). The CRISPR-Cas, Cas12a ( known as Cpf1) and, recently discovered, CasX have proven to be highly efficient valuable tools for precise genome editing across a wide range of cell types and organisms. The CRISPR-Cas system is becoming an increasingly important tool for a wide range of targeted mutagenesis, gene replacement, and other novel applications The presence of selectable markers and nuclease genes (e.g., CRISPR-Cas9) that were used for the targeted mutations, limit their further application in subsequent transformations and may pose excessive metabolic burden on the cell’s machinery.
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