Sequential Genome Editing and Induced Excision of the Transgene in N. tabacum BY2 Cells

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.

Characterization of the novelSmsHSP24.1Promoter: Unveiling its abiotic stress-inducible expression in transgenic Eggplant (Solanum melongenaL.)

“…This enables targeted overexpression or knockdown of genes associated with pivotal agroclimatic traits. Recent studies demonstrated a comparative analysis of using CRISPR/Cas9 system under a heat shock inducible promoter produces less off-target mutations compared to constitutive ubiquitin promoter in Arabidopsis, rice, and tobacco (Liang et al, 2023; Nandy et al, 2019; Sheva et al, 2020).…”

Temporally gene knockout using heat shock–inducible genome‐editing system in plants

“…Compared to the constitutive rice ( Oryza sativa ) UBIQUITIN promoter, the use of the heat‐inducible heat‐shock protein 17.5E promoter from soybean ( Glycine max ) resulted in much fewer off‐target effects when used to express Cas9 (Nandy et al., 2019). Likewise, the Arabidopsis heat‐shock protein 18.2 ( HSP18.2) promoter was used to express a specific sgRNA targeting the T‐DNA boundaries for a transgene excision in a tobacco ( Nicotiana tabacum ) BY2 cell suspension (Sheva et al., 2020). The target efficiency, specificity, and programmability of IGE systems are dependent on the inducible strategies adopted.…”

CRISPR/Cas9‐mediated knockout of a prolyl‐4‐hydroxylase subfamily in Nicotiana benthamiana using DsRed2 for plant selection

“…Typically, the CRISPR/Cas9 cassette is left in the genome, and further work is required to segregate the transgene from the mutations and thus generate transgene-free lines. [35,36 ] The workload becomes more difficult to manage for the introduction of multiple mutations, and ideally, it would be possible to introduce all mutations and remove the transgene cassette in the T0 and T1 generations, respectively. Fluorescent proteins can be used for the noninvasive confirmation of transgene removal, and this has been applied to CRISPR/Cas9 editing in many species.…”