Sequential Genome Editing and Induced Excision of the Transgene in N. tabacum BY2 Cells
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 designa...
Main Authors: | , , , , , , , |
---|---|
Format: | Article |
Language: | English |
Published: |
Frontiers Media S.A.
2020-11-01
|
Series: | Frontiers in Plant Science |
Subjects: | |
Online Access: | https://www.frontiersin.org/articles/10.3389/fpls.2020.607174/full |
id |
doaj-658f67c60f784860bcf490b63026a503 |
---|---|
record_format |
Article |
spelling |
doaj-658f67c60f784860bcf490b63026a5032020-12-08T08:43:56ZengFrontiers Media S.A.Frontiers in Plant Science1664-462X2020-11-011110.3389/fpls.2020.607174607174Sequential Genome Editing and Induced Excision of the Transgene in N. tabacum BY2 CellsMaor ShevaUri HananiaTami ArielAlbina TurbovskiVishal Kumar Rameshchandra RathodDina OzYoram TekoahYoseph ShaaltielWhile 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.https://www.frontiersin.org/articles/10.3389/fpls.2020.607174/fullCRISPR/Cas9genome editingglyco-engineeringN. tabacum BY2 cellsplant glycanstransgene-free |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Maor Sheva Uri Hanania Tami Ariel Albina Turbovski Vishal Kumar Rameshchandra Rathod Dina Oz Yoram Tekoah Yoseph Shaaltiel |
spellingShingle |
Maor Sheva Uri Hanania Tami Ariel Albina Turbovski Vishal Kumar Rameshchandra Rathod Dina Oz Yoram Tekoah Yoseph Shaaltiel Sequential Genome Editing and Induced Excision of the Transgene in N. tabacum BY2 Cells Frontiers in Plant Science CRISPR/Cas9 genome editing glyco-engineering N. tabacum BY2 cells plant glycans transgene-free |
author_facet |
Maor Sheva Uri Hanania Tami Ariel Albina Turbovski Vishal Kumar Rameshchandra Rathod Dina Oz Yoram Tekoah Yoseph Shaaltiel |
author_sort |
Maor Sheva |
title |
Sequential Genome Editing and Induced Excision of the Transgene in N. tabacum BY2 Cells |
title_short |
Sequential Genome Editing and Induced Excision of the Transgene in N. tabacum BY2 Cells |
title_full |
Sequential Genome Editing and Induced Excision of the Transgene in N. tabacum BY2 Cells |
title_fullStr |
Sequential Genome Editing and Induced Excision of the Transgene in N. tabacum BY2 Cells |
title_full_unstemmed |
Sequential Genome Editing and Induced Excision of the Transgene in N. tabacum BY2 Cells |
title_sort |
sequential genome editing and induced excision of the transgene in n. tabacum by2 cells |
publisher |
Frontiers Media S.A. |
series |
Frontiers in Plant Science |
issn |
1664-462X |
publishDate |
2020-11-01 |
description |
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. |
topic |
CRISPR/Cas9 genome editing glyco-engineering N. tabacum BY2 cells plant glycans transgene-free |
url |
https://www.frontiersin.org/articles/10.3389/fpls.2020.607174/full |
work_keys_str_mv |
AT maorsheva sequentialgenomeeditingandinducedexcisionofthetransgeneinntabacumby2cells AT urihanania sequentialgenomeeditingandinducedexcisionofthetransgeneinntabacumby2cells AT tamiariel sequentialgenomeeditingandinducedexcisionofthetransgeneinntabacumby2cells AT albinaturbovski sequentialgenomeeditingandinducedexcisionofthetransgeneinntabacumby2cells AT vishalkumarrameshchandrarathod sequentialgenomeeditingandinducedexcisionofthetransgeneinntabacumby2cells AT dinaoz sequentialgenomeeditingandinducedexcisionofthetransgeneinntabacumby2cells AT yoramtekoah sequentialgenomeeditingandinducedexcisionofthetransgeneinntabacumby2cells AT yosephshaaltiel sequentialgenomeeditingandinducedexcisionofthetransgeneinntabacumby2cells |
_version_ |
1724390209420787712 |