CRISPR/Cas9‐mediated knockout of a prolyl‐4‐hydroxylase subfamily in Nicotiana benthamiana using DsRed2 for plant selection. Issue 7 (20th May 2022)

• Record Type: Journal Article
• Authors: Uetz, Pia; Melnik, Stanislav; Grünwald‐Gruber, Clemens; Strasser, Richard; Stoger, Eva
• Is Part Of: Biotechnology journal. Volume 17:Issue 7(2022)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2022-05-20
• Subjects: CRISPR‐based gene targeting -- host plant engineering -- molecular farming -- Nicotiana benthamiana -- plant‐made proteins; Biotechnology -- Periodicals; 660.605
• DOI: 10.1002/biot.202100698
• Languages: English
• ISSNs: 1860-6768

Abstract

Abstract: The properties of host plants used for molecular farming can be modified by CRISPR/Cas9 genome editing to improve the quality and yield of recombinant proteins. However, it is often necessary to target multiple genes simultaneously, particularly when using host plants with large and complex genomes. This is the case for Nicotiana benthamiana, an allotetraploid relative of tobacco frequently used for transient protein expression. A multiplex genome editing system incorporating the DsRed2 fluorescent marker for the identification and selection of transgenic plants was established. As proof of principle, NbP4H4 was targeted encoding a prolyl‐4‐hydroxylase involved in protein O ‐linked glycosylation. Using preselected gRNAs with efficiencies confirmed by transient expression, transgenic plant lines with knockout mutations in all four Nb P4H4 genes were obtained. Leaf fluorescence was then used to screen for the absence of the SpCas9 transgene in T1 plants, and transgene‐free lines with homozygous or biallelic mutations were identified. The analysis of plant‐produced recombinant IgA1 as a reporter protein revealed changes in the number of peptides containing hydroxyproline residues and pentoses in the knockout plants. The selection of efficient gRNAs combined with the DsRed2 marker reduces the effort needed to generate N. benthamiana mutants and simplifies the screening processes to obtain transgene‐free progeny. Graphical Abstract and Lay Summary: Genetic engineering via CRISPR/Cas9 is a widely used method to modify many different organisms, including the plant Nicotiana benthamiana, to improve traits or delete undesired ones. In this study, the authors improve existing vectors rendering the process more efficient and easier by providing a selection‐mechanism of modified material through detection of a red fluorescent protein. This work provides accessible tools for genome editing in plants while also investigating the role of a specific set of genes.