LAUSR

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 wild relative of tobacco used for transient protein expression in laboratory and commercial settings. To improve the performance of this species, we established a multiplex genome editing system incorporating the DsRed2 fluorescent marker for the identification and selection of transgenic plants. As proof of principle, we targeted the P4H4 gene encoding a prolyl-4-hydroxylase involved in protein O-linked glycosylation. Using preselected gRNAs with efficiencies confirmed by transient expression, we established transgenic plant lines with knockout mutations in all four P4H4 genes simultaneously. Leaf fluorescence was then used to screen for the absence of the SpCas9 transgene in T1 plants, and we subsequently identified transgene-free lines with homozygous or biallelic mutations. The analysis of plant-produced recombinant IgA1 as a reporter protein revealed changes in the amount of peptides containing hydroxyproline residues and pentoses in the knockout plants. The multiplex expression of efficient gRNAs combined with the DsRed2 marker in a binary vector system reduces the effort needed to generate N. benthamiana mutants and simplifies the screening processes needed to obtain transgene-free progeny. This article is protected by copyright. All rights reserved.