Lamiaa M. Mahmoud, Prabhjot Kaur, Daniel Stanton, Jude W. Grosser & Manjul Dutt

Plant Methods: Vol. 18, Article number: 33 (2022)

Background

The genetic engineering of crops has enhanced productivity in the face of climate change and a growing global population by conferring desirable genetic traits, including the enhancement of biotic and abiotic stress tolerance, to improve agriculture. The clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system has been found to be a promising technology for genomic editing. Protoplasts are often utilized for the development of genetically modified plants through in vitro integration of a recombinant DNA fragment into the plant genome. We targeted the citrus Nonexpressor of Pathogenesis-Related 3 (CsNPR3) gene, a negative regulator of systemic acquired resistance (SAR) that governs the proteasome-mediated degradation of NPR1 and developed a genome editing technique targeting citrus protoplast DNA to produce stable genome-edited citrus plants.

Results

Here, we determined the best cationic lipid nanoparticles to deliver donor DNA and described a protocol using Lipofectamine™ LTX Reagent with PLUS Reagent to mediate DNA delivery into citrus protoplasts. A Cas9 construct containing a gRNA targeting the CsNPR3 gene was transfected into citrus protoplasts using the cationic lipid transfection agent Lipofectamine with or without polyethylene glycol (PEG, MW 6000). The optimal transfection efficiency for the encapsulation was 30% in Lipofectamine, 51% in Lipofectamine with PEG, and 2% with PEG only. Additionally, plasmid encapsulation in Lipofectamine resulted in the highest cell viability percentage (45%) compared with PEG. Nine edited plants were obtained and identified based on the T7EI assay and Sanger sequencing. The developed edited lines exhibited downregulation of CsNPR3 expression and upregulation of CsPR1.

Conclusions

Our results demonstrate that utilization of the cationic lipid-based transfection agent Lipofectamine is a viable option for the successful delivery of donor DNA and subsequent successful genome editing in citrus.

See: https://plantmethods.biomedcentral.com/articles/10.1186/s13007-022-00870-6

Figure 2: Transient gene expression in Citrus sinensis protoplasts. The average number of EGFP positive cells in ‘N7-3’ protoplast cultures were greater in those treated with Lipofectamine LTX + the Arg9 CCP compared to those treated with Lipofectamine LTX only (A). Error bars represent standard error. Brightfield (B) and fluorescent (C) images showed that some of the protoplasts were EGFP positive 72 h after transfection. Confocal image of a protoplast cell indicating that the Arg9 CPP conjugated with TAMRA penetrated the cell membrane but not the nuclear membrane. There was no colocalization between the nucleus stained with DAPI (D) and the TAMARA signal (E) indicated by the merge (F). Scale bar indicates 100 µM in length