A cationic lipid mediated CRISPR/Cas9 technique for the production of stable genome edited citrus plants

Abstract

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.

Keywords: CRISPR/Cas9; Citrus; Genome editing; Lipofection; NPR3; Protoplast; Systemic acquired resistance (SAR).

Fig. 1

Schematic illustration of protoplast isolation and CRISPR/Cas9-mediated genome-editing steps via Lipofectamine. Protoplasts isolated from in vitro grown cell suspension (A). Protoplast layer represents protoplasts between solutions of sucrose 25% and mannitol 13%. Bright field of protoplast 1 h after isolation. Mixing of lipofectamine and plasmid constructs targeting NPR3 and protoplast transfection (B). Characterization steps by monitoring EGFP expression and cas9 presence in the genomic DNA, T7EI assay and sanger sequencing and gene expression analysis (C). GFP-expressed cell was monitored by a confocal laser scanning microscope. The figure was created in BioRender.com

Fig. 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

Fig. 3

Effect of different concentrations of CRISPR/Cas9 plasmid encapsulated in different amounts of Lipofectamine (Lipo) mediated DNA delivery into citrus protoplasts. Four concentrations of DNA were used (25, 50, 75 and 100 µg) and four amounts of Lipo (0.5, 1, 2 and 4%). ***Expressed p value < 0.0001. Transfection efficiency was calculated as the percentage of GFP-expressed cells by the total number of the viable cells within 48 h of each treatment ± standard errors (vertical bars). Different letters represent significant differences by Tukey’s honestly test (p ≤ 0.05)

Fig. 4

Evaluation of transfection agents on the transient transformation efficiency (A), and cell viability (B) of citrus protoplasts

Fig. 5

Development of genome edited transgenic plants following lipofectamine-mediated plasmid delivery into protoplast. A Colonies of developed embryos, (B) EGFP-expressing embryos. A germinating EGFP expressing seedling visualized under white light (C) and the same seedling exhibiting EGFP expression under an epi-fluorescence stereomicroscope (D). E Regenerated plants in tissue culture medium before micrografting and (F) Micrografted plant well acclimated in the greenhouse. Inset shows a section of the leaf exhibiting EGFP fluorescence as visualized under the dissecting microscope fitted with a NIGHTSEA fluorescence adapter. Scale bar indicates 1 cm in length

Fig. 6

Targeted mutagenesis of NPR3. T7 Endonuclease I (T7EI) assay targeting CsNPR3 indicating cleavage products from targeted mutagenesis (A). Positive sign ( +) is used to indicate T7EI reaction by incubate the annealed PCR product at 37 ºC for 15 min. The untreated samples are marked with negative sign (−). Sanger sequencing chromatograms of four selected lines. Arrows point to the editing site and the modifications are indicated alongside (B)

Fig. 7

Changes in relative expression of CsNPR3 (A), and CsPR1 (B) of four selected and edited sweet orange ‘N7-3’ plants. Means compared using Tukey–Kramer HSD test, means followed by the same letter were not different at (p < 0.05)