CRISPR/Cas9 Mutagenesis through Introducing a Nanoparticle Complex Made of a Cationic Polymer and Nucleic Acids into Maize Protoplasts

Abstract

Presently, targeted gene mutagenesis attracts increasing attention both in plant research and crop improvement. In these approaches, successes are largely dependent on the efficiency of the delivery of gene editing components into plant cells. Here, we report the optimization of the cationic polymer poly(2-hydroxypropylene imine) (PHPI)-mediated delivery of plasmid DNAs, or single-stranded oligonucleotides labelled with Cyanine3 (Cy3) or 6-Carboxyfluorescein (6-FAM)-fluorescent dyes into maize protoplasts. Co-delivery of the GFP-expressing plasmid and the Cy3-conjugated oligonucleotides has resulted in the cytoplasmic and nuclear accumulation of the green fluorescent protein and a preferential nuclear localization of oligonucleotides. We show the application of nanoparticle complexes, i.e., "polyplexes" that comprise cationic polymers and nucleic acids, for CRISPR/Cas9 editing of maize cells. Knocking out the functional EGFP gene in transgenic maize protoplasts was achieved through the co-delivery of plasmids encoding components of the editing factors Cas9 (pFGC-pcoCas9) and gRNA (pZmU3-gRNA) after complexing with a cationic polymer (PHPI). Several edited microcalli were identified based on the lack of a GFP fluorescence signal. Multi-base and single-base deletions in the EGFP gene were confirmed using Sanger sequencing. The presented results support the use of the PHPI cationic polymer in plant protoplast-mediated genome editing approaches.

Keywords: CRISPR/Cas9; DNA oligonucleotide; DsRed fluorescent marker protein; GFP expression; maize protoplasts; mutagenesis; nanoparticle; plasmid uptake; poly (2-hydroxypropylene imine); polyplexes.

Figure 1

(A) The refractive index of the synthesized polymer samples as a function of the concentration. (B) The Debye plot used to estimate the molecular weight of the polymer.

Figure 2

Delivery efficiencies of the pGFP plasmid into maize protoplasts depends on the polymer concentration. Maize protoplasts were treated with polyplexes formed using 1 µg pGFP plasmid and cationic polymer (PHPI) in the indicated quantity. Delivery efficiencies were analyzed 24 h after the polyplex treatment through counting the number of GFP expressing protoplasts under confocal laser scanning microscopy. Graph shows mean values (±SD) from three separate experiments. Significance was tested using a Student’s t-test (ns: non-significant, *: 0.01 < p < 0.05, **: p < 0.01).

Figure 3

Maize protoplasts were incubated with polyplexes formed using 1 µg GFP plasmid and 2 µg cationic polymer for the indicated durations. At the end of these incubation periods, supernatants were removed and fresh protoplast media (ppN6M) were added. Delivery efficiencies were analyzed 24 h after the polyplex treatment through counting the number of GFP-expressing protoplasts under confocal laser scanning microscopy. Graph shows mean values (±SD) from three separate experiments. Significance was tested using a Student’s t-test (*: 0.01 < p < 0.05, **: p < 0.01).

Figure 4

(A) Detection of green maize protoplasts indicates the uptake of the pGFP plasmid. (B) Merged image of brightfield and fluorescence is shown 24 h after treatment. Insets show a dividing cell after 5 days post treatment. In this experiment, 3 µL PCV of maize protoplasts was treated with polyplexes formed of 1 µg pGFP plasmid and 2 µg cationic polymer. After 1 d or 5 d (inset) following the polyplex treatment, GFP-expressing protoplasts were visualized under confocal laser scanning microscopy. (C) An example of the formation of a multicellular colony from GFP-positive protoplasts as an indication of a stable transformation event induced through polyplex treatment. The picture was taken after two weeks of culture in ppN6M protoplast-culturing medium. Scale bars are 25 µm.

Figure 5

(A) Detection of the FAM signals in membranes and nuclei of the maize protoplasts after treatment with polyplexes formed using 1 μg oligonucleotide and 2 µg cationic polymer (PHPI) in a highly dense protoplast culture. (B) Brightfield image superimposed onto a fluorescence image to indicate the total number of protoplasts in the same field. (C) Inset shows the accumulation of oligonucleotides in membranes and nuclei of maize protoplasts after treatment with polyplexes formed using 0.5 µg Cy3-labeled oligonucleotide and 1 µg cationic polymer (PHPI). Arrowheads highlight two protoplasts with Cy3 accumulation in the nuclei. Scale bars are 25 µm.

Figure 6

Cationic polymer PHPI can co-deliver both plasmid and oligonucleotide molecules into maize protoplasts. (A) GFP-expressing protoplast 1 day after polyplex treatment. (B) Delivery and retention of Cy3-labeled oligonucleotides and their nuclear accumulation. (C) Brightfield-merged green and red fluorescence images indicating successful co-delivery of both the plasmid and the oligonucleotide to the same protoplast. Maize protoplasts were treated with polyplexes formed using 0.5 µg GFP plasmid, 0.5 µg Cy3-labeled oligonucleotide, and 2 µg cationic polymer. Protoplasts were visualized under confocal laser scanning microscopy 24 h after the polyplex treatment. Scale bar is 10 µm.

Figure 7

Phenotypic identification of maize microcalli with a knockout EGFP gene after the introduction of CRISPR/Cas9 plasmid vectors through cationic polymer (PHPI) treatment of transgenic maize protoplasts carrying the EGFP gene. A half microgram of each of the plasmids expressing Cas9 (pFGC-pcoCas9) and gRNA (pZmU3-gRNA) was complexed with 2 µg PHPI cationic polymer. (A) Protoplast-derived microcalli expressing EGFP (mother culture) and DsRed (transformed/edited) imaged using confocal microscopy. (B) The same microcalli population that shows a lack of green signal for the DsRed-positive callus. (C1) White light epi-illumination image of maize protoplast-derived microcalli under stereo dissection microscopy. (C2) Fluorescence image of the same microcalli expressing the DsRed marker gene showing the origin of these cells from a protoplast carrying the modified pZmU3-gRNA plasmid introduced via cationic polymer treatment. (C3) The same field of microcalli that shows an absence of green signal for the corresponding red callus. (D) Multi-base deletion and single-base deletion in the EGFP gene confirmed using Sanger sequencing of the EGFP fragment from knockout microcalli. Red GGG: PAM sequences; blue letters: CRISPR target sequence; black letters: wild type (wt) sequences, red dash lines: deleted nucleotides; red letters: inserted nucleotides/mismatch.