Application of protoplast technology to CRISPR/Cas9 mutagenesis: from single-cell mutation detection to mutant plant regeneration

Abstract

Plant protoplasts are useful for assessing the efficiency of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) mutagenesis. We improved the process of protoplast isolation and transfection of several plant species. We also developed a method to isolate and regenerate single mutagenized Nicotianna tabacum protoplasts into mature plants. Following transfection of protoplasts with constructs encoding Cas9 and sgRNAs, target gene DNA could be amplified for further analysis to determine mutagenesis efficiency. We investigated N. tabacum protoplasts and derived regenerated plants for targeted mutagenesis of the phytoene desaturase (NtPDS) gene. Genotyping of albino regenerants indicated that all four NtPDS alleles were mutated in amphidiploid tobacco, and no Cas9 DNA could be detected in most regenerated plants.

Keywords: CRISPR/Cas9; protoplast isolation; protoplast regeneration; single-cell analysis.

Figure 1

Improved protoplast isolation. (a) A razor blade was divided into four pieces and the pieces assembled in parallel in a scalpel handle. (b) Seedlings of rice were cut in cross section and placed in digestion solution. Bar = 1 cm. (c) Seedlings of rice were cut longitudinally (in the same direction as the veins) and placed in digestion solution. Bar = 1 cm. (d) Microscopic image of rice seedlings cut in cross section (perpendicular to vascular bundles) after 3‐h digestion. Bar = 10 μm. (e) Microscopic image of rice seedlings cut in longitudinally (parallel to vascular bundles) after 3‐h digestion. Bar = 10 μm. (f) Tomato hypocotyl sections were incubated in medium supplemented with 10 mg/L NAA and photographed after 1 month. Bar = 1 cm. (g) Microscopic image of tomato suspension cells. Bar = 40 μm. (h) Tomato suspension cells grown in 1 mg/L 2,4‐D after 7 days. Bar = 1 cm. (i) Tomato suspension cells formed calli on solid medium supplemented with 1 mg/L 2,4‐D. Bar = 1 cm. (j) mRFP‐NLS plasmid DNA was delivered to rice protoplasts using a PEG‐mediated method. Protoplasts were photographed after 24 h. Red colour indicates RFP epifluorescence. Bar = 50 μm. (k) Overlay of epifluorescence and bright field images of transfected rice protoplasts. Bar = 50 μm. (l) mRFP‐NLS plasmid DNA was delivered into tomato Micro‐Tom protoplasts using a PEG‐mediated method. Protoplasts were photographed after 24 h. Red colour indicates RFP epifluorescence. Bar = 50 μm. (m) Overlay of epifluorescence and bright field images of transfected tomato protoplasts. Bar = 50 μm.

Figure 2

Schematic showing mutation of target site sequences after genome‐editing using a protoplast transfection system. (a) The genomic sequence of the target gene is used to design an sgRNA sequence and primers. Target sites containing a restriction enzyme site are chosen. Blue, Target site; red, protospacer adjacent motif (PAM). The single guide RNA (sgRNA) was cloned into pCAMBIA1300‐OsU3‐Cas9 (or OsU6). Plasmid DNA is delivered into protoplasts by a PEG‐mediated method. (b) Fluorescence image of transfected millet protoplasts. The plasmid mRFP‐NLS is used as a marker to determine the transfection efficiency. Cells fluorescing red are successfully transfected. (c) DNA from nontransfected (WT) or transfected (OsU3‐24 h or OsU3‐48 h) protoplasts are isolated and used as a PCR template for target gene amplification ( PDS in this case). PCR products with (+) or without (−) PstI digestion. (d) Sanger method sequencing result of one aliquot pool of PCR product. The PCR product pool contains a mixture of wild‐type and differentially mutagenized DNA [SNPs (single nucleotide polymorphisms), insertions and deletions]. (e) An aliquot of mutagenized PCR products is subcloned into a T/A cloning vector. The inserts of individual colonies are amplified by colony PCR and the mutation confirmed by digestion with PstI. Clones containing a putative mutagenized target gene (indicated by star marks) are subjected to sequencing. (f) DNA sequencing results of mutated clones. First line, wild type; blue, target site; red, PAM; purple, insertion; −, deleted nucleotides.

Figure 3

Single‐protoplast analysis of the effect of plasmid dosage on NtPDS target mutagenesis. Different amounts of plasmid DNA (containing the expression cassette of NtPDS sgRNA and SaCas9; Kaya et al., 2016) were transfected into tobacco mesophyll protoplasts. After three days, the target mutations were analysed by RFLP. S, N. sylvestris form; T, N. tomentosiformis form. Green box, wild‐type RFLP control; red box, albino mutant RFLP control. Numbers above the gel lanes indicate protoplast number.

Figure 4

Single‐protoplast analysis of the effect of incubation times on NtPDS target mutagenesis. Tobacco protoplasts were transfected with 20 μg plasmid DNA (containing the expression cassette of NtPDS sgRNA and SaCas9; Kaya et al., 2016) and incubated for various number of days. The target mutation was analysed by RFLP. S, sylvestris form; T, tomentosiformis form. Green box, wild‐type RFLP control; red box, albino mutant RFLP control. Numbers above the gel lanes indicate protoplast number.

Figure 5

Targeted mutagenesis of the ZmIPK gene in protoplasts. (a) The gene structure of ZmIPK . Blue, gRNA positions; green, two pairs of primers for single‐cell PCR analysis; red, size of PCR product from the wild‐type gene; orange, size between two gRNAs; purple, theoretical size after precise deletion. (b) Maize protoplasts were transfected with two sgRNAs that targeted different regions of the ZmIPK gene. DNA from several single protoplasts was amplified by PCR 72 h after transfection. P, pooled DNA PCR product; −, no plasmid DNA transfected. Numbers above the gel lanes indicate protoplast number.

Figure 6

NtPDS mutant plants derived from CRISPR mutagenesis of transfected protoplasts. (a) Growing calli were embedded in shooting medium for 1.5 months. (a*) Magnified view of one portion of the plate shown in (a). Bar = 1 cm. (b) Green shoot clusters were subcultured in shooting medium for 3 weeks. (b*) Magnified view of one portion of the plate shown in (b). Bar = 1 cm. (c) Albino calli were subcultured in shooting medium for 3 weeks. (c*) Magnified view of one portion of the plate shown in (c). Bar = 1 cm. (d) Albino calli were subcultured in shooting medium after 7 weeks. (d*) Magnified view of one portion of the plate shown in (d). Bar = 1 cm.

Figure 7

Targeted mutagenesis of NtPDS genes from regenerated tobacco protoplasts. Albino (A) and green (G) plantlets from regenerated protoplasts derived from 20 μg plasmid DNA treatment. Twenty plantlets of each type were analysed for NtPDS mutagenesis. The target sequences were amplified and validated using RFLP. S, N. sylvestris form; T, N. tomentosiformis form. Green box, wild‐type RFLP control; red box, albino mutant RFLP control.