Potato Virus X Vector-Mediated DNA-Free Genome Editing in Plants

Abstract

Genome editing technology is important for plant science and crop breeding. Genome-edited plants prepared using general CRISPR-Cas9 methods usually contain foreign DNA, which is problematic for the production of genome-edited transgene-free plants for vegetative propagation or highly heterozygous hybrid cultivars. Here, we describe a method for highly efficient targeted mutagenesis in Nicotiana benthamiana through the expression of Cas9 and single-guide (sg)RNA using a potato virus X (PVX) vector. Following Agrobacterium-mediated introduction of virus vector cDNA, >60% of shoots regenerated without antibiotic selection carried targeted mutations, while ≤18% of shoots contained T-DNA. The PVX vector was also used to express a base editor consisting of modified Cas9 fused with cytidine deaminase to introduce targeted nucleotide substitution in regenerated shoots. We also report exogenous DNA-free genome editing by mechanical inoculation of virions comprising the PVX vector expressing Cas9. This simple and efficient virus vector-mediated delivery of CRISPR-Cas9 could facilitate transgene-free gene editing in plants.

Keywords: Nicotiana benthamiana; CRISPR-Cas9; Plant genome editing; RNA virus; Virus vector.

Fig. 1

Targeted mutagenesis by agroinoculation with PVX vector expressing Cas9 and sgRNA. (A) Schematic diagram of a PVX-based SpCas9 expression vector. A full-length SpCas9 sequence was placed after a duplicated subgenomic promoter for coat protein (shown by arrows). The sgRNA was fused to the 3′ end of the SpCas9 coding sequence. 35S, Cauliflower mosaic virus 35S RNA promoter; RdRp, RNA-dependent RNA polymerase; TGB, triple gene block; CP, coat protein; nosT, nopaline synthase terminator; LB, left border of T-DNA; RB, right border of T-DNA. (B) Western blot analysis of SpCas9 accumulation in PVX-Cas9-inoculated Nicotiana benthamiana leaves at 5 dai. Lanes 1 and 2 are independent plants. Lane M, protein size markers; n.c., non-inoculated control N.�benthamiana leaf. (C) CAPS analysis for the detection of introduced mutations in agroinfiltrated N.�benthamiana leaves. Mixtures of Agrobacterium strains harboring pRI-p19, pDe-Cas9 and pPZP2028_AtU6Sp_NbTOM1, or pRI-p19 and pPZPVX-Cas9_NbTOM1 were infiltrated and DNA was extracted from four independent plants at 7�dai. PCR products containing the target site in NbTOM1a/b genes were digested with AvaI. Black triangle indicates undigested bands. Green letters indicate the PAM sequence. n.c., nontreatment control.

Fig. 2

Transgene integration-free genome editing using PVX vector through regeneration without selection. (A) Flowchart for PVX-mediated genome editing of N.�benthamiana. Throughout the process, no antibiotics for the selection of genome-edited cells were added to the medium. (B) Characterization of regenerated shoots. Mutations in the target site within the NbTOM1a/b gene were detected by CAPS analysis. PCR products were digested with AvaI (top). Presence of T-DNA derived from pPZPVX-Cas9_NbTOM1 was detected by PCR (middle). PVX RNA was detected by RT-PCR (bottom). WT, untreated N. benthamiana; pc, pPZPVX-Cas9 plasmid DNA was used as a PCR template for positive control; PVX, RNA extracted from PVX-infected N. benthamiana was used as a template for RT-PCR. (C) Summary of the mutation and T-DNA integration rates in shoots regenerated from PVX-Cas9-inoculated leaves from three independent experiments. These rates were calculated from the sum of three independent experiments. (D) DNA sequences around the target region in NbTOM1a/b in regenerated shoots. WT, wild-type sequence. Underlined bases indicate the target sequence and green letters indicate the PAM sequence. Red letters represent DNA insertion or substitution, and dashes indicate deletion.

Fig. 3

C-to-T base editing using a PVX vector. Schematic diagram of pPZPVX-AID. 35S, Cauliflower mosaic virus 35S promoter; RdRp, RNA-dependent RNA polymerase; TGB, Triple gene block; CP, coat protein; nosT, nopaline synthase terminator; PmCDA1, Petromyzon marinus cytidine deaminase 1; LB, left border of T-DNA; RB, right border of T-DNA. (B) Detection of mutations in regenerated shoots by CAPS analysis. Shoots were regenerated from pPZPVX-AID_NbTOM1-inoculated N. benthamiana leaves as in Fig.�2A. PCR products encompassing the target site in the NbTOM1a/b gene were digested with AvaI (upper panel). Presence of T-DNA derived from pPZPVX-AID_NbTOM1 was detected by PCR (lower panel). Black triangle indicates undigested bands. (C) Summary of the mutation and T-DNA integration rates of shoots regenerated from pPZPVX-AID-inoculated leaves. (D) DNA sequences around the target region of NbTOM1a/b (analyzed collectively) in regenerated shoots. WT, wild-type sequence. Underlined bases indicate the target sequence, and green letters indicate the PAM sequence. Red letters represent nucleotide substitutions.

Fig. 4

DNA-free genome editing using a PVX vector. (A) Flowchart for PVX-mediated DNA-free genome editing of N.�benthamiana. Filter-sterilized leaf sap from PVX-Cas9 agroinfiltrated leaves was used for mechanical inoculation. Throughout the process, no antibiotics for the selection of genome-edited cells were added to the medium. (B) CAPS analysis for the detection of introduced mutations in mechanically inoculated N.�benthamiana leaves. DNA was extracted from three independent plants at 18�dai. PCR products containing the target site in NbTOM1a/b genes were digested with AvaI. Black triangle indicates undigested bands. (C) Characterization of regenerated shoots. Mutations in the target site within the NbTOM1a and NbTOM1b genes were independently detected by CAPS analysis. PCR products were digested with AvaI. (D) Summary of the mutation rates in shoots regenerated from PVX-Cas9-inoculated leaves in two independent experiments. (E) DNA sequences around the target region.