Gene editing by co-inoculation of TRSV-SpCas9 and TRV carrying sgRNA. (A) Detection of mutations in the NbTOM1a/b, NbAGb, NbFLS2a/b target site 1 or 2 and NbPDSa/b by CAPS analysis in the 10th upper leaves from TRSV (and TRV)-inoculated leaves of N. benthamiana plants inoculated with virus vectors denoted. The 20-nt sgRNA target sequence and the NGG PAM sequence are shown in blue and brown, respectively, and the recognition sites of restriction enzymes (AvaI, MfeI, TspRI, AvaI and NcoI) are underlined. Lanes represent individual plants. The positions of undigested bands are indicated as ‘edited’. Lanes of the fifth panel were rearranged. (B) Schematic representation of TRV vectors. 35S, CaMV 35S promoter; Rz, self-cleaving ribozyme; nos, nopaline synthase terminator; PEBV Pro, subgenomic RNA promoter of pea early-browning virus; FT, full-length Arabidopsis thaliana Flowering locus T open reading frame sequence; 134K, 194K, MP, 16K and CP are TRV-encoded proteins. (C) Heritability of the introduced mutations in the NbTOM1 or NbAG genes. Mutation frequencies of each allele and nucleotide sequences of the target sites with mutations are shown in Supplementary Table S3 and S4, respectively. (D) Detection of TRSV, TRV and ALSV RNAs in the progenies produced by seeds from plants inoculated with ALSV-NbRDR6, TRSV-SpCas9 and TRV-NbTOM1 by RT-PCR. The endogenous N. benthamiana Actin gene was used as an internal control. Progeny number 36 had the mutation. ‘Uninoc’ and ‘inoc’ indicate an uninoculated (negative control) and inoculated (positive control) N. benthamiana plant, respectively.