The plant codon-optimized Streptococcus pyogenes Cas9 (spCas9) was expressed under the 35S promoter in the binary expression vector pK2GW7. Two constructs (PK2GW7-Cas9-E1 and PK2GW7-Cas9-E2) were developed to target the eIF4E gene (Table 1). The eIF4E gene was amplified by specific primers (Figure 1A), and gRNAs were designed on the conserved region of exon-1 (sequencing data in Supplementary Figure S1A). The PCR product confirmed the presence of the gRNA cassette (U6 promoter, gRNA, and gRNA scaffold) in the vector (Figure 1B). The 545-bp bands were eluted from a chimeric plasmid cloning vector and transferred to the expression vector pK2GW7 by HindIII restriction-ligation reactions (Figures 1C,D). The PCR results confirmed the presence of Cas9 (∼4.1 kb; Supplementary Figure S4A). The final constructs were confirmed by Sanger sequencing (Figures 1E,F). A complete diagrammatic illustration of the vector map is shown in Figure 1G.

Agrobacterium-mediated transformation (GV3101 strain) generated 25 transgenic lines for gRNA1 and 15 lines for gRNA2 targeting the eIF4E gene. The mutated lines were confirmed by Sanger sequencing and multiplied before shifting to soil (Figure 2).

The editing by Cas9 was tested by PCR amplification of the targeted regions of eIF4E and Sanger sequencing. Initially, PCR was performed to confirm the integration of Cas9 and the gRNA cassette into the plant genome and was followed by sequencing. We sequenced a total of 40 PCR-positive transgenic lines. In 25 of the 40 lines, eIF4E was targeted with gRNA1, and with gRNA2 in the other 15. The targeting efficiency of the CRISPR/Cas9 system in “Kruda” cultivar was around 15%. The targeted region of eIF4E was sequenced to determine the mutations. The purified PCR product was ligated into the pTZ57R/T vector by TA cloning, and 20 clones from each line were sequenced. The target regions were successfully mutated by insertion/deletion (Indels) and incorporation of single-nucleotide polymorphisms (SNPs). The gRNA1 targeted the most conserved region of the eIF4E gene near 5′-UTR. In the mutated lines K_E1.8, K_E1.9, K_E1.16, and K_E1.46, eIF4E was targeted with gRNA1, and the editing events were characterized by Sanger sequencing, which confirmed large deletions in one of the alleles of 25 bp, 15 bp, and 139 bp and insertion of 10 bp, 35 bp, and 42 bp in the K_E1.16 and K_E1.46 lines, respectively (shown in Table 2; Supplementary Figures S1A–D). We found that the SNPs in the coding regions in K_E1.46 generate stop codons that ultimately truncate the protein synthesis. The gRNA2 targeting eIF4E generated SNPs in K_E2.9 and K_E2.13 lines (Supplementary Figures S1E,F). In K_E2.9, a nucleotide with single base pair polymorphism and 3-bp deletions was observed, while in K_E2.13, three different events (a 1-bp addition and 3- and 1-bp deletions) were observed (Table 2, with gRNAs in red, PAM in purple, and editing events in green). These are synonymous mutations. We validated the resistance/tolerance capacity of these mutated alleles by ELISA and reverse transcription–polymerase chain reaction (RT-PCR).

The resistance level of the mutated alleles was validated by ELISA and RT-PCR. Sixty-day mature mutated and wild-type lines were inoculated with PVY. Visual symptoms such as mosaic pattern on the leaves were observed on the wild-type plants (Figure 3). At 7, 15, 30, and 60 days post-infection (dpi), DAS-ELISA was performed. The analysis provided co-relating results with phenotypic observations: the wild-type lines exhibited common symptoms of PVY infection such as stunting, leaf mottling, crinkling, yellowing, and necrosis of leaves, while the mutated lines remained healthy without any visual symptoms of infection (Figure 3). The specificity and sensitivity of DAS-ELISA for PVY detection were confirmed by testing the extracts from PLRV-, PVX-, PVA-, PVS-, and PVM-infected potato samples along with negative controls. The negative results indicated the specificity and sensitivity of DAS-ELISA for PVY. The PVY titer was estimated through visual (yellow) observation and by ELISA-reading at 405 nm (Figure 3). Compared to the wild-type plants, all mutated lines showed a gradual decrease in virus titer and showed resistance at 60 dpi of PVY inoculation. The mutated lines K_E1.8, K_E1.9, K_E1.16, and K_E1.46 showed stronger resistance compared to K_E2.9 and K_E2.13 (Figure 3). Tubers were collected from PVY-resistant and PVY-tolerant lines, as shown in Supplementary Figures S2A,B. Phenotypically strong resistance was achieved by gRNA1 in lines K_E1.8, K_E1.9, K_E1.16, and K_E1.46, while tolerance was gained against PVY by gRNA2 in lines K_E2.9 and K_E2.13.

Real-time PCR (RT-PCR) was performed to determine the functional expression of eIF4E and Cas9 in tetraploid plants. The plants were treated with PVY and classified into the following: 1) non-inoculated wild-type Kruda as negative control; 2) PVY-inoculated wild-type Kruda as positive control; and 3) PVY-inoculated K_E1.8, K_E1.9, K_E1.16, K_E1.46, K_E2.9, and K_E2.13 eIF4E mutated lines. After 20 days of inoculation, symptoms appeared on the inoculated wild-type Kruda plants. Samples were collected at 20 and 30 dpi from PVY-resistant mutated and wild-type lines. The presence of virus in the samples was confirmed by DAS-ELISA. Total RNA was isolated, and cDNA was synthesized for the quantification of the relative expression of eIF4E and Cas9 genes (Supplementary Figure S3A). To determine the functional expression of Cas9 in these mutated lines, the relative expression of Cas9 in mutated lines K_E1.8, K_E1.9, K_E1.16, K_E1.46, K_E2.9, and K_E2.13 was determined by using specific primers (shown in Table 3). The relative expression of Cas9 gene is shown in Figure 4. The relative expression of eIF4E was higher in the wild-type control plant than in mutated lines, as determined by the 2^−ΔΔCT method (using primers shown in Table 3). The quantitative expression of eIF4E in mutated lines K_E1.8, K_E1.9, K_E1.16, K_E1.46, K_E2.9, and K_E2.13 was many folds lower than that in the wild-type (Figure 4), as indels caused the frameshift for the expression of eFI4E.

The PVY pathosystem in ‘Kruda’ cultivar was validated by determining the viral load in the mutated lines and quantified by quantitative reverse transcription PCR (RT-qPCR). The optimal thermal cycling conditions were used to obtain the standard curve, with almost equal efficiency of amplification of the samples as shown in Supplementary Figure S3B. The mutated lines K_E1.8, K_E1.9, K_E1.16, K_E1.46, K_E2.9, and K_E2.13 of eIF4E and wild-type plants were inoculated with PVY. The copy number of PVY was determined by RT-qPCR, which indicated low or very low titer in mutated lines, compared to inoculated wild-type plants. The serial dilutions for RT-qPCR are shown in Table 4. The mutated lines E1.46 and E1.16 showed very low to zero virus accumulation and transmission at regular intervals. Interestingly, a decrease in the intensity of viral titer was observed in the E1.8 line at 45 dpi, compared to the wild-type plants (Figure 4). The confirmed resistant mutated lines were shifted from pots to soil to collect the tubers.

The tetraploid Kruda cultivar of potato was selected for establishing PVY resistance. Control plants were obtained from the Gene Transformation Lab (National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan). Two days prior to transformation, the internodal stem cuttings from control plants were placed on MS medium (Murashige and Skoog, 1962) in a growth chamber with a photoperiod of 16 h and a temperature of 22°C (±1°C). DAS-ELISA (Andersen and Johansen, 1998) was performed against single-stranded RNA (ssRNA) viruses for confirmation of virus-free parental lines.

In this study, Solanum tuberosum cv. Kruda plants were used. To find out the exact sequence of the susceptibility gene eIF4E, primers were designed on the NCBI-available sequence (NM_001288431) to amplify the coding region of the gene (Table 5). CTAB and Trizole (Invitrogen, United States) were used to extract genomic DNA (Doyle, 1990) and RNA, respectively, for the amplification of the coding region. PCR products of 635 bp for the coding sequence of eIF4E was amplified by using specific primers (Table 5, P1) according to the manufacturer’s standard PCR conditions (Catalog # K1082).

This coding sequence was cloned into Ptz57R/T (Catalog# K1213, Thermo Fisher Scientific, United States) for sequencing. The 20-bp gRNAs were designed on the conserved regions of exon1 of the eIF4E gene. All gRNAs were designed manually and screened for potential off-targets using the online Cas-OFFinder tool (http://www.rgenome.net/cas-offinder/). The gRNAs were cloned into the BbsI site of the p. chimera vector under the A. thaliana-U6 promoter. The Pk2GW7-Cas9 construct was obtained from the Laboratory for Genome Engineering and Synthetic Biology, Centre for Desert Agriculture, KAUST, Saudi Arabia. The binary PK2GW7-Cas9 vector was designed with a unique Hind III site, and the gRNA cassette was inserted into the Pk2GW7-SpCas9 vector using Hind III restriction and ligation methods. Plant codon-optimized spCas9 (approximately 4.1 kb) was amplified by using high-fidelity polymerase (M0530S) following the manufacturer’s instructions, and further confirmation of Cas9 was performed using the diagnostic primer for Cas9 (Table 5).

The Kruda internode cuttings were placed on MS medium for 2 days prior to transformation by Agrobacterium-mediated transformation (GV³¹⁰¹ strain) harboring constructs. For each gRNA, approximately 100 cuttings were transformed. Each construct was grown independently with rifampicin and spectinomycin antibiotic selection. Optical density (O.D₆₀₀) of the bacterial liquid culture was measured, and 0.6 O.D. was used for the transformation of the internodes. The liquid culture was pelleted down with 4430 g for 5 min and washed three times with liquid MS medium, and final O.D. was measured for transformation. A time duration of 20–25 min was used for the incubation of internodes in the liquid MS medium with the Agrobacterium construct. The cuttings were placed in a dark room overnight at 25–28°C for potato transformation. After overnight incubation, the transformed internodal cuttings were washed with timentin and then placed on kanamycin medium (50 mg/L) for selection. The transformed lines were transferred to callus induction medium (CIM) for callus induction. The cuttings were transferred continuously on a weekly basis for up to 40 days. The surviving calli were then transferred to regeneration medium, and regenerated plantlets were transferred to shooting medium followed by rooting medium with variable amounts of plant growth hormones. Before transferring to soil (clay and sand), the mutated lines were multiplied in a growth chamber, and three clonal replicates of each mutated line were transferred to sand and kept in a greenhouse under controlled conditions (25°C (±1°C) temperature, light period for 16 h, and with insect/pest proof). After 2 weeks of hardening, the lines were shifted into large pots for tuber development and phenotypic assays.

The transgenic lines were confirmed for the presence of Cas9 and the gRNA cassette. DNA was extracted from the mutated and control lines using the CTAB method. P. chimera forward (PCF) and reverse (PCR) primers (Table 5), Cas9 primers, and pNeomycin phosphotransferase II (NptII) gene (responsible for kanamycin resistance) primers (Table 5) were used for the gRNA cassette, Cas9 gene, and NptII confirmation, respectively. For the identification of insertion/deletions (Indels), 188 bp sequences consisting of eIF4E target regions were amplified by primers (Table 5) and cloned into the pTZ57R/T vector, and 20 clones from each line were sent for sequencing.

The screening of mutated lines for resistance against PVY was carried out in a glass house under controlled environmental conditions. The mutated and wild-type lines were hardened for 2 weeks in pots containing a mixture of sand and peat moss. Triple superphosphate (CaH₂PO₄·2H₂O) at 128 kg per hectare and muriate of potash (KCl) at 485 kg per hectare were applied to strengthen the potato lines. After 2 months of maturation, mutated plants from each line were selected for inoculation of PVY virus by applying viral sap mixed with carborundum powder on the lower side of the leaves. The experiment was conducted in three batches. The control wild-type lines were arranged with six mutated lines and treated with PVY, despite the fact that the control line was PVY susceptible (Hull, 2009). All these lines were screened for PVY resistance using a local virulent PVY strain. The inoculum was prepared to infect the wild-type control and transgenic plants with the virus. The leaf sap of PVY-infected plants was extracted by crushing the leaves in 0.5% sodium acetate solution. The transgenic plants were inoculated with the filtered leaf sap after mixing with carborundum (1 mg/ml) powder. The control lines were inoculated in the same pattern as the mutated lines. PVY from the crude extract was applied to the wild-type plants at various concentrations (1:60 dilutions) to determine the sensitivity of PVY detection. At 7, 15, 30, and 60 dpi, the response of the mutated plants was examined, and data were recorded by observing the mosaic pattern on leaves. Further confirmation was carried out by DAS-ELISA, and the quantitative expression of eIF4E in mutated lines was analyzed by RT-qPCR.

Screening of PVY was performed by DAS-ELISA in the wild-type and mutated Kruda lines. Leaves from the infected non-transgenic control plants and tolerant/resistant plants mutated for PVY resistance were collected. About 1 g of leaves was ground in distilled water and then the kit-protocol (Catalog# V093) was followed for titer confirmation of PVY in the mutated and control lines. The DAS-ELISA was performed at regular intervals of 7, 15, 30, and 60 dpi. DAS-ELISA was performed for PVS, PLRV, PVA, and PVM with PVY to find out the screening sensitivity of DAS-ELISA.

Total RNA was extracted from the inoculated potato leaves using Trizol reagent (Invitrogen, United States) following the manufacturer’s instructions. The extracted RNA was treated with gradient DNase I (Thermo Fisher Scientific, United States) as per the manufacturer’s instructions for the removal of DNA contamination. Complementary DNA (cDNA) was synthesized using the RevertAid First Strand cDNA synthesis kit (Thermo Fisher Scientific, United States). The cDNA was synthesized by using oligo (dT₁₈) primers and gene-specific Cas9-reverse primers for eIF4E and Cas9, respectively. The expression of eIF4E upon PVY treatment was analyzed in both mutated and control plants by observing relative expression via RT-PCR. The eIF4E transcripts were amplified using St-IF4E RT-F and eIF4E RT-R primers (Table 3), and the functional Cas9 transcripts were analyzed by using Cas-RT-F and Cas-RT-R primers (Table 3). Reaction mixtures of a volume of 25 µl were prepared using 12.5 µl SYBR Green Real-Time PCR Master Mix (Thermo Fisher Scientific, United States), 0.1 pmole of F&R primers, 2 µl cDNA, and 9.5 µl water. The reaction was performed under optimized conditions using a Bio-Rad iQ5 thermal cycler (Bio-Rad, United States). The actin-97 gene was used as an endogenous control for expression using an actin-F/actin-R primer set (Table 3). The quantification results were analyzed by the 2^−ΔΔCT method (Livak and Schmittgen, 2001). To find the virus copy number, analysis of the absolute expression was performed and each treatment was replicated three times.