Impacts of RNA Mobility Signals on Virus Induced Somatic and Germline Gene Editing

Figures

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  FIGURE 1. Effects of RNA mobility signals on FoMV-induced gene editing of NbPDS in Nicotiana benthamiana. (A) Schematic representation of the foxtail mosaic virus (FoMV) vector. The FoMV genome is transcribed under the control of a 2X cauliflower mosaic virus 35S promoter (35S) and the nopaline synthase (NOS) terminator. The FoMV genome consists of an RNA-dependent RNA polymerase (RdRP), triple gene block proteins (TGB1, 2, 3), and coat protein (CP), flanked by 5′ and 3′ untranslated regions (UTRs). sgRNAs or sgRNAs fused to RNA mobility signals are located in the insertion site (IS) between TGB3 and CP. Small black arrows above the FoMV genome represent the positions of the subgenomic promoters. (B) Images of systemic leaves of wild type or Cas9 N. benthamiana plants that were either mock-inoculated or inoculated with FoMV:sgNbPDS (PDS) 5 weeks post-inoculation. (C) RT-PCR amplification of FoMV in systemic leaf 8, approximately 5 weeks after inoculation. Primers flanking the IS of FoMV were used to demonstrate the stability of sgRNAs and sgRNAs fused to AtFT or AttRNAIle. Intact FoMV:sgRNA is represented by a 571 bp amplicon, FoMV:sgRNA-AtFT is represented by a 673 bp amplicon, and…

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  FIGURE 2. Effects of RNA mobility signals on FoMV-induced mutations in ZmHKT1. (A) Percentage of Cas9 maize plants displaying systemic FoMV infection 6 weeks post-injection, as determined by RT-PCR. Data are represented as box plots indicating the 25%–75% interquartile range, split by a median line. Whiskers represent maximum and minimum values. A one-way ANOVA indicated no significant differences in infection rates between treatments (p = 0.7024). (B) RT-PCR analysis of FoMV in systemic Z. mays leaves 6 weeks post-infection. Primers were designed to flank the insertion site to assess stability of sgRNA and sgRNA-mobility signal insertions. Intact FoMV:sgRNA is represented by a 571 bp amplicon, FoMV:sgRNA-AtFT, FoMV:sgRNA-ZCN8, FoMV:sgRNA-ZCN16, and FoMV:sgRNA-ZCN19 are represented by a 673 bp amplicon, and FoMV:sgRNA-AttRNAIle is represented by a 646 bp amplicon. ZmActin was used as an internal control, and NTC is the no template control. (C) Representative gel of FoMV-induced somatic mutations in ZmHKT1 (see Supplementary Figure S2 for additional gels). A fragment of ZmHKT1 containing the sgZmHKT target site was PCR amplified from genomic DNA isolated from systemic leaf tissue, 6 weeks…

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  FIGURE 3. Somatic and germline mutations in NbPDS induced by BSMV, PVX or TRV containing a sgRNA alone or fused to truncated AtFT. (A) Systemic leaves of Cas9 N. benthamiana plants infected with BSMV:sgNbPDS, PVX:sgNbPDS, and TRV:sgNbPDS. (B) RT-PCR amplification of the indicated virus spanning sgRNA insertion sites from systemic parental leaf tissue. NbPP2A was used as an internal control. (C) NbPDS amplicons from systemic leaf DNA of plants infected with the indicated viral constructs following digestion with NcoI. Digestion of unedited NbPDS amplicons generates two fragments migrating to 256 and 541 bp. Non-digested fragments represent NbPDS editing due to disruption of the NcoI site, and have similar size to the non-digested control (NDC) amplicon (797 bp). For PVX and TRV, PVX:sgPDS and TRV:sgPDS infection of wild type N. benthamiana represents the negative control for somatic mutations. For BSMV experiments, the negative control for somatic mutations was Cas9 N. benthamiana infected with BSMV with no sgPDS. (D) Representative images of progeny from NbCas9 plants infected with BSMV:sgNbPDS, PVX:sgNbPDS, and TRV:sgNbPDS, with or without mobility signals. White seedlings represent…