Fig. 2: Somatic and heritable editing in Arabidopsis using TRV to deliver ISYmu1 TnpB and guide RNA targeting AtPDS3.
From: Viral delivery of an RNA-guided genome editor for transgene-free germline editing in Arabidopsis
a, Schematic of the TRV1 and TRV2 plasmids. Green arrows indicate the RNA-dependent RNA polymerase (RDRP) and pPEBV promoters for TRV1 and TRV2, respectively; the grey boxes in TRV1 and TRV2 indicate the native TRV components; the red Cargo box in TRV2 indicates the location of either Architecture_A or Architecture_B; below TRV2 are schematics of the components, Architecture_A or Architecture_B, cloned into the TRV2 Cargo slot. b, Representative picture of a plant displaying white sectors in leaves (yellow arrows) ~3 weeks after TRV delivery. c,d, Barplot displaying the somatic editing efficiencies (percent indel reads) (Y axis) for ISYmu1 gRNA2 in WT and ku70 genetic backgrounds (c) and for ISYmu1 gRNA12 in WT (d). The TRV2 cargo architectures are plotted along the X axis with either room or HS treatment. Each dot represents an individual plant that underwent agroflood TRV delivery. The s.e.m. was calculated for each experiment. e,f, DNA indel repair profile for an individual WT plant that underwent delivery of TRV Cargo Architecture_B with ISYmu1 gRNA2 (e) or with ISYmu1 gRNA12 (f) under the heat-shock treatment. The top five most common indel types are listed on the left. The read counts for each indel are listed on the right. The PAM is identified by the red box, and the target site is outlined by the black box, in the Reference sequence. The total read number and editing efficiency are listed below each indel profile. g, Representative image of albino and green progeny seedlings from a WT plant showing 54.54% somatic editing using the TRV2 Architecture_B design with gRNA2 that underwent heat-shock treatment. h, Sanger sequencing trace file screenshot from one of the albino plants in Fig. 3a. Top: sequence of the wild-type reverse complement. Middle: the ISYmu1 gRNA2 target and PAM (grey box). Bottom: the ab1 trace file displaying a homozygous 4 bp deletion. i, Table summarizing the transmission of edited alleles from two individual plants that underwent agroflood delivery using ISYmu1 gRNA12. The ‘Progeny screened’ column indicates the number of seedlings genotyped; the ‘Biallelic edits (%)’ column indicates the number of seedlings containing biallelic edits; and the ‘Monoallelic edits (%)’ column indicates the number of plants harbouring monoallelic edits.
