Fig. 3: Chimeric Cas9-VirD2 fusions mediate efficient editing of ALS in rice calli.

a Co-immunoprecipitation of the repair template with VirD2-Cas9 from rice protoplasts. Rice protoplasts transfected with Cy5-labeled repair templates (T-RB, templates with RB and T-NRB, templates without RB) and plasmid expressing VirD2-Cas9 using PEG-mediated delivery. VirD2-Cas9 was immunoprecipitated with anti-Flag bound agarose beads. Part of each sample was run on a 6% agarose gel and imaged (upper panel) for Cy5. The unlabeled (T-RB) and Cy5-labeled (Cy5-T-RB) repair templates were used as controls. A part of the sample was run on SDS-PAGE and the immunoblot was developed for the Flag tag (VirD2-Cas9-Flag) as input with anti-Flag and anti-mouse secondary antibody. b Schematic diagram of the in planta expression plasmids. The plant codon-optimized Cas9-VirD2 and VirD2-Cas9 complexes were cloned into a plant expression vector under the control of the Ubiquitin promoter followed by a terminator, and sgRNAs were cloned under the U6 promoter followed by a termination signal. c Confirmation of the in vivo endonuclease activity of the Cas9-VirD2 and VirD2-Cas9 fusions. The T7EI assay was conducted with 200 ng of target-flanking purified PCR product. A high rate of Indels was detected by the T7EI digestion assay of the samples expressing Cas9 (58%), Cas9-VirD2 (53%), or VirD2-Cas9 (47%) and sgRNA. T7EI-treated samples were separated on a 2% agarose gel. Arrowheads indicate the corresponding T7EI digestion at the Indel created by the endonuclease activity of the Cas9 fusions. d Alignments of the Sanger sequencing reads of the target-flanked PCR product cloned into the pJet2.1 plasmid. The top line shows the wild-type sequence with the target sequence in green and the PAM sequence underlined. Alignment analysis shows the presence of Indels at the target sites. The induced Indels are represented by numbers to the right of each lane.