Fig. 4: Chimeric Cas9-VirD2 fusions with phosphorothioate-modified repair templates make efficient edits at the ALS target locus.

a Schematic of the desired edit (green) in the wild-type OsALS allele. The desired modification (W548L), the MfeI recognition site, and the modified PAM sequence in the repair template are shown. The repair template (red line, right and left homology arms) with the desired modification (green) was used to make edits at the target site (black line). The forward allele-specific PCR primer is represented by an arrow corresponding to the desired modification and the reverse primer outside the homology arm sequence. b Allele-specific confirmation of successful editing in the rice callus. The allele-specific PCR from the DNA extracted from 24 individual calli bombarded with phosphorothioate-modified repair templates and unmodified repair templates with and without RB sequence (T-NRB, mT-NRB, T-RB and mT-RB) with Cas9 or Cas9-VirD. The phosphorothioate-modified repair templates are more stable than the unmodified repair templates and enhance the rate of HDR in combination with Cas9-VirD2. The amplification of the exact size 417 bp fragment only in the VirD2-Cas9 or Cas9-VirD2 with mT-RB samples by allele specific PCR (indicated by the arrow head) confirmed the presence of the herbicide resistance allele. The lower band is a nonspecific PCR amplification and present in all samples. c Molecular confirmation of the exact insertion of the desired nucleotide sequence at the target locus via MfeI digestion. Restriction digestion of the target-flanked PCR product from the DNA of pooled selected calli (N = 100). Using ImageJ software, the rate (in %) of the exact repair (MfeI recognition site insertion) was calculated and is represented below the corresponding lane. Cas9 alone was used as the baseline control. The arrowheads represent the MfeI-digested fragments.