Prgeb32

To create a single guide (sg) RNA-Cas vectors, one set of oligomers targeting the second exon of OsSNDP3 was synthesized. The synthesized 24 bp oligos were ligated with a Cas9 expression backbone vector pRGEB32 (Addgene plasmid ID: 63,142). Ligation products were transformed into E. coli. And then, the construct was transformed into A. tumefaciens LBA4404. The Agrobacterium-mediated stable transformation was used to create transgenic plants. Genomic DNA was extracted from transgenic plants, and PCRs were carried out with gene-specific primers. To check for mutations, PCR products were purified and sequenced.

The VirD2 open reading frame was PCR amplified from the total DNA isolated from Agrobacterium tumefacien strain GV3101 and cloned into the pENTER-D-Topo vector, and the sequence was confirmed by Sanger sequencing. To express Cas9-VirD2 or VirD2-Cas9 under the T7 promoter, the VirD2 open reading frame was fused in-frame to the N or C terminus of Cas9 in pET28a (Addgene plasmid #53261) following the Gibson assembly protocol. For in planta expression, the plant codon-optimized sequence of VirD2 was custom synthesized and cloned in-frame to either the N or C terminus of Cas9 in pRGE32 and pRGEB32 (Addgene plasmid #63142) following the Gibson assembly protocol. sgRNAs as a primer dimer were first cloned into the entry clone under the U3 promoter by Golden Gate cloning with BsaI. The whole cassette, U3::sgRNA, was PCR amplified and cloned into the HindIII and SbfI sites in the pRGEB-Cas9-VirD2 and pRGEB-VirD2-Cas9 vectors. The sequences of all primers used in PCR amplification are provided in Supplementary Table 3.