2.1 Cloning of the SaCas9 expression construct
The coding sequence for Cas9 nuclease from S. aureus (GenBank HE980450) has been supplemented with nuclear localization sequences (NLS) derived from SV40 large T antigen and from nucleoplasmin according to Li et al. [10] for subsequent use in planta and a Strep-tag II sequence [11] to facilitate purification of the recombinant protein. The coding sequence was codon-optimized for expression in Zea mays L. using the tool offered by Integrated DNA Technologies Inc. (IDT; https://eu.idtdna.com/CodonOpt). Synthetic DNA fragments (gBlocks) were purchased from IDT, and the coding sequence was merged by Gibson assembly using NEBuilder HiFi DNA Assembly Master Mix (NEB, Frankfurt/Main, Germany). The construct was subcloned into a modified pIVEX vector behind the T7 promoter [12]. The integrity of the construct was verified by Sanger sequencing. The nucleotide and amino acid sequences of rSaCas9 are shown in Figure S1 and S2, respectively.