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.