2.3 Cloning of the sgRNA expression construct
Target sites for SaCas9 in the upstream region of the maize CCD8 gene
were identified using the Cas-designer tool [14]. Ten target sites
were selected (Figure S3), with six sequences derived from the plus
strand (T2-T7) and four derived from the minus strand (T1 and T8-T10).
Synthetic DNA (gBlocks) comprising the T7 promoter, single guide RNAs
(sgRNAs) and a 3’ Dra I site (Figure S4) were purchased from IDT
(Coralville, Iowa, USA) and subcloned into a modified pIVEX vector
[12].