Fig. 1

A newly developed RNA-guided Cas9 system. Diagrams depict modification of a previously reported RNA-guided Cas9 tool to an efficient multiplex editing vector system. Two adjacent cutting sites, KpnI and SpeI, and two other closely linked cutting sites, XbaI and SbfI, are located at 5′ and 3′ end of the sgRNA expression cassette, respectively (①). Two independent sgRNA expression cassettes can be combined when one of them is digested by KpnI and SpeI while the other one is digested by KpnI and XbaI (②). More sgRNA expression cassettes can be combined in one plasmid by repeating the same procedure (③). The combined sgRNA expression cassettes containing different guide sequences against different loci can be entirely isolated by cutting with KpnI and SbfI by which the binary vector containing the Cas9 expression cassette was also digested (④, ⑤). The UBIQUTIN 10 (UBQ10) promoter was used to drive the expression of Arabidopsis codon optimized Cas9 (pcoCas9) gene (④). pUBQ10: promoter of Arabidopsis UBIQUTIN 10 (UBQ10; AT4G05320) gene; pcoCas9: Arabidopsis codon optimized Cas9 gene; Atu6: any of Arabidopsis U6-1, U6-26 or U6-29 gene promoters