0.05 μL Taq Polymerase
0.4 μL Forward primer (Plasmid Specific; e.g., AtU6–26sgRNA Forward) (10 μM)
0.4 μL Reverse primer (Reverse Oligo) (10 μM)
Fig. 6 Preparation of CRISPR/Cas9 vector containing single sgRNA. The flow charts show preparation of
CRISPR/Cas9 T-DNA construct containing single sgRNA by using (a) two plasmids or (b) one plasmid (see
Fig. 2, top panel). To prepare single sgRNA containing construct from two plasmids AtU6-sgRNA cassette is
digested with restriction enzyme, followed by ligation with annealed oligo, digestion of binary vector, and
lastly cloning into Cas9 harboring pCAMBIA1300 vector. However, for one plasmid system, annealed oligo is
directly cloned into the AtU6 sgRNA cassette harboring binary vector by using suitable restriction enzymes
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0.4 μL Forward primer (Plasmid Specific; e.g., AtU6–26sgRNA Forward) (10 μM)
0.4 μL Reverse primer (Reverse Oligo) (10 μM)
Fig. 6 Preparation of CRISPR/Cas9 vector containing single sgRNA. The flow charts show preparation of
CRISPR/Cas9 T-DNA construct containing single sgRNA by using (a) two plasmids or (b) one plasmid (see
Fig. 2, top panel). To prepare single sgRNA containing construct from two plasmids AtU6-sgRNA cassette is
digested with restriction enzyme, followed by ligation with annealed oligo, digestion of binary vector, and
lastly cloning into Cas9 harboring pCAMBIA1300 vector. However, for one plasmid system, annealed oligo is
directly cloned into the AtU6 sgRNA cassette harboring binary vector by using suitable restriction enzymes
CRISPR/Cas9-Based Genome Editing in Arabidopsis
133