16. Agarose, loading buffer and TAE (40 mM Tris (pH 7.6),
20 mM acetic acid, 1 mM EDTA).
17. Agarose gel electrophoresis tank and power supply.
2.2 Agrobacterium
Transformation
1. Agrobacterium tumefaciens strain GV3101 competent cells.
2. T-DNA plasmid (Fig. 2).
3. LB liquid and solid medium with antibiotics.
Fig. 3 Detailed sgRNA design. (a) Partial sequence of AtU6-26 promoter, sgRNA cloning site, and scaffold. BbsI
restriction enzyme recognition sites are represented in green letters. The annealed oligo is cloned into the
restriction sites. (b) Arabidopsis endogenous GLABRA 2 (GL2; At1g79840) gene and targeting sgRNA
sequence. Upper schematic represents genomic sequence structure of GL2 gene, and two arrows indicate
PCR primers used for genotyping (see Fig. 8b, Table 1). Red bases indicate PAM sequence of designed sgRNA,
horizontal arrow indicates sgRNA target site, and square represents PstI site used for mutation screening in
RFLP assay (see Fig. 8a, b). (c) Designed sgRNA oligomer to target GL2 gene
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