4. Cloning sgRNA cassettes into Cas9 expression binary vector.
Procedure is described above (see Subheading 3.1.3, steps 15–
18) (Fig. 7) (see Note 4).
3.1.5 AgrobacteriumMediated Arabidopsis
Transformation
1. Transform sgRNA with Cas9 binary vector, which was constructed at Subheadings 3.1.3, step 18 or Subheading 3.1.4,
step 4, into Agrobacterium tumefaciens competent cells (strain
GV3101). Add 1 μL T-DNA binary vector construct into
50 μL of Agrobacterium competent cells.
2. Incubate on ice for 5 min.
3. Incubate in liquid nitrogen for 5 min.
4. Incubate at 37
C on heat block or water bath for 5 min.
5. Add 1 mL of LB, mix well, and incubate at 28
C for 1 h.
6. Spread 100 μL on a LB plate with 50 mg/L kanamycin and
50 mg/L rifampicin (or gentamicin) at 28
C for 2 days.
7. Pick a single colony of Agrobacterium and culture it in 8 mL LB
liquid medium with 50 mg/L kanamycin and 50 mg/L rifampicin (or gentamicin) at 28
C overnight.
Fig. 7 Preparation of CRISPR/Cas9 vector containing multiple sgRNAs. The flow
chart shows construction of CRISPR/Cas9 T-DNA construct for multiple sgRNA by
using one plasmid (see Fig. 2, middle panel). One or some sgRNA expression
cassette(s), AtU3b-sgRNA in this Figure, are cloned into the AtU6 sgRNA cassette
and Cas9 harboring binary vector by using suitable restriction enzymes
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Daisuke Miki et al.
Procedure is described above (see Subheading 3.1.3, steps 15–
18) (Fig. 7) (see Note 4).
3.1.5 AgrobacteriumMediated Arabidopsis
Transformation
1. Transform sgRNA with Cas9 binary vector, which was constructed at Subheadings 3.1.3, step 18 or Subheading 3.1.4,
step 4, into Agrobacterium tumefaciens competent cells (strain
GV3101). Add 1 μL T-DNA binary vector construct into
50 μL of Agrobacterium competent cells.
2. Incubate on ice for 5 min.
3. Incubate in liquid nitrogen for 5 min.
4. Incubate at 37
C on heat block or water bath for 5 min.
5. Add 1 mL of LB, mix well, and incubate at 28
C for 1 h.
6. Spread 100 μL on a LB plate with 50 mg/L kanamycin and
50 mg/L rifampicin (or gentamicin) at 28
C for 2 days.
7. Pick a single colony of Agrobacterium and culture it in 8 mL LB
liquid medium with 50 mg/L kanamycin and 50 mg/L rifampicin (or gentamicin) at 28
C overnight.
Fig. 7 Preparation of CRISPR/Cas9 vector containing multiple sgRNAs. The flow
chart shows construction of CRISPR/Cas9 T-DNA construct for multiple sgRNA by
using one plasmid (see Fig. 2, middle panel). One or some sgRNA expression
cassette(s), AtU3b-sgRNA in this Figure, are cloned into the AtU6 sgRNA cassette
and Cas9 harboring binary vector by using suitable restriction enzymes
136
Daisuke Miki et al.