8. Clone AtU6-sgRNA cassette and donor DNA into the
pCAMBIA3301:
X μL AtU6-sgRNA cassette (insert).
Y μL Donor DNA (insert).
10 ng pCAMBIA3301.
1 μL 10Â T4 DNA Ligase buffer.
0.5 μL T4 DNA Ligase
Add ddH 2 O to 10 μL.
The ratio of insert DNA mass (insert:vector ¼ 3 ~ 5:1) can be
calculated from the website https://nebiocalculator.neb.
com/#!/ligation. Incubate at 16
C overnight or room
temperature for 10 min.
9. Transform ligation product into E. coli as described above (see
Subheading 3.1.3, steps 7–9).
10. Screen for correct clones by colony PCR using
pCAMBIA3301-specific R primer and reverse oligo for
sgRNA (see Table 1). Correct clones will give PCR products
of approximately 650 bp. The procedure is described above (see
Subheading 3.1.3, step 10).
11. Miniprep 2–4 clones using the QIAprep Spin Miniprep Kit and
confirm the AtU6-sgRNA cassette and donor DNA in pCAMBIA3301 by DNA sequencing using pCAMBIA3301-specific
F and R primers (Figs. 4c and 5c, see Table 1).
12. Transform sgRNA and donor DNA in pCAMBIA3301 binary
vector into Agrobacterium competent cells (strain GV3101) as
described above (see Subheading 3.1.5, steps 1–6).
13. Transform the donor construct into the DD45 pro::Cas9 harboring parental line (CS69955 or CS69956) as described
above (see Subheadings 3.1.5, steps 7–13).
3.2.4 Screening of GeneTargeted Arabidopsis
Plants
1. Select T1 transgenic Arabidopsis plants by 50 mg/L glufosinate
ammonium antibiotic containing 0.5Â MS plate as described
above (see Subheading 3.1.6, steps 1–5).
2. Extract genomic DNA from T1 transgenic Arabidopsis leaves as
described above (see Subheadings 3.1.6, steps 6–18).
3. For knock-in, the positive gene targeting plants can be identified by PCR. False-positive bands would be detected when
GT-specific primer sets are used, due to hypersensitivity of the
primer sets. The external primer sets seem better option for
screening of true GT-positive plants (Fig. 9a, b). Perform PCR
including a WT DNA sample as a control:
0.5 μL Template extracted genome DNA.
2 μL 10Â Taq buffer.
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Daisuke Miki et al.
pCAMBIA3301:
X μL AtU6-sgRNA cassette (insert).
Y μL Donor DNA (insert).
10 ng pCAMBIA3301.
1 μL 10Â T4 DNA Ligase buffer.
0.5 μL T4 DNA Ligase
Add ddH 2 O to 10 μL.
The ratio of insert DNA mass (insert:vector ¼ 3 ~ 5:1) can be
calculated from the website https://nebiocalculator.neb.
com/#!/ligation. Incubate at 16
C overnight or room
temperature for 10 min.
9. Transform ligation product into E. coli as described above (see
Subheading 3.1.3, steps 7–9).
10. Screen for correct clones by colony PCR using
pCAMBIA3301-specific R primer and reverse oligo for
sgRNA (see Table 1). Correct clones will give PCR products
of approximately 650 bp. The procedure is described above (see
Subheading 3.1.3, step 10).
11. Miniprep 2–4 clones using the QIAprep Spin Miniprep Kit and
confirm the AtU6-sgRNA cassette and donor DNA in pCAMBIA3301 by DNA sequencing using pCAMBIA3301-specific
F and R primers (Figs. 4c and 5c, see Table 1).
12. Transform sgRNA and donor DNA in pCAMBIA3301 binary
vector into Agrobacterium competent cells (strain GV3101) as
described above (see Subheading 3.1.5, steps 1–6).
13. Transform the donor construct into the DD45 pro::Cas9 harboring parental line (CS69955 or CS69956) as described
above (see Subheadings 3.1.5, steps 7–13).
3.2.4 Screening of GeneTargeted Arabidopsis
Plants
1. Select T1 transgenic Arabidopsis plants by 50 mg/L glufosinate
ammonium antibiotic containing 0.5Â MS plate as described
above (see Subheading 3.1.6, steps 1–5).
2. Extract genomic DNA from T1 transgenic Arabidopsis leaves as
described above (see Subheadings 3.1.6, steps 6–18).
3. For knock-in, the positive gene targeting plants can be identified by PCR. False-positive bands would be detected when
GT-specific primer sets are used, due to hypersensitivity of the
primer sets. The external primer sets seem better option for
screening of true GT-positive plants (Fig. 9a, b). Perform PCR
including a WT DNA sample as a control:
0.5 μL Template extracted genome DNA.
2 μL 10Â Taq buffer.
142
Daisuke Miki et al.