selected for the next generation. To confirm heritable gene
mutations, an additional screening and genotyping should be
performed in the T2 generation. Furthermore, if necessary, the
obtained mutants should be backcrossed with WT to remove
Cas9 transgene and to purify genetic background.
3.2 Gene Targeting
3.2.1 Design of sgRNA
1. Design sgRNA by following the instructions given above (see
Subheading 3.1.1). The sgRNA should be designed close to
the knock-in or amino acid substitution site (Figs. 4a and 5a)
(see Note 1).
2. Prepare AtU6-sgRNA expression cassette in the pBluescript SK
background, if it is necessary amplify it by using certain restriction enzyme harboring primers, as described above (see Subheadings 3.1.3 and 3.1.4).
3.2.2 Design of
Donor DNA
Designing homology arms of 500–800 bp should be enough for
efficient gene targeting (Figs. 4a and 5b) (see Note 6). Silent
mutations must be incorporated at the PAM sequence to avoid
additional DSBs [25]. If it is impossible, at least three silent mutations should be integrated at the protospacer target sequence
(Fig. 5a).
3.2.3 Preparation of
Donor T-DNA Construct for
Agrobacterium
Transformation
1. Prepare donor DNA using high-fidelity PCR. The donor DNA
fragment is prepared by two rounds of PCR (Figs. 4b and 5b).
Homology arms and knock-in sequence fragments are amplified in the first round and they are fused in the second round:
0.5 μL Template genomic DNA or plasmid.
PCR reaction buffer (final concentration 1Â).
1.6 μL dNTP (2.5 mM).
0.5 μL High-fidelity polymerase (e.g., KOD DNA
polymerase).
0.4 μL Forward primer (10 μM).
0.4 μL Reverse primer (10 μM)
Add ddH 2 O to 20 μL.
Perform PCR: 95
C for 5 min, 30 cycles of 94
C for 30 s,
60
C for 30 s and 72
C for 1 min, and additional 5 min of
72
C.
2. Run 1.2% agarose gel and gel-purify the PCR product by using
QIAquick Gel Extraction Kit; elute the PCR product into
30 μL elution buffer.
3. Carry out the second round PCR (Figs. 4b and 5b):
0.5 μL Each purified template first round PCR products (see
Note 7).
PCR reaction buffer (final concentration 1Â).
140
Daisuke Miki et al.
mutations, an additional screening and genotyping should be
performed in the T2 generation. Furthermore, if necessary, the
obtained mutants should be backcrossed with WT to remove
Cas9 transgene and to purify genetic background.
3.2 Gene Targeting
3.2.1 Design of sgRNA
1. Design sgRNA by following the instructions given above (see
Subheading 3.1.1). The sgRNA should be designed close to
the knock-in or amino acid substitution site (Figs. 4a and 5a)
(see Note 1).
2. Prepare AtU6-sgRNA expression cassette in the pBluescript SK
background, if it is necessary amplify it by using certain restriction enzyme harboring primers, as described above (see Subheadings 3.1.3 and 3.1.4).
3.2.2 Design of
Donor DNA
Designing homology arms of 500–800 bp should be enough for
efficient gene targeting (Figs. 4a and 5b) (see Note 6). Silent
mutations must be incorporated at the PAM sequence to avoid
additional DSBs [25]. If it is impossible, at least three silent mutations should be integrated at the protospacer target sequence
(Fig. 5a).
3.2.3 Preparation of
Donor T-DNA Construct for
Agrobacterium
Transformation
1. Prepare donor DNA using high-fidelity PCR. The donor DNA
fragment is prepared by two rounds of PCR (Figs. 4b and 5b).
Homology arms and knock-in sequence fragments are amplified in the first round and they are fused in the second round:
0.5 μL Template genomic DNA or plasmid.
PCR reaction buffer (final concentration 1Â).
1.6 μL dNTP (2.5 mM).
0.5 μL High-fidelity polymerase (e.g., KOD DNA
polymerase).
0.4 μL Forward primer (10 μM).
0.4 μL Reverse primer (10 μM)
Add ddH 2 O to 20 μL.
Perform PCR: 95
C for 5 min, 30 cycles of 94
C for 30 s,
60
C for 30 s and 72
C for 1 min, and additional 5 min of
72
C.
2. Run 1.2% agarose gel and gel-purify the PCR product by using
QIAquick Gel Extraction Kit; elute the PCR product into
30 μL elution buffer.
3. Carry out the second round PCR (Figs. 4b and 5b):
0.5 μL Each purified template first round PCR products (see
Note 7).
PCR reaction buffer (final concentration 1Â).
140
Daisuke Miki et al.