off-target scores (see Notes 20 and 21) and (b) does not
contain a BpiI or BsaI site (see Note 22).
3. Design and synthesize a pair of PCR primers to amplify a
genomic region of approximately 700 bp that includes the
gRNA target site (see Notes 23–25). Extract genomic DNA
from your wild-type cultivar following the steps in Subheading
3.5.1 and Sanger sequence an amplified PCR product as
described in Subheading 3.5.3 to ensure the reference
sequence is correct and the designed primers are accurate (see
Note 26).
3.2 MultiSite-Puchta
Vector Construction
for One or Two
gRNA(s)
This protocol has been adapted from Schiml et al. [8].
3.2.1 Annealing
and Transfer
of Oligonucleotides into
the Entry Vector
1. For one gRNA target site, order two complementary oligonucleotides with 4-bp overhangs (see Notes 27–29):
Forward: 5
0 -ATTG-protospacer-3
0 ,
Reverse: 5
0 -AAAC-reverse complement of protospacer-3
0 .
2. Mix 1 μL of each 100 μM complementary oligonucleotide with
48 μL of Milli-Q water in a PCR tube.
3. Place in a thermal cycler and use the following parameters:
95
C for 5 min and 25
C for 20 min.
4. Mix 1 μL of the annealed oligonucleotides with 1 μL of
100 ng/μL entry vector (see Note 30), 3 μL of 10Â T4 DNA
ligase buffer, 3 μL of 1 mg/mL BSA, 5 units of BpiI, 5 units of
T4 DNA ligase and 20.5 μL of Milli-Q water in a PCR tube.
5. Place in a thermal cycler and run the following program to
perform a Golden Gate reaction: 37
C for 5 min, (20
C for
5 min, 37
C for 5 min) Â 50 cycles, 50
C for 10 min, 80
C
for 10 min, and hold at 12
C.
6. Transform 5 μL of the Golden Gate reaction into competent
E. coli DH5α and plate on LB medium with the appropriate
antibiotic (see Note 31).
7. Set up a colony-PCR for four colonies using pEn-F and your
protospacer reverse oligonucleotide as primers and anneal at
56
C. The expected band size is 280 bp.
8. Isolate pEn-Chimera DNA from two colonies using a plasmid
miniprep kit.
9. Conduct Sanger sequencing with primer pEn-F to verify the
presence of your spacer.
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Gwen Swinnen et al.
contain a BpiI or BsaI site (see Note 22).
3. Design and synthesize a pair of PCR primers to amplify a
genomic region of approximately 700 bp that includes the
gRNA target site (see Notes 23–25). Extract genomic DNA
from your wild-type cultivar following the steps in Subheading
3.5.1 and Sanger sequence an amplified PCR product as
described in Subheading 3.5.3 to ensure the reference
sequence is correct and the designed primers are accurate (see
Note 26).
3.2 MultiSite-Puchta
Vector Construction
for One or Two
gRNA(s)
This protocol has been adapted from Schiml et al. [8].
3.2.1 Annealing
and Transfer
of Oligonucleotides into
the Entry Vector
1. For one gRNA target site, order two complementary oligonucleotides with 4-bp overhangs (see Notes 27–29):
Forward: 5
0 -ATTG-protospacer-3
0 ,
Reverse: 5
0 -AAAC-reverse complement of protospacer-3
0 .
2. Mix 1 μL of each 100 μM complementary oligonucleotide with
48 μL of Milli-Q water in a PCR tube.
3. Place in a thermal cycler and use the following parameters:
95
C for 5 min and 25
C for 20 min.
4. Mix 1 μL of the annealed oligonucleotides with 1 μL of
100 ng/μL entry vector (see Note 30), 3 μL of 10Â T4 DNA
ligase buffer, 3 μL of 1 mg/mL BSA, 5 units of BpiI, 5 units of
T4 DNA ligase and 20.5 μL of Milli-Q water in a PCR tube.
5. Place in a thermal cycler and run the following program to
perform a Golden Gate reaction: 37
C for 5 min, (20
C for
5 min, 37
C for 5 min) Â 50 cycles, 50
C for 10 min, 80
C
for 10 min, and hold at 12
C.
6. Transform 5 μL of the Golden Gate reaction into competent
E. coli DH5α and plate on LB medium with the appropriate
antibiotic (see Note 31).
7. Set up a colony-PCR for four colonies using pEn-F and your
protospacer reverse oligonucleotide as primers and anneal at
56
C. The expected band size is 280 bp.
8. Isolate pEn-Chimera DNA from two colonies using a plasmid
miniprep kit.
9. Conduct Sanger sequencing with primer pEn-F to verify the
presence of your spacer.
330
Gwen Swinnen et al.