3.2.2 Transfer
of gRNA(s) into
the Binary Vector
1. To transfer one gRNA, set up a Gateway reaction with 1 μL of
100 ng/μL pEn-Chimera, 1 μL of 100 ng/μL pDe-Cas9 (see
Note 32) and 0.5 μL of LR Clonase II. Proceed to step 3.
2. To transfer two gRNAs, set up a MultiSite Gateway reaction
with 1 μL of each pEn-Chimera (50 ng/μL), 4 μL of 50 ng/μL
pDe-Cas9 (see Note 32), 2 μL of Milli-Q water, and 2 μL of LR
Clonase II Plus.
3. Incubate at 25
C overnight.
4. Add 1 μL of 2 μg/μL Proteinase K.
5. Incubate at 37
C for 10 min.
6. Transform 2.5–5 μL of the Gateway reaction into competent
E. coli DH5α and plate on LB medium with 75 mg/L
spectinomycin.
7. Set up a colony-PCR for three colonies using pDe-F and
pDe-R as primers and anneal at 55
C. The expected band
size is 1180 bp and 1660 bp if one or two gRNAs are transferred, respectively.
8. Isolate binary vector DNA from two colonies using a plasmid
miniprep kit.
9. Conduct Sanger sequencing with primers pDe-F and pDe-R to
verify the presence of your gRNA(s).
3.3 Modular Vector
Construction for Up
to 12 gRNAs
3.3.1 Golden Gate Entry
Module for One gRNA
1. For one gRNA target site, order two complementary oligonucleotides with 4-bp overhangs (see Notes 27, 28 and 33):
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 50 ng/
μL entry vector (see Note 34), 5 units of BbsI-HF, 200 units of
T4 DNA ligase, 1.5 μL of 10 mM ATP, 1.5 μL of 10Â recommended restriction enzyme buffer and 9 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 3 min, 16
C for
3 min) Â 30 cycles, 50
C for 5 min, 80
C for 5 min, hold at
12
C.
6. Transform 5 μL of the Golden Gate reaction into competent
E. coli DH5α and plate on LB medium with 100 μg/mL
carbenicillin.
CRISPR-Cas-Mediated Gene Knockout
331
of gRNA(s) into
the Binary Vector
1. To transfer one gRNA, set up a Gateway reaction with 1 μL of
100 ng/μL pEn-Chimera, 1 μL of 100 ng/μL pDe-Cas9 (see
Note 32) and 0.5 μL of LR Clonase II. Proceed to step 3.
2. To transfer two gRNAs, set up a MultiSite Gateway reaction
with 1 μL of each pEn-Chimera (50 ng/μL), 4 μL of 50 ng/μL
pDe-Cas9 (see Note 32), 2 μL of Milli-Q water, and 2 μL of LR
Clonase II Plus.
3. Incubate at 25
C overnight.
4. Add 1 μL of 2 μg/μL Proteinase K.
5. Incubate at 37
C for 10 min.
6. Transform 2.5–5 μL of the Gateway reaction into competent
E. coli DH5α and plate on LB medium with 75 mg/L
spectinomycin.
7. Set up a colony-PCR for three colonies using pDe-F and
pDe-R as primers and anneal at 55
C. The expected band
size is 1180 bp and 1660 bp if one or two gRNAs are transferred, respectively.
8. Isolate binary vector DNA from two colonies using a plasmid
miniprep kit.
9. Conduct Sanger sequencing with primers pDe-F and pDe-R to
verify the presence of your gRNA(s).
3.3 Modular Vector
Construction for Up
to 12 gRNAs
3.3.1 Golden Gate Entry
Module for One gRNA
1. For one gRNA target site, order two complementary oligonucleotides with 4-bp overhangs (see Notes 27, 28 and 33):
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 50 ng/
μL entry vector (see Note 34), 5 units of BbsI-HF, 200 units of
T4 DNA ligase, 1.5 μL of 10 mM ATP, 1.5 μL of 10Â recommended restriction enzyme buffer and 9 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 3 min, 16
C for
3 min) Â 30 cycles, 50
C for 5 min, 80
C for 5 min, hold at
12
C.
6. Transform 5 μL of the Golden Gate reaction into competent
E. coli DH5α and plate on LB medium with 100 μg/mL
carbenicillin.
CRISPR-Cas-Mediated Gene Knockout
331