2 Materials
2.1 Vector
Construction for
Single/Multiple Gene
Mutations and Base
Editing
1. AtU6 sgRNA cassette plasmid (pBluescript SK background or
binary vector background for one plasmid strategy).
2. Cas9 nuclease expression cassette harboring binary vector
(we preferentially use YAO or CDC45 promoter to drive
Cas9 expression in pCAMBIA1300 binary vector (Fig. 2)
[11, 14].
3. Forward and reverse oligo of sgRNA (100 μmol/L) (Fig. 3, see
Table 1).
4. Restriction enzymes BbsI, HindIII, and SalI.
5. T4 DNA Ligase and reaction buffer.
6. DH5α E. coli chemical competent cells.
7. Luria Bertani (LB) liquid medium (1% tryptone, 0.5% yeast
extract, 1% sodium chloride) and solid medium (with
1.5% agar).
8. Antibiotics: carbenicillin (or ampicillin) and kanamycin.
9. QIAquick Gel Extraction Kit (Qiagen, #28704).
10. QIAprep Spin Miniprep Kit (Qiagen, #27104).
11. Thermocycler (e.g., Applied BiosytemsTM).
12. TaKaRa Taq DNA Polymerase (TaKaRa, #R001A).
13. Incubators (37 and 16
C).
14. Zero Blunt TOPO PCR Cloning kit (Thermo Fisher Scientific,
#450245).
15. Absorption spectrometer (e.g., NanoDrop).
Fig. 2 CRISPR/Cas9 constructs. Schematic view of T-DNA constructs containing single (top) or multiple
(middle) sgRNAs, and base editing system (bottom). The expression of single sgRNA is driven by AtU6-26 Pol III
promoter. The expression of multiple sgRNAs is driven by a combination of other promoters such as AtU3b and
At7SL-2 harbored on a same T-DNA construct with Cas9 expression cassette. The base editing construct
consists of d/nCas9 fused with the base editors (CBE or ABE)
CRISPR/Cas9-Based Genome Editing in Arabidopsis
125
2.1 Vector
Construction for
Single/Multiple Gene
Mutations and Base
Editing
1. AtU6 sgRNA cassette plasmid (pBluescript SK background or
binary vector background for one plasmid strategy).
2. Cas9 nuclease expression cassette harboring binary vector
(we preferentially use YAO or CDC45 promoter to drive
Cas9 expression in pCAMBIA1300 binary vector (Fig. 2)
[11, 14].
3. Forward and reverse oligo of sgRNA (100 μmol/L) (Fig. 3, see
Table 1).
4. Restriction enzymes BbsI, HindIII, and SalI.
5. T4 DNA Ligase and reaction buffer.
6. DH5α E. coli chemical competent cells.
7. Luria Bertani (LB) liquid medium (1% tryptone, 0.5% yeast
extract, 1% sodium chloride) and solid medium (with
1.5% agar).
8. Antibiotics: carbenicillin (or ampicillin) and kanamycin.
9. QIAquick Gel Extraction Kit (Qiagen, #28704).
10. QIAprep Spin Miniprep Kit (Qiagen, #27104).
11. Thermocycler (e.g., Applied BiosytemsTM).
12. TaKaRa Taq DNA Polymerase (TaKaRa, #R001A).
13. Incubators (37 and 16
C).
14. Zero Blunt TOPO PCR Cloning kit (Thermo Fisher Scientific,
#450245).
15. Absorption spectrometer (e.g., NanoDrop).
Fig. 2 CRISPR/Cas9 constructs. Schematic view of T-DNA constructs containing single (top) or multiple
(middle) sgRNAs, and base editing system (bottom). The expression of single sgRNA is driven by AtU6-26 Pol III
promoter. The expression of multiple sgRNAs is driven by a combination of other promoters such as AtU3b and
At7SL-2 harbored on a same T-DNA construct with Cas9 expression cassette. The base editing construct
consists of d/nCas9 fused with the base editors (CBE or ABE)
CRISPR/Cas9-Based Genome Editing in Arabidopsis
125