Materials and Reagents

1. 1.5 ml microcentrifuge tubes (SARSTEDT, catalog number: 72.690.001 )
   
2. 200 µl PCR tubes (Labomedic, catalog number: 2081644AA )
   
3. Petri dishes (SARSTEDT, catalog number: 82.1472 )
   
4. 2 ml microcentrifuge tubes (SARSTEDT, catalog number: 72.691 )
   
5. 20 µl pipette tips (SARSTEDT, catalog number: 70.1116 )
   
6. 200 µl pipette tips (SARSTEDT, catalog number: 70.760.012 )
   
7. 1,000 µl pipette tips (SARSTEDT, catalog number: 70.762.010 )
   
8. Agrobacterium tumefaciens (A. tumefaciens) strain GV3101::pMP90
   
9. Arabidopsis thaliana seeds (Col-0)
   
10. Vectors (see Figure 1)
    
    
    Figure 1. Vector maps of pUB-Cas9 (A) and pFH6 (B). pFH6 is used to integrate the 20 bp target sequence upstream of the sgRNA scaffold and under the control of the Arabidopsis U6-26 RNA polymerase III promoter. The whole sgRNA cassette is then transferred via Gibson cloning into the binary T-DNA vector pUB-Cas9 (contains the Cas9 gene under the control of the Ubiquitin10 promoter) for plant transformation. Maps were generated using SnapGene Viewer (http://www.snapgene.com/products/snapgene_viewer/).
    
    
    1. Plant T-DNA Cas9 vector pUB-Cas9 (GenBank accession number KY080691), containing a Chlamydomonas reinhardtii codon-optimized ubiquitously (UBIQUITIN10 promoter) expressed Cas9 gene, a kanamycin resistance cassette for bacterial selection and a hygromycin resistance cassette as plant selection marker (Hahn et al., [2017]; available at Addgene, catalog number: 86556 )
       
    2. sgRNA subcloning vector pFH6 (GenBank accession number KY080689) containing the Arabidopsis U6-26 promoter, the integration site for the 20 bp protospacer sequence, the sgRNA scaffold and an ampicillin resistance cassette (Hahn et al. [2017]; available at Addgene, catalog number: 86555 )
       Note: pFH6 contains an additional 9 bp fragment (GTCCCTTCG) between the 3’ end of the U6-26 promoter and the protospacer integration site. In several experiments, we could show that this does not affect gene editing activity. However, we have also cloned a new version of the subcloning vector without the additional fragment (pFH6_new), which shows high cleavage activity in preliminary experiments and can be obtained from us. This version only contains an additional guanine (G) between the 3’ end of the U6-26 promoter and the protospacer integration site, which allows the integration of any 20 bp protospacer without restriction of G as first bp (compare e.g., Fauser et al. [2014]). The cloning strategy for pFH6_new is analogous to the one described in this protocol, the only difference is that the forward primer for cloning your 20 bp protospacer sequence contains a different overlap and lacks the need for an initial G at the beginning (ATTG-N20, compare procedure section).
       
11. Competent Escherichia coli (E. coli) cells (e.g., Mach1^TM competent cells, Thermo Fisher Scientific, Invitrogen^TM, catalog number: C862003 )
    
12. BbsI-HF + CutSmart buffer (New England Biolabs, catalog number: R3539S )
    
13. Distilled H₂O
    
14. Plasmid mini prep kit and agarose gel extraction kit (e.g., GeneMATRIX 3 in 1–Basic DNA Purification Kit, Roboklon, catalog number: E3545 )
    
15. T4 DNA ligase with 10x ligation buffer (New England Biolabs, catalog number: M0202S )
    
16. Ampicillin (Amp) (Carl Roth, catalog number: K029.2 )
    
17. Primers (see Table 1)
    
    Table 1. List of oligonucleotides
    
    
    
18. 5x Green GoTaq Reaction buffer (Promega, catalog number: M791A )
    
19. dNTPs (10 mM each) (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: R0192 )
    
20. GoTaq G2 polymerase (Promega, catalog number: M7841 ) or other standard PCR polymerase
    
21. HindIII-HF + CutSmart buffer (New England Biolabs, catalog number: R3104S )
    
22. KpnI-HF + CutSmart buffer (New England Biolabs, catalog number: R3142S )
    
23. Phusion High-Fidelity polymerase (New England Biolabs, catalog number: M0530S ) or other proofreading polymerases
    
24. Gibson Assembly Cloning Kit (New England Biolabs, catalog number: E5510S )
    
25. Kanamycin sulfate (Kan) (Carl Roth, catalog number: T832.4 )
    
26. Rifampicin (Rif) (Molekula, catalog number: 32609202 )
    
27. Gentamycin sulfate (Gent) (Carl Roth, catalog number: 0233.3 )
    
28. Hygromycin B (Hyg) (Carl Roth, catalog number: CP12.2 )
    
29. T7 Endonuclease I (optional; New England Biolabs, catalog number: M0302S )
    
30. LB medium (agar plates and liquid), supplemented with 200 μg/ml ampicillin (see Recipes)
    
31. LB medium (agar plates and liquid), supplemented with 30 μg/ml kanamycin sulfate (see Recipes)
    
32. YEP medium (agar plates and liquid), supplemented with 150 μg/ml rifampicin, 50 μg/ml gentamycin sulfate, 50 μg/ml kanamycin (see Recipes)
    
33. ½ MS medium agar plates, supplemented with 33.3 μg/ml hygromycin B (see Recipes)
    

Equipment

1. Agarose gel electrophoresis equipment (e.g., VWR, Peqlab, model: PerfectBlue^TM Gel System Mini M, catalog number: 700-0434 )
   
2. Bacteria plate incubators (28 °C, 37 °C, e.g., Memmert, model: IN55 ) and shaker (28 °C, 37 °C; e.g., Eppendorf, New Brunswick^TM, model: Innova^® 44 , catalog number: M1282-0002)
   
3. Heating blocks (e.g., Eppendorf, model: Thermomixer Compact , catalog number: T1317-1EA)
   
4. PCR cycler (e.g., Thermo Fisher Scientific, Applied Biosytems^TM, model: Veriti^TM 96-well Thermal Cycler, catalog number: 4375786 )
   
5. 10 µl pipette (e.g., Pipetman Neo P10N, Gilson, catalog number: F144562 )
   
6. 20 µl pipette (e.g., P20N, Gilson, catalog number: F144563 )
   
7. 200 µl pipette (e.g., P200N, Gilson, catalog number: F144565 )
   
8. 1,000 µl pipette (e.g., P1000N, Gilson, catalog number: F144566 )
   
9. Plant growth chamber (e.g., CLF Plant Climatics, Percival Scientifici, model: AR-66L )
   

Software

1. SerialCloner
   
2. Cas-OFFinder
   
3. 4Peaks software
   
4. MUSCLE (Optional)
   

Procedure