Materials and Reagents

1-ml syringe
0.22-μm filter
Wild type N. benthamiana plants; six to eight-leaf stage transgenic N. benthamiana plants stably expressing oCas9 (Arabidopsis codon optimized Cas9) - KQ334 plant
Agrobacterium tumefaciens strain GV3101, E. coli DH5α, E. coli DB3.1 (for propagating vectors pJG081)
The following plasmids are required: pJG081, pKQ334 (Yin et al., 2015), pT-U6p-scaffold-U6t (Yin et al., 2015), pCAMBIA1301, pCAMBIA2300 (www.cambia.org), pCVA (Tang et al., 2010), pCVB (Tang et al., 2010), and pMD18-T vector (Takara Bio, catalog number: D101A)
Notes:

To generate a construct for constitutive expression of oCas9, oCas9 is PCR amplified and subsequently cloned into pJG081 by ligation independent cloning (LIC) technique (Aslanidis and de Jong, 1990) to generate pKQ334.
pKQ334 is then used to transform N. benthamiana to generate Cas9 transgenic plant - KQ334 plant. pKQ334 can also be used in transient expression of Cas9 in N. benthamiana.
The pT-U6p-scaffold-U6t is used to generate p-T-U6p-gRNA-scaffold containing gRNA of the target gene by PCR mutagenesis.
pCVA and pCVB are CaLCuV-based T-DNA vectors. pCVA is used to generate pCVA-gRNA or pCVA-scaffold. pCVB is used in combination with pCVA or its derivatives to generate CaLCuV. pMD18-T vector is used to clone PCR amplicons.
These vectors can be obtained by contacting Dr. Yule Liu.

Primers (Table 1)

Table 1. Primers used in this protocol
TransStart FastPfu DNA polymerase (Beijing TransGen Biotech, catalog number: AP221 )
Restriction enzymes (New England Biolabs, stored at -20 °C)

DpnI (New England Biolabs, catalog number: R0176 )
ApaI (New England Biolabs, catalog number: R0114 )
SacI (New England Biolabs, catalog number: R0156 )
PstI (New England Biolabs, catalog number: R3140S )
KpnI (New England Biolabs, catalog number: R3142S )
XbaI (New England Biolabs, catalog number: R0145S )
MlyI (New England Biolabs, catalog number: R0610 )

10x Cutsmart buffer
Antibiotics
Ampicillin (AMRESCO, catalog number: 0339 )
Kanamycin (AMRESCO, catalog number: 0408 )
Rifampicin (Sigma-Aldrich, catalog number: R3501 )
TIANprep Mini Plasmid Kit (TIANGEN Biotech, catalog number: DP103 ) for plasmid DNA extraction
Ethanol (Beijing Chemical Factory, catalog number: B0301002 )
T4 DNA polymerase (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: EP0062 )
dATP (Takara Bio, catalog number: 4026 )
Chloroform (Sigma-Aldrich, catalog number: 1601383 )
Phenol (Sigma-Aldrich, catalog number: P4557 )
3 M NaAc (Biovision, catalog number: 2118 )
dTTP (Takara Bio, catalog number: 4029 )
DNA Purification Kit (BioMED, catalog number: DH103-01 )
T4 DNA ligase (Takara Bio, catalog number: 2011A )
Tryptone (BD, Bacto^TM, catalog number: 211705 )
Yeast extract (BD, Bacto^TM, catalog number: 212750 )
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 31434 )
Magnesium chloride hexahydrate (MgCl₂·6H₂O) (Sigma-Aldrich, catalog number: M7304 )
MES (Sigma-Aldrich, catalog number: M3671 )
Potassium hydroxide (KOH)
Acetosyringone (Solarbio, catalog number: A8110-1 )
DMSO
Sodium phosphate monobasic (NaH₂PO₄) (Sigma-Aldrich, catalog number: S3139 )
Sodium phosphate dibasic (Na₂HPO₄) (Sigma-Aldrich, catalog number: S3264 )
Sodium phosphate monobasic (NaH₂PO₄) (AMRESCO, catalog number: 0571 )
Sodium phosphate dibasic (Na₂HPO₄) (AMRESCO, catalog number: 0404 )
Sodium phosphate monobasic monohydrate (NaH₂PO₄·H₂O)
Sodium phosphate dibasic heptahydrate (Na₂HPO₄·7H₂O)
X-gluc (AMRESCO, catalog number: 0919 )
Methanol (Beijing Chemical Factory, catalog number: B0301005 )
Potassium ferricyanide (AMRESCO, catalog number: 0713 )
Triton X-100 (Sigma-Aldrich, catalog number: T9284 )
PEG 8000 (Sigma-Aldrich, catalog number: 89510 )
Magnesium chloride hexahydrate (MgCl₂·6H₂O) (AMRESCO, catalog number: 0288 )
Magnesium chloride hexahydrate (MgCl₂·6H₂O) (Sigma-Aldrich, catalog number: 63068 )
MES (Sigma-Aldrich, catalog number: M3671 )
Acetosyringone (Sigma-Aldrich, catalog number: D134406 )
EasyTaq DNA polymerase (Beijing TransGen Biotech, catalog number: AP111 )
DNAsecure Plant Kit (TIANGEN Biotech, catalog number: DP320 ) for genomic DNA extraction
Luria-Bertani medium (see Recipes)
MgCl₂ (1 M stock) (see Recipes)
MES (0.5 M stock) (see Recipes)
Acetosyringone (1 M stock) (see Recipes)
Infiltration buffer (see Recipes)
2x phosphate buffer (pH 7) (see Recipes)
X-gluc substrate solution (see Recipes)
PEG-MgCl₂ solution (see Recipes)

Equipment

Microcentrifuge (Eppendorf)
PCR cycler (Bio-Rad Laboratories)
37 °C and 28 °C incubator; 37 °C and 28 °C shaker
NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, model: NanoDrop 1000 ) (for measuring the concentration of the DNA)
Electrophoresis apparatus (Junyi, Beijing)
Vacuum apparatus (WILMAD-LABGLASS)
Glass/plastic beaker
Autoclave

Software

ImageJ (https://imagej.nih.gov/ij)

Procedure

gRNA design for specific gene/site of interest
For knocking out a gene of interest, choose gRNAs located in the exons. For editing the non CDS region in the genome, choose a region where disruption would have this desired effect. We strongly recommend using CRISPR-P (cbi.hzau.edu.cn) to design gRNAs (Lei et al., 2014). It provides a specificity score for each possible gRNA and lists the potential off-targets. Additionally, it provides a list of useful restriction enzymes that would recognize the gRNA spacer for RFLP analysis of this specific site.
Construct gRNA plasmid
To drive gRNA expression in plants, we constructed a gRNA plasmid targeting NbPDS. Any other genes-of-interest can also be targeted. To clone the gRNA easily, we first used a small vector (T-U6p-scaffold-U6t). Then, the promoter-gRNA-terminator construct is transferred to a larger binary vector, such as pCAMBIA2300 and pCVA.
1. Perform inverse PCR (Stemmer and Morris, 1992) to add gRNA spacer between the AtU6 promoter and gRNA scaffold necessary for Cas9 binding (Figure 1). Setup reactions as follows:
  
  Figure 1. Illustration of pT-U6p-scaffold-U6t (Yin et al., 2015) and the use of inverse PCR for inserting gRNA spacer into pT-U6p-scaffold-U6t to generate pT-U6p-gRNA-scaffold-U6t. A. AtU6 promoter, scaffold, and AtU6 terminator are shown in orange boxes on the plasmid map. Two primers used for inserting gRNA spacer (targeting NbPDS) are indicated on the plasmid map with their respective directions. Parallel blacklines on the primers are complementary sequences. SacI and PstI are used to cut the gRNA expression cassette which is then cloned into a binary vector. The small arrow here is to indicate scaffold. B. Sequence information for inverse PCR. Here, gRNA spacer targeting NbPDS is used as an example. Partial sequence of the NbPDS locus is shown in black. PAM and MlyI site are shown. gRNA spacer is also shown in black above the NbPDS locus. Two primers (oYK1056 and oYK1057) for PCR are shown, the sequence from AtU6 is shown in red, and the sequence from scaffold is shown in blue. The two primers that bind to the template (vector) during the PCR process are also shown with primers and vector indicated. The change after inverse PCR is shown below the arrow.
2. Perform PCR amplification using the following parameters (T_m represents the annealing temperature of the primer pair):
3. Setup a restriction enzyme digest to remove the plasmid template in the above PCR product as follows:
  Note: DpnI is a methylation sensitive enzyme, and plasmid purified from E. coli DH5α is generally methylated. Thus, DpnI is often used to remove plasmid template. Here, a ligation or a denaturation/reannealing step is not necessary to connect the ends of the vector, because the PCR product with overlapping ends will re-circularize to form a doubly-nicked plasmid and will be repaired further in bacteria to form an intact plasmid.
  
  Mix well and incubate at 37 °C for 2 h.
4. Transform E.coli DH5α using 1-2 μl product of the above reaction with heat shock at 42 °C for 90 sec, recover at 37 °C for 1 h, plate on LB plate with 100 mg/L ampicillin, and then incubate overnight at 37 °C (Im, 2011).
5. Select one to five transformants and culture overnight in LB with 100 mg/L ampicillin at 37 °C, 220 rpm. Extract plasmids from the transformants respectively with TIANprep Mini Plasmid Kit, and Sanger sequence the plasmid pT-U6p-gRNA:NbPDS-scaffold-U6t with sequencing primer M13-47 to confirm the presence of the gRNA spacer.
Check Cas9 activity and gRNA efficiency using fsGUS (frameshift GUS) reporter system
It’s necessary to confirm if KQ334 plants retain Cas9 activity and that Cas9 cleaves DNA under the guidance of gRNA. In this system, a 23 bp sequence with a PAM (AGG) at the end is inserted downstream of the start codon of the GUS gene, which causes a frame shift of the GUS gene (fsGUS). We co-expressed two vectors: one harboring the fsGUS gene driven by a 35S promoter termed pJG081-fsGUS and the other p2300-gRNA:fsGUS containing an Arabidopsis U6-26 gene promoter (AtU6 promoter)-driven gRNA containing a 20 bp sequence that matches the first 20 bp of the sequence inserted into the fsGUS gene.
There is another highly efficient method to check gRNA efficiency based on protoplast transfection. Please refer to Li and co-workers’ protocol (Li et al., 2015)
1. Setup a PCR reaction to amplify an fsGUS fragment in which a short sequence containing the gRNA spacer sequence and a MlyI recognition site is inserted after the GUS start codon (Figure 2). Primers used at this step should be designed specifically to flank the cut sites of different gRNAs.
  
  Figure 2. Illustration of the fsGUS reporter system used to check gRNA efficiency. Illustration of the fsGUS reporter system. A 23 bp sequence containing a MlyI site is inserted after the start codon of the GUS gene, and the resulting sequence is designated as fsGUS. Due to the inserted sequence, fsGUS cannot be translated into a functional enzyme. fsGUS is driven by a CaMV35S promoter and placed in a binary vector. Another binary vector contains AtU6 promoter-driven gRNA that targets the first 20 bp of the inserted sequence in fsGUS. Two A. tumefaciens lines transformed with each binary vector are mixed together and infiltrated into KQ334 plant leaves. After two T-DNAs enter into the same cell, the first 20 bp of the inserted sequence in fsGUS is targeted by the Cas9/gRNA complex for cleavage. Repair via NHEJ will create in-dels at the cleavage site and, in some cases, recover the normal coding frame for fsGUS, thus creating a functional GUS which can be detected by GUS staining. (This figure is adapted and modified from Yin et al., 2015.)
  
  Perform PCR amplification using the following parameters (T_m represents the annealing temperature of the primer pair):
2. Purify the PCR product using PEG/MgCl₂ method (Paithankar and Prasad, 1991)
3. Construct pJG081-fsGUS using LIC method.
  Note: LIC is a fast, simple, and very cheap cloning technique. It utilizes annealing of complementary overhangs of a vector and a PCR fragment consisting of 9-15 bases.
4. Digest pJG081 for LIC reaction:
5. Setup T4 DNA polymerase treatment for this linear pJG081:
  
  The resulting product is designated as pJG081-LIC.
6. Construct pJG081-fsGUS:
7. Construct p2300-gRNA:fsGUS.
8. Agrobacterium transformation
9. Transient expression
  Note: Either Cas9 transgenic plants (KQ334 plants) or another Agrobacterium culture containing pKQ334 plasmid (constitutive expression of Cas9) can be used for this experiment.
10. GUS staining
  The GUS staining assay is based on Kabbage et al. (2011) and performed as follows:
Construct pCVA-gRNA and pCVA-scaffold
To perform VIGE of the target gene, pCVA-gRNA is first generated by inserting a cassette containing an AtU6 promoter and gRNA::NbPDS into pCVA. The construct pCVA-scaffold is taken as a negative control.
1. Setup PCR reactions as follows:
2. Perform PCR amplification using the following parameters (T_m represents the annealing temperature of the primer pair):
3. Then purify the product with the DNA Purification Kit according to manufacturer recommendations.
4. Setup a restriction enzyme reaction as follows:
5. Incubate at 37 °C for 3 h. Then purify the product with the DNA Purification Kit. The final products are U6p-gRNA-scaffold_KpnI/XbaI and U6p-scaffold_KpnI/XbaI.
6. At the same time, setup a restriction enzyme reaction as follows:
7. Incubate at 37 °C for 3 h. Then purify the product with the DNA Purification Kit according to manufacturer recommendations.
8. Next, setup a ligation reaction as follows to generate pCVA-gRNA and pCVA-scaffold
9. Incubate at 16 °C for 8 h and transform it into E. coli DH5α, which is then selected on medium with kanamycin.
VIGE of a gene/site of interest
1. First, transform pCVA-gRNA, pCVA-scaffold and pCVB respectively into Agrobacterium tumefaciens strain GV3101 by heat shock method. Select the transformants on medium with kanamycin and rifampicin.
2. Next, the Agrobacterium cultures are inoculated to 5 ml Luria-Bertani medium containing appropriate antibiotics (50 mg L^-1 rifampicin, and 50 mg L^-1 kanamycin) and grown overnight in a 28 °C shaker.
3. Agrobacterium cells are harvested by centrifugation at 5,000 x g for 5 min and resuspended in infiltration buffer, adjusted to an optical density at 600 nm of 1.0 and incubated at room temperature for 3 to 4 h before infiltration.
4. Agrobacterium cultures containing pCVB and pCVA or their derivatives are mixed at a 1:1 ratio and infiltrated into petioles of six to eight-leaf stage KQ334 plants using a 1-ml syringe (Tang et al., 2010) (Figure 3 and Video 2). As a control, they are also infiltrated into petioles of six to eight-leaf stage wild-type plants using the same method.
  
  Figure 3. Illustration of VIGE. The Agrobacterium containing either pCVA-gRNA or pCVB are mixed with each other and infiltrated into petiole of KQ334 plants (Cas9 transgenic plants). Systemic leaves are used to check the genome editing events. AtU6 promoter is used to drive the expression of gRNA. The gRNA spacer is shown in red. CR, common region; AL1, replication-associated protein; AL2, transcription activator; AL3, replication enhancer; AL4, putative pathogenesis-related protein; BL1 and BR1, movement proteins; LB, left border; RB, right border. (This figure is adapted and modified from Yin et al., 2015.)
  
  Video 2. Agro-infiltration into petiole of N. benthamiana
Detection of VIGE mediated mutations in plant genomic DNA
1. For the detection of VIGE results, genomic DNA extracted from systemic leaves according to the manufacturer’s instructions is used as template for PCR amplification (see below) of a targeted locus with primers (PDS_MlyIF and PDS_MlyIR) flanking the target site using EasyTaq DNA polymerase. PCR product is purified and digested with selected restriction enzyme (i.e., MlyI for NbPDS) and run on a 2% agarose gel.
2. Mutation rate is estimated by comparing band intensities using ImageJ (https://imagej.nih.gov/ij). The uncut band is then cut from the gel and the DNA is recovered by using DNA Purification Kit according to the manufacturer’s instructions. The undigested purified DNA is subsequently cloned into pMD18-T vector (TAKARA) and transformed into E. coli DH5α. Clones are sequenced with M13-47 primer to check for mutations (Figure 3). An example of VIGE for NbPDS gene is shown in Figure 4, in which the PCR product of a specific primer pair targeting the NbPDS gene is digested with MlyI.
  PCR amplification is performed using the following parameters (T_m represents the annealing temperature of the primer pair):
  
  Figure 4. VIGE of NbPDS. A. illustration of VIGE targeted gene NbPDS. Cut site of Cas9/gRNA is under the red scissor. MlyI is used for discriminating the edited and wild-type sequence. B. schematic of the gel assay used to estimate the editing efficiency of VIGE of NbPDS gene.

Data analysis

To investigate whether endogenous NbPDS gene is edited, ImageJ is used to evaluate the editing efficiency by VIGE.

Select Lanes for analysis and add frames to include bands with ImageJ (https://imagej.nih.gov/ij/).
Measure the intensity of each band in each lane with ImageJ.
The relative editing efficiency is calculated by dividing the intensity of uncut band by the total intensities of all the bands.

Recipes

Luria-Bertani medium
10 g/L tryptone
5 g/L yeast extract
10 g/L NaCl
Adjust pH to 7, autoclave at 121 °C for 20 min and store at room temperature
MgCl₂ (1 M stock)
Dissolve 203.3 g of MgCl₂·6H₂O in 800 ml of deionized H₂O
Adjust the volume to 1 L with deionized water
MES (0.5 M stock)
Dissolve 19.52 g MES in 200 ml deionized water
Adjust to pH 5.6 with 1 N KOH
Acetosyringone (1 M stock)
Dissolve 1.962 g acetosyringone in 10 ml of DMSO
Infiltration buffer
Note: The stock buffers above (Recipes 2-4) are filter sterilized with a 0.22-μm filter and stored at room temperature. The infiltration buffer is prepared freshly for each time.
10 mM MgCl₂
10 mM MES
200 μM acetosyringone
2x phosphate buffer (pH 7)
0.2 M NaH₂PO₄
0.2 M Na₂HPO₄

First, make two solutions:
Solution A: dissolve 55.2 g NaH₂PO₄·H₂O in 1 L deionized water
Solution B: dissolve 107.3 g Na₂HPO₄·7H₂O in 1 L deionized water
Autoclave at 121 °C for 20 min and store them at room temperature
To make 200 ml 2x phosphate buffer (pH 7), add 39.0 ml Solution A and 61.0 ml Solution B and then dilute with deionized water to 200 ml

X-gluc substrate solution (Kabbage et al., 2011)

Add 1 mg X-gluc in 0.1 ml methanol
Then add 1 ml 2x phosphate buffer, 20 μl 0.1 M potassium ferricyanide, 10 μl 10% Triton X-100 and 850 μl deionized water

PEG-MgCl₂ solution

Dissolve 30 g of PEG 8000 in 100 ml of 30 mM MgCl₂
Filter sterilize the solution with a 0.22-μm filter and store it at room temperature

Acknowledgments

The VIGE method is based on the report by Yin et al. (2015). The method of inoculating CaLCuV is based on Tang et al. (2010). The GUS staining assay is based on Kabbage et al. (2011). This work is supported by the National Basic Research Program of China (2014CB138400), the National Transgenic Program of China (2014ZX0800104B, 2014ZX08010-002 and 2014ZX08005-001) and the National Natural Science Foundation of China (31421001, 3142100007). We thank Alice L. Yu from University of North Carolina at Chapel Hill for proofreading the manuscript.

References

Ali, Z., Abul-faraj, A., Li, L., Ghosh, N., Piatek, M., Mahjoub, A., Aouida, M., Piatek, A., Baltes, N. J., Voytas, D. F., Dinesh-Kumar, S. and Mahfouz, M. M. (2015). Efficient virus-mediated genome editing in plants using the CRISPR/Cas9 system. Mol Plant 8(8): 1288-1291.
Aslanidis, C. and de Jong, P. J. (1990). Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res 18(20): 6069-6074.
Baltes, N. J., Gil-Humanes, J., Cermak, T., Atkins, P. A. and Voytas, D. F. (2014). DNA replicons for plant genome engineering. Plant Cell 26(1): 151-163.
Höfgen, R. and Willmitzer, L. (1988). Storage of competent cells for Agrobacterium transformation. Nucleic Acids Res 16(20): 9877.
Im, H. (2011). The inoue method for preparation and transformation of competent E. coli: “Ultra Competent” cells. Bio-protocol Bio101: e143.
Kabbage, M., Ek-Ramos, M. and Dickman, M. (2011). A β-glucuronidase (GUS) based cell death assay. J Vis Exp 6(51).
Lei, Y., Lu, L., Liu, H. Y., Li, S., Xing, F. and Chen, L. L. (2014). CRISPR-P: a web tool for synthetic single-guide RNA design of CRISPR-system in plants. Mol Plant 7(9): 1494-1496.
Li, J. F., Zhang, D. and Sheen, J. (2015). Targeted plant genome editing via the CRISPR/Cas9 technology. Methods Mol Biol 1284: 239-255.
Nekrasov, V., Staskawicz, B., Weigel, D., Jones, J. D. and Kamoun, S. (2013). Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nat Biotechnol 31(8): 691-693.
Paithankar, K. R. and Prasad, K. S. (1991). Precipitation of DNA by polyethylene glycol and ethanol. Nucleic Acids Res 19(6): 1346.
Stemmer, W. P. and Morris, S. K. (1992). Enzymatic inverse PCR: a restriction site independent, single-fragment method for high-efficiency, site-directed mutagenesis. Biotechniques 13(2): 214-220.
Tang, Y., Wang, F., Zhao, J., Xie, K., Hong, Y. and Liu, Y. (2010). Virus-based microRNA expression for gene functional analysis in plants. Plant Physiol 153(2): 632-641.
Wang, H., La Russa, M. and Qi, L. S. (2016). CRISPR/Cas9 in genome editing and beyond. Annu Rev Biochem 85: 227-264.
Yin, K., Han, T., Liu, G., Chen, T., Wang, Y., Yu, A. Y. and Liu, Y. (2015). A geminivirus-based guide RNA delivery system for CRISPR/Cas9 mediated plant genome editing. Sci Rep 5: 14926.