Materials and Reagents

Pipette tips (Thermo Fisher Scientific, Thermo Scientific^TM, catalog numbers: 2140 , 2149 , 2065E , 2069 , and 2079E )
75 cm² U-shaped canted neck cell culture flask with vent cap (Corning, catalog number: 430641U )
Microcentrifuge tubes, 1.5 ml (Sigma-Aldrich, catalog number: T6649 )
Tissue culture plate, 6-well (Corning, Costar^®, catalog number: 3516 )
Serological pipettes
5 ml (Corning, Costar^®, catalog number: 4051 )
10 ml (Corning, Costar^®, catalog number: 4488 )
25 ml (Corning, Costar^®, catalog number: 4489 )
Cuvettes
Coverslip
50-ml conical centrifuge tube (Corning, catalog number: 430828 )
Human K562 cells (ATCC, catalog number: CCL-243 )
Catalytically inactive Streptococcus pyogenes Cas9 (SpdCas9) plasmid DNA (from MilliporeSigma; see Figure 1A)
SpCas9 sgRNA plasmid constructs with the guide sequences 5’-CCAAGGGTGAGGCCGGGAAG-3’ and 5’-CATCTCCCCCATGTACACCT-3’ for binding two human POR target sites (from MilliporeSigma; see Figure 1B)
Campylobacter jejuni Cas9 (CjCas9) plasmid DNA (from MilliporeSigma; see Figure 1C)
CjCas9 sgRNA plasmid construct with the guide sequence 5’-TTCGCCAGTACGAGCTTGTG-3’ for binding a human POR target site (from MilliporeSigma; see Figure 1D)

Figure 1. Vector maps
Single stranded DNA oligo donor for introducing a diagnostic EcoRI site at the CjCas9 cleavage site in POR. The oligo sequence is: 5’-CACCCTTGGTCTCCCCTTTCCAGCATTCGCCAGTACGAGCGAATTCTTGTGGTCCACACCGACATAGATGCGGCCAAGGTGTACATGG-3’ (Underlined: EcoRI site)
Note: Synthesize the oligo at 0.2 µmol scale and purify by PAGE. Re-suspend the oligo in 10 mM Tris buffer (pH 7.6) at 200 µM.
PCR primers for amplification of the targeted POR genomic region: forward 5’-CTCCCCTGCTTCTTGTCGTAT-3’, reverse 5’-ACAGGTCGTGGACACTCACA-3’
Alcohol
Hank’s balanced salt solution (Sigma-Aldrich, catalog number: H6648 )
Nucleofector instrument (Lonza, catalog number: AAB-1001 )
Amaxa Cell Line Nucleofector Kit V (Lonza, catalog number: VCA-1003 )
GenElute Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich, catalog number: NA2010 )
JumpStart Taq ReadyMix (Sigma-Aldrich, catalog number: P2893 )
GenElute PCR Clean-Up Kit (Sigma-Aldrich, catalog number: NA1020 )
EcoRI restriction enzyme (New England Biolabs, catalog number: R0101S )
10% Mini-PROTEAN TGX Precast Protein Gels, 15-well, 15 µl (Bio-Rad Laboratories, catalog number: 4561036 )
Tris buffered saline (Sigma-Aldrich, catalog number: T5912 )
Gel loading buffer (Sigma-Aldrich, catalog number: G2526 )
DirectLoad Wide Range DNA Marker (Sigma-Aldrich, catalog number: D7058 )
Iscove’s modified Dulbecco’s medium (Sigma-Aldrich, catalog number: I3390 )
Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F2442 )
L-glutamine, 200 mM (Sigma-Aldrich, catalog number: G7513 )
K562 culture medium (see Recipes)

Equipment

Water bath (Polyscience)
Laminar flow hood for sterile tissue culture, biosafety level 2 approved (Thermo Fisher Scientific)
Microbiological incubator with 37 °C, atmospheric CO₂ (Thermo Fisher Scientific)
Pipettes (Gilson)
Bench-top centrifuge (Eppendorf, model: 5417 C )
Mini-PROTEAN Tetra Vertical Electrophoresis Cell (Bio-Rad Laboratories, catalog number: 1658004 )
PCR thermocycler (Bio-Rad Laboratories, catalog number: 1861096 )

Software

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

Procedure

Target site selection and oligo design for sgRNA expression
1. Nuclease cleavage site selection will be dependent on gene editing objective. For gene knockout, it is recommended to select cleavage sites in the first two coding exons or in protein functional domains. For gene correction or homologous recombination (HR), the cleavage site should be as close as possible to the nucleotide to be changed or to the target integration site to maximize the efficiency. For cytidine deamination-based editing, the nicking site will be dependent on the cytidine deamination window.
2. Select a proximal dCas9 binding site upstream and downstream of the cleavage site. The dCas9 binding sites each must be separated from the nuclease binding site by about 10 bp to avoid potential steric hindrance, but beyond the 10 bp separation the dCas9 binding sites also should be kept as close as possible to the nuclease binding site to maximize the effects. The dCas9 binding sites can be on either strand of the DNA.
3. One of the advantages of CRISPR-Cas effector nucleases over previous generations of programmable nucleases as genome editing tools is the simplicity of nuclease retargeting via straightforward oligo design and sgRNA expression plasmid cloning once target sites are selected. In this illustrative experiment, the two SpdCas9 proximal binding sites are: 5’-CCAAGGGTGAGGCCGGGAAGCGG-3’, and 5’-CAAGGTGTACATGGGGGAGATGG-3’; and the CjCas9 binding site is: 5’-TTCGCCAGTACGAGCTTGTGGTCCACAC-3’; where the underlined nucleotides are protospacer adjacent motifs (PAMs). Therefore, the DNA oligo sequences for sgRNA expression plasmid cloning are: forward 5’-ACCGCCAAGGGTGAGGCCGGGAAG-3’ and reverse 5’-AAACCTTCCCGGCCTCACCCTTGG-3’ for SpdCas9 binding site 1; forward 5’-ACCGCAAGGTGTACATGGGGGAGA-3’ and reverse 5’-AAACTCTCCCCCATGTACACCTTG-3’ for SpdCas9 binding site 2; and forward 5’-ACCGTTCGCCAGTACGAGCTTGTG-3’ and reverse 5’-AAACCACAAGCTCGTACTGGCGAA-3’ for the CjCas9 binding site; where the underlined nucleotides in the forward sequences are a constant overhang for ligation to a human U6 promoter and the underlined nucleotides in the reverse sequences are a constant overhang for ligation to a SpdCas9 sgRNA scaffold or a CjCas9 sgRNA scaffold in cloning vectors.
Transfection and gene editing analysis
1. Culture K562 cells in Iscove’s modified Dulbecco’s medium, supplemented with 10% FBS and 2 mM L-glutamine. Maintain the culture at 37 °C and 5% CO₂. Split the culture every 2-3 days or when the culture reaches 1-2 million cells per ml by transferring 1/10 or 1/5 of the culture to a new culture flask containing fresh medium pre-warmed to 37 °C to make a 1:10 or a 1:5 split (see Note 1).
2. Split the culture to 0.25 million cells per ml one day before transfection. The culture will reach about 0.5 million cells per ml for transfection the next day (see Note 1).
3. Prepare three plasmid DNA solutions for transfection in 1.5-ml microcentrifuge tubes (see Note 2):
4. Add 2 ml of fresh culture medium to each well of a 6-well plate and equilibrate the medium at 37 °C and 5% CO₂for at least 20 min before transfection.
5. Set the Nucleofector program to T-016 (see Note 3). Unseal the cuvettes and disposable transfer pipettes and place them inside a cell culture hood.
6. Count the cells with a glass hemocytometer:
7. Transfer the required amount of cells to a sterile 50-ml conical centrifuge tube. Each transfection reaction will require 1.0 million cells (see Note 4). Centrifuge the cells at 200 x g at room temperature for 5 min. Carefully remove the culture medium by aspiration.
8. Resuspend the cell pellet in 20 ml of Hank’s balanced salt solution. Centrifuge at 200 x g at room temperature for 5 min and carefully remove the wash fluid by aspiration. Gently re-suspend the cell pellet in nucleofection solution V to 1.0 million cells per 100 µl (see Note 5).
9. Pipette 100 µl of the cells into a 1.5-ml tube containing a plasmid DNA solution and mix thoroughly but gently by pipetting up and down 3-5 times. Transfer the whole content to a nucleofection cuvette and perform the transfection immediately using the T-016 program.
10. Add a pipette full of pre-equilibrated culture medium to the transfected cells inside the cuvette with a disposable transfer pipette and immediately transfer the whole content into a well containing pre-equilibrated culture medium. Culture the cells at 37 °C and 5% CO₂ immediately after nucleofection.
11. Extract genomic DNA 3 days after transfection using GenElute Mammalian Genomic DNA Miniprep Kit. PCR amplify the POR genomic region using JumpStart Taq ReadyMix with the forward primer 5’-CTCCCCTGCTTCTTGTCGTAT-3’ and the reverse primer 5’-ACAGGTCGTGGACACTCACA-3’ and the following cycling condition: 98 °C for 2 min for initial denaturation; 34 cycles of 98 °C for 15 sec, 62 °C for 30 sec, and 72 °C for 45 sec; and a final extension at 72 °C for 5 min.
12. Purify PCR products with GenElute PCR Clean-Up Kit. Digest purified PCR products (about 500 ng per digestion) with EcoRI (20 U) at 37 °C for 2 h. Resolve digestion products on a pre-cast 10% acrylamide gel from Bio-Rad Laboratories using DirectLoad Wide Range DNA Marker as markers. Figure 2 illustrates an example of expected results.
  
  Figure 2. ssDNA oligo templated gene editing in K562 cells by CjCas9 using proximal dCas9 targeting. A. Campylobacter jejuni Cas9 (CjCas9) and catalytically inactive Streptococcus pyogenes Cas9 (SpdCas9) target sites in the human POR locus and an ssDNA oligo donor carrying a diagnostic EcoRI site. Targets are indicated by bars and PAMs are highlighted in dark blue (CjCas9) and purple (SpdCas9). B. EcoRI digestion analysis of ssDNA oligo templated gene editing. EcoRI site integration efficiency (%) was determined by ImageJ. The two EcoRI restriction fragments are indicated by yellow arrows. The sgRNA numbers correspond to the target numbers in (A). ND, not determined. M, wide-range DNA markers.

Data analysis

Use ImageJ to measure the band intensity for the uncut band and EcoRI digested bands. Sequence integration efficiency (%) is determined by the formula: 100 x (b + c)/(a + b + c), where a is the integrated intensity of the undigested PCR product, and b and c are the integrated intensities of each restriction digestion product.

Notes

When working with adherent cells, split the culture every 2-3 days or when the culture reaches approximately 80% confluency (approximately 80% of the surface is occupied by cells). Split the culture two days before transfection. Seed the culture at about 20% confluency so that the culture will be at about 60-80% confluency at the time of cell preparation for transfection.
Prepare plasmid DNA at a concentration ≥ 2 µg/µl to limit the total volume to ≤ 10 µl for each transfection.
Different cell lines require different nucleofection programs. Follow the instructions from the manufacturer when working with other cell lines.
Up to 2 million K562 cells can be used per nucleofection, but it may reduce the transfection efficiency slightly.
Do not leave cells in the nucleofection solution for a prolonged time (30 min).

Recipes

K562 culture medium
500 ml Iscove’s modified Dulbecco’s medium
50 ml fetal bovine serum
5 ml 200 mM L-glutamine

Acknowledgments

The authors wish to thank Patrick Sullivan for R&D administrative support and the financial support from MilliporeSigma, a business of Merck KGaA, Darmstadt, Germany. This protocol was originally published as part of Chen et al. (2017)

References

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