Protocol for Using CRISPR-Cas9 to Generate a Monocyte Cell Line Harboring a Single-Nucleotide Polymorphism

Materials and reagents

Biological materials

1. THP-1 cell line (ATCC)

2. Bacterial strain (XL10-Gold ultracompetent cells) (VWR)

Reagents

1. Anza^TM T4 DNA ligase master mix (Invitrogen, catalog number: IVGN2102)

2. BbsI (New England BioLabs, catalog number: R0539S)

3. T4 polynucleotide kinase (PNK) (Thermoscientific, catalog number: EK0031)

4. rCutSmart buffer, 10× (New England BioLabs, catalog number: B6004S)

5. T4 DNA ligase reaction buffer, 5× (Invitrogen, catalog number: 15224-017)

6. Alkaline phosphatase (rSAP) (New England BioLabs, catalog number: M0371S)

7. RPMI-1640 (Sigma-Aldrich, catalog number: R8758)

8. Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: 0926)

9. Penicillin-streptomycin (10,000 U/mL) (Sigma-Aldrich, catalog number: P0781)

10. Trypsin-EDTA solution (Sigma-Aldrich, catalog number: T3924)

11. Opti-MEM^TM I reduced serum medium, no phenol red (Gibco, catalog number: 31985-070)

12. Lipofectamine 3000 (Invitrogen, catalog number: L3000-015)

13. Puromycin (Sigma-Aldrich, catalog number: P8833)

14. Taq DNA Polymerase kit (Invitrogen, catalog number: 18038-042)

15. Agarose (Sigma-Aldrich, catalog number: A9539)

16. Dulbecco's phosphate-buffered saline (DPBS) (Sigma-Aldrich, catalog number: D8537)

17. Gene Ruler 1 kb Plus DNA ladder (Thermoscientific, catalog number: SM1333)

18. Ampicillin (Sigma-Aldrich, catalog number: A9518)

19. Gene Elute Gel Extraction and PCR Purification kit (Sigma-Aldrich, catalog number: NA1111-1KT/NA1020-KT)

20. Gene Elute Plasmid Miniprep kit (Sigma-Aldrich, catalog number: PLN350-1KT)

21. Gene Elute Mammalian Genomic DNA Miniprep kit Gene Elutes (Sigma-Aldrich, catalog number: G1N70-1KT)

22. LB medium (Sigma-Aldrich, catalog number: L3022)

23. Agar, bacteriological (Amresco, catalog number: J637)

24. PvuII-HF (New England BioLabs, catalog number: R3151S)

25. 2-Beta-ME or 2-ME (Sigma-Aldrich, catalog number: M6250)

26. S.O.C. medium (Invitrogen, catalog number: 15544-034)

27. Trizma-base (Sigma-Aldrich, catalog number: T6066)

28. Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA) (Sigma-Aldrich, catalog number: E5134)

29. Glacial acetic acid (Sigma-Aldrich, catalog number: A6283)

30. L-Glutamine (Sigma-Aldrich, catalog number: G7513)

Solutions

1. 50× TAE buffer (see Recipes)

2. LB medium (see Recipes)

3. LB broth medium (see Recipes)

4. Complete RPMI 1640 (see Recipes)

5. Puromycin solution (1 mg/mL) (see Recipes)

Recipes

1. 50× TAE buffer

Weigh 242 g of Trizma-base (molecular weight 121.14 g/mol) and add to a 1 L Duran bottle. Dissolve it in 700 mL of MilliQ water. Add 100 mL of 0.5 M EDTA and carefully add 57.1 mL of glacial acetic acid. Finally, top up the solution to 1 L with MilliQ water.

2. LB agar medium

Add 2 g of agar powder and 2 g of LB medium to 100 mL of distilled water. Autoclave it before use.

3. LB broth medium

Add 2 g of LB broth powder to 100 mL of distilled water and autoclave it before use.

4. Complete RPMI 1640

To RPMI-1640, add 10% FBS (heat-inactivated), 1% penicillin-streptomycin antibiotics, and 1% glutamine.

5. Puromycin solution (1 mg/mL)

Dissolve 10 mg of puromycin in 1 mL of water and then dilute 1:10 to make a 1 mg/mL concentration.

Laboratory supplies

1. 96-well cell culture plate (Cellstar, catalog number: 655180)

2. 24-well cell culture plate (Cellstar catalog number: 665180)

3. 15 mL centrifuge tube (VWR, catalog number: 525-1069)

4. 50 mL centrifuge tube (VWR, catalog number: 525-0610)

5. 1.7 mL centrifuge tube (VWR, catalog number: 87003-294)

6. 10 μL tip (VWR, catalog number: 76323-388)

7. 200 μL tip (VWR, catalog number: 76323-390)

8. 1,250 μL tip (VWR, catalog number: 76323-404)

9. 10 mL serological pipette (Corning, catalog number: 75816-100)

10. 10 cm cell culture dish (Corning, catalog number: 430167)

11. 14 mL polystyrene round-bottom tube (Falcon, catalog number: 352057)

Plasmid and DNA sequences (Table 1)

Table 1. Plasmids, primers, sgRNAs, and ssODNs used for CRISPR-Cas9 editing of rs1024611

Equipment

1. Pipettes (Eppendorf)

2. PCR thermocycler (Bio-Rad, model: T100)

3. DNA electrophoresis apparatus (Bio-Rad, model: Wide-mini subcell GT)

4. Gel image analysis system (Invitrogen, model: iBright1500)

5. Nanodrop spectrophotometer (Thermo Scientific, model: Nanodrop One)

6. Centrifuge (Eppendorf, model: 5415 R)

7. 37 °C CO₂ incubator (Thermo Scientific, model: 3578)

8. Shaker (New Brunswick Scientific I2400 incubator shaker)

9. Water bath (Fisher Scientific, model: ISOTEMP 215)

Software and datasets

1. Online sgRNA designing tools (https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design) (free tool)

Procedure

A. Genotyping THP-1 cells

236 bp encompassing the CCL2-2518 polymorphic region was Sanger-sequenced using GENEWIZ Pvt Ltd. Sequencing results indicate that the polymorphic region is heterozygous with A/G nucleotide in parental THP-1 cells. To generate the THP-1 cells having only one nucleotide at the -2518 position of the CCL2 gene, we performed the CRISPR-based genomic editing in the THP-1 cells using the following method.

B. Cloning of sgRNA sequences into the pSPcas9 (BB)-2A-puro vector

Note: We designed and ordered sgRNAs from a qualified company (e.g., Integrated DNA Technology, IDT), listed in Table 1.

1. Preparation of the sgRNA oligos for vector insertion: Design sgRNAs that have a cut site near the SNP using online sgRNA designing tools (https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design).

2. Order 100 μM top and bottom sgRNA from IDT and reconstitute with IDT buffer in 0.2 mL tubes according to Table 2.

Table 2. Components and concentrations for sgRNA annealing and phosphorylation reaction

3. Put the tubes into the thermocycler for oligo annealing: 37 °C for 30 min, 95 °C for 5 min, ramp down to 25 °C at 0.1 °C/s.

4. Dilute the annealed oligos 200× using ddH₂O.

5. Digest plasmid pSpCas9 (BB)-2A-Puro. Prepare the mixture in the 1.5 mL tubes according to Table 3 and incubate the tubes at 37 °C for 12–16 h.

Table 3. Reagents and volumes for BbsI-mediated plasmid digestion

6. Transfer 42 μL of the mixture to a new tube and perform dephosphorylation using rSAP in 1× CutSmart buffer, following the manufacturer’s protocol. Incubate the tube at 37 °C for 15 min and at 50 °C for 15 min. Run the digested product on 1% agarose gel, purify the plasmid using the GeneElute Gel Extraction kit, and dilute the digested plasmid to 25 ng/μL for the next step.

7. Ligate the sgRNA and vector according to Table 4 and incubate at room temperature for 1 h.

Table 4. Amount of sgRNA, vector, and reagents for the ligation mixture

Critical: 1 μL of ddH₂O instead of sgRNA in the ligation system serves as a negative control.

C. Transformation

1. Mix 4 μL of 2-Beta-ME to each aliquot of XL10-Gold ultracompetent cells and keep on ice for 10 min.

2. Add 20–40 μL of competent cells + 5 μL of ligation mix into an ice-cold 14 mL Falcon tube and incubate on ice for 30 min.

3. Subject cells to heat-shock at 42 °C for 30 s, followed by incubation on ice for 2 min. Add 250–450 μL of prewarmed S.O.C. medium and allow to grow at 37 °C under shaking conditions at 250 rpm for 1 h.

4. Plate 50–100 μL of ligation mixture (Table 4) on an Amp-LB-agar plate and place the plate in the incubator for 14–16 h at 37 °C. Use a 100 μg/mL concentration of ampicillin to make the LB-agar plates.

5. Pick a single clone and place it in the tube containing 3 mL of LB medium with 100 μg/mL ampicillin. Then, put the tube in the shaker at 37 °C for 12–16 h.

Notes:

1. More clones from one plate can be picked up at the same time to ensure positive clones.

2. Plasmids were purified and verified using the U6-Fwd primer for sequencing.

D. Cell culture and transfection

Note: THP-1 cells were cultured in complete RPMI-1640 medium at 37 °C with 5% CO₂. One 10 cm plate of 70%–80% confluent cells was prepared for this experiment. At the same time, the SNP-centered repair templates should be designed and ordered (ssODNs) from IDT.

D1. Prepare cells for transfection

1. Remove the old medium and rinse the cells once with DPBS. Add 10 mL of ice-cold DPBS to a 10 cm plate, and detach superficially attached cells using gentle pipetting or scraping.

2. Pipette the cells gently, transfer the cell suspension into 15 mL tubes, and spin at 500× g for 5 min.

3. Remove supernatant. Homogenize cells in 5 mL of fresh media and transfer 0.5 mL of the mixture to each well in a 24-well plate. Note that one extra well should be added as control cells. Put the 24-well plate in the cell culture incubator at 37 °C with 5% CO₂.

4. Sixteen hours later, replace media with 0.5 mL of fresh media.

D2. Plasmid transfection

1. Dilute Lipofectamine 3000 reagent and plasmid DNAs with Opti-MEM, according to Table 5.

Table 5. Volume of plasmids, ssODN, and reagents for the transfection mixture

2. Mix the solution from mixture #2 with mixture #1 and incubate at room temperature for 10–15 min. Add 50 μL to each well and mix gently by rocking.

3. Sixteen hours later, replace the transfection medium with 0.5 mL of fresh warm complete RPMI medium. At 48 h post-transfection, apply puromycin selection at a final concentration of 3 μg/mL.

E. Isolating clonal lines

Note: After 3–5 days, when the control cells all died in the medium with 3 μg/mL puromycin, the experiments are ready for isolating single-cell clones. For seeding single cells on a 10 cm plate, remove the spent culture medium, rinse the cells twice with DPBS, and then dissociate cells using gentle scraping or pipetting and transfer to a 10 cm Petri dish, followed by incubation at 37 °C in a CO₂ incubator.

1. Pick single-cell clones for genotyping, as shown in Figure 1. When the clones are visible to the naked eye, they can be picked up from the 10 cm plate using the following steps:

Figure 1. Workflow of picking a single clone for genotyping. (A) Aspirate 2.5 μL of trypsin in the pipette tip. (B and C) Scrape the clone from the plate using the pipette tips (B), then slowly release the pipette to carefully aspirate the clone into the tip (C). (D) Transfer the single clone into one well of a 96-well plate with 20 μL of trypsin and incubate for 5 min at 37 °C. (E) Inactivate trypsin and separate the cells by pipetting; transfer 10 μL of cell suspension from each well into a 96-well PCR plate.

a. Add 20 μL of trypsin-EDTA into each well of the 96-well plate.

b. Take out the 10 cm plate from the incubator, discard the old medium completely, and add 4 mL of DPBS into the plate. Be sure that the DPBS will not spill out while rotating the plate.

c. Use 10 μL pipette tips to pick clones of moderate size. When picking clones from a 10 cm plate, we recommend aspirating 2.5 μL of trypsin first in the pipette tip and then pipetting it out on the clone and holding the push-button.

d. Scrape the clone from the plate with the pipette tip. Then, carefully aspirate the cell into the tip for transfer.

e. Transfer these single clones quickly into the wells of a 96-well plate.

f. Pick 10–20 single clones from each plate with different genotypes (GG or AA repair template) and incubate at 37 °C for 5 min. Then, add 30 μL of medium supplemented with 10% (v/v) FBS to each well of the 96-well plate to inactivate trypsin. g. Pipette the cells to separate the adherent cells. Transfer 10 μL of cell suspension from each well into an 8-tube PCR strip (for genotyping). Then, add 100 μL of fresh medium to each well. Place the 96-well plate back in the cell incubator and, 24 h later, change the old medium of the 96-well plate with fresh medium to remove the trypsin.

F. Genotyping cell clones

1. After collecting all samples in an 8-tube PCR strip, perform DNA extraction using Gene Elute Mammalian Genomic DNA miniprep kit solution and column, followed by PCR for genotype.

Note: We run the following PCR program: an initial denaturation at 94 °C for 3 min; 35 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 30 s; and a final extension at 72 °C for 10 min, followed by a hold at 4 °C. We use the PCR mix in Table 6 to amplify a specific PCR product of 236 bp harboring the CCL2 promoter polymorphism at the -2518 position (rs1024611).

Table 6. PCR reaction mixture for amplification of the CCL2 promoter (rs1024611 genotyping in THP-1 clones)

2. Purify the PCR product using the Gene Elute DNA Extraction and PCR Purification kit. Take the purified DNA from each clone as a template for genotyping PCR.

3. Genotype the clones as soon as possible, before they reach confluence in the 96-well plate. The clones in the 96-well plate should be checked every day.

G. Expansion of cell clones

1. After the genotyping step, culture-expand the cells with the successfully converted SNP genotypes: When cells reach 50%–70% confluence in the 96-well plate, remove the old medium, rinse the cells twice with DPBS, add 100 μL of fresh complete RPMI medium, and dissociate cells by pipetting. Transfer the entire cell suspension into wells prefilled with 400 μL of fresh medium in a 24-well plate.

Data analysis

To assess genome editing efficiency, 10 single-cell clones were isolated from the transfected THP-1 population and expanded. Genomic DNA was extracted from each clone, and the target locus was PCR-amplified and sequenced. Two out of the 10 clones carried the desired CCL2-2518 SNP edit, corresponding to an editing efficiency of approximately 20%. This clonal analysis provides a direct and precise measure of successful genome editing in THP-1 cells.

Validation of protocol

We have confirmed the single-nucleotide mutation using either Sanger sequencing (Figure 2A–C) or using restriction fragment length polymorphism (PvuII restriction enzyme used to digest the 321 bp of amplified DNA from parental THP-1 carrying G/A, mutant THP-1 cells carrying G/G or A/A allele in the CCL2 promoter at -2518 position, also referred to as rs1024611 SNP) (Figure 2D). After PvuII restriction digestion of THP-1 cells, parental G/A cells show three DNA bands of 321, 182, and 139 bp. G/G mutant cells display two bands of 182 and 139 bp, as the G nucleotide creates the PvuII recognition site (CAGCTG). A/A mutant cells show a single intact band of 321 bp, indicating loss of the restriction site. Using this method, the mutated efficiency at rs1024611 in THP-1 cells is approximately –20%. Thus, the efficiency of this method can vary at different SNP sites and cell lines. This is probably due to the differences in local genomic sequence features, chromatin structure, and cellular properties.

Figure 2. Genotype at rs1024611 in THP-1 cells was successfully converted from G/A to G/G and A/A. (A–C) DNA sequencing chromatogram peak analysis, indicating that the genotype at rs1024611 in THP-1 cells was successfully converted from G/A (A) to G/G (B) and A/A (C). (D) PCR-amplified 321 bp fragments spanning the SNP site were digested with PvuII to distinguish parental and edited alleles. Parental cells (G/A heterozygous) show three bands of 321, 182, and 139 bp, reflecting the presence of both alleles. G/G mutant clones display two bands of 182 and 139 bp, indicating homozygosity for the G allele. A/A mutant clones show a single 321 bp band, consistent with homozygosity for the A allele. This restriction digest confirms the genotypes of the edited THP-1 clones. The GeneRuler 1 kb Plus DNA Ladder was used as a molecular size marker. The chromatograms shown in Figure 2 were generated and analyzed using Chromas version 2.6.6 software or Geneious R8 version.

General notes and troubleshooting

General notes

1. Cells should be initially seeded at a lower density (40%–50%); they must reach 70%–80% confluence at the time of transfection.

2. Change the position after selecting a single clone, or remember to rotate the plate frequently to prevent the plate from drying out.

3. The cells on the 96-well plate usually grow very slowly. Remember to change the medium every three days until the cells are confluent for passaging. Earlier transfer of the cells to a bigger plate may cause cell death when the density of the cells is too low.

Troubleshooting

Problem 1: Genotyping THP-1 cells.

Possible cause: Primers have low specificity for genotyping.

Solution: The SNP genotype of the target cells should be determined before performing CRISPR/Cas9-mediated SNP editing. Accurate genotyping is essential for the proper design of sgRNAs and ssODN repair templates. If sequencing results do not show a single, unambiguous peak, the genotyping primers should be redesigned and revalidated. Careful design of allele-specific primers is critical to ensure reliable and accurate final genotyping results.

Problem 2: Inserting sgRNAs into vector pSpcas9 (BB)-2A-Puro using BbsI restriction digestion.

Possible cause: Ligation efficiency was low.

Solution: Digest vector pSpCas9 (BB)-2A-Puro with BbsI by incubating at 37 °C for 12–16 h to ensure complete digestion. A shorter digestion time results in incomplete digestion of vector DNA, which can significantly reduce the ligation efficiency of sgRNAs into the vector.

Problem 3: THP-1 cell culture and transfection.

Possible cause: Low transfection efficiency.

Solution: THP-1 cells were transfected using Lipofectamine 3000 following the manufacturer’s protocol. Optimization experiments were performed using this reagent; alternative transfection reagents were not comparatively evaluated in this study.

Problem 4: Pick a single clone for genotyping.

Possible cause: The single clone died after being transferred to the 96-well plate.

Solution: Once clones are visible to the naked eye, individual clones can be picked from the 10 cm plate. Select the moderate-sized clones using a pipette with a 10 μL tip. Very small clones fail to proliferate after transferring clones from a 10 cm plate to a 96-well plate and are likely to undergo apoptosis. In addition, large clones are unlikely to be single clones, likely being a mixture of two or more single clones.

Problem 5: ≥90% cell death is not observed in non-transfected controls within 3–5 days.

Possible cause: The puromycin concentration may be insufficient.

Solution: In such cases, a puromycin kill curve should be performed to determine the minimal effective concentration required to eliminate non-transfected cells within 72 h. Antibiotic potency and cell density at the start of selection should also be verified.

Acknowledgments

This work was supported in part by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Numbers R21AI151936, R21AI171734, and R01AI168543 to J.L. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

pSpCas9(BB)-2A-Puro (PX459) V2.0 was a gift from Feng Zhang (Addgene plasmid #62988; http://n2t.net/addgene:62988; RRID: Addgene_62988). The graphical abstract was created using BioRender. This protocol is adapted from [9].

Competing interests

The authors declare that they have no competing interests.

Ethical considerations

No human or animal subjects were involved in this protocol.

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