Experiment Summary

The CRISPR/Cas9 system is a powerful tool for genome editing and is adaptable for a wide range of applications. Here, we have put together a step-by-step protocol for generating knockout cell lines (coding or non-coding region) using CRISPR/Cas9 tool. The protocol below has been tested on adherent cell lines such as HeLa and MCF7. However, it may easily be adapted to other adherent cell lines with minor variations.

Materials

a) Experimental models: cell lines

1. HeLa
2. HEK293FT

b) Recombinant DNA

1. lentiCas9-Blast for Cas9
2. pgRNA-humanized
3. pCMV-VSVG
4. psPAX2

c) Bacterial and viral strains

1. DH5α Competent Cells

d) Chemicals, peptides and recombinant proteins

1. DMEM
2. FBS
3. Penicillin-Streptomycin
4. DPBS
5. Puromycin
6. Poly-D-Lysine
7. 0.25% Trypsin-EDTA (1×)
8. BstXI
9. XhoI
10. Buffer 3.1
11. 1 Kb Plus DNA Ladder
12. Ampicillin sodium salt
13. Hexadimethrine bromide (Polybrene)
14. LB broth
15. LB broth with agar
16. Lipofectamine 2000
17. T4 DNA Ligase
18. Opti-MEM
19. Kapa Taq DNA Polymerase
20. Agarose
21. KOD Hot Start DNA Polymerase
22. Proteinase K, recombinant, PCR Grade
23. Phenol: chloroform: isoamyl (PCI) alcohol
24. T4 DNA Ligase Buffer

e) Critical commercial assays

1. Plasmid Midi Kit
2. Spin Miniprep Kit
3. Gel Extraction Kit
4. PCR Purification Kit
5. DNA Mini Kit

Procedure

A. Designing and cloning of sgRNAs in pgRNA-humanized vector

1. Designing guide sequences for region of interest.
2. Oligos designing for validation of knockout clones.
3. Cloning of guide sequences in pgRNA-humanized vector.

Fig. 1 Graphical representation of the PCR amplification and cloning of sgRNAs in pgRNA-humanized vector.

B. Production of lentiviruses in HEK293FT cells

1. Cell culture. HEK293FT and HeLa cell lines are maintained/cultured in DMEM supplemented with 10% FBS (v/v) and 5% PenStrep (v/v) at 37°C with 5% CO₂. Passage both cell lines every 3 days.

2. Cell preparation for transfection (Late Evening)

a) Aspirate medium and add 2 mL of 0.25% Trypsin-EDTA to 10 cm dish of HEK293FT cells. Incubate the plate for 1–2 min at 37°C.

b) Remove trypsin from the plate carefully to avoid disturbing the cells. Gently tap the plate to dislodge the cells.

c) Collect and resuspend cells in 3 mL of fresh culture media.

d) Seed 0.8 × 106 cells on poly-D lysine-coated 35 mm dishes ∼14 h before transfection. Place the plate back into incubator.

3. Plasmid transfection (Early Morning)

Transfection mixture of DNA and Lipofectamine 2000 is to be prepared in Opti-MEM medium as follows:

a) Add 100 μL Opti-MEM and 3 μg (0.6 μg each of psPAX2, pCMV-VSVG, pgRNA-humanized sgRNA1, pgRNA-humanized sgRNA2 and lentiCas9-Blast) DNA to tube A.

b) Add 150 μL Opti-MEM and 6 μL lipofectamine 2000 to tube B.

c) Mix the contents of both tubes thoroughly by pipetting.

d) Add the contents of tube B to tube A and mix thoroughly.

e) Short spin tube A to collect the liquid from the tube walls.

f) Incubate the transfection mixture at room temperature (25°C) for 25 min.

g) Meanwhile, remove the spent medium from the plate to be transfected and add 0.75 mL of fresh Opti-MEM to it.

h) Add the transfection mixture (tube A) to the cells after 25 min. Swirl the plate to evenly distribute the mixture. Incubate the cells for 6 h at 37°C.

i) Remove the transfection mixture from the cells after 6 h of transfection. Add 2 mL fresh media to the plate and incubate it for 48 h in the incubator.

4. Viral Particle collection and transduction

a) After 48 h, take the plates out of the incubator and transfer the medium containing viral particles to a sterile 15 mL falcon tube (Falcon A).

b) Store the Falcon A at 4°C and incubate the cells for next 24 h with 1 mL of fresh media at 37°C.

c) Seed 0.7 × 106 HeLa cells (or any cell line of interest) in 35 mm dish approx. 14 h prior to transduction.Remove the media from HEK293FT cell plate after 24 h and transfer it to Falcon A.

d) Filter the entire viral particle-containing media by passing it through 0.45-micron syringe-driven filters. Collect the filtered media in fresh sterile 15 mL tubes.

e) Check the volume of filtered media you have and add 8 μg/mL polybrene.

f) Add 2 mL of this filtered media to HeLa cells in 35 mm dishes (or any other cell line of interest).

g) Remove the medium after 12–14 h of transduction. Incubate the cells for 24 h with 2 mL fresh media.

C. Selection of sgRNA-positive cells and seeding of single cells

1. Selection of cells transfected with sgRNAs

a) To select the sgRNA positive cells, add 3 μg/mL puromycin to the transduced cells for 48 h.

b) Remove the media and wash the cells twice with DPBS to remove any dead cells.

c) Add 0.3 mL trypsin and incubate at 37°C for 1–2 min.

d) Carefully remove the trypsin and tap the plate to dislodge the cells.

e) Collect the cells and resuspend them in 2 mL of fresh media without puromycin.

2. Single cells seeding in 96 well plates

a) Using a cell counter, count the number of cells per mL of media. Count the cells at least three times and if the difference between the three readings is small, the average should be used to minimize the variation.

b) Add the media and cells to the sterile reagent reservoirs.

c) Dilute the cells such that 100 μL of DMEM contains only one cell.

d) Using the 100 μL multi-channel pipette, add 100 μL of DMEM-containing cells to the 96 well plate.

e) The next day, observe each well under light microscope at 20× resolution to label the wells containing single cell.

f) Disregard the wells that have no cell or more than one cell.

g) Allow the single cells to form colonies.

h) Change the media every 3–4 days, taking care not to lose the cells.

D. Screening of knockout clones

1. Genomic DNA isolation

a) Add 50 μL of trypsin to the colonies and transfer them to 48-well plates with 0.5 mL media. Allow the cells to grow for 2-3 days.

b) Trypsinize the cells once they are 80–90% confluent by adding 100 μL trypsin to each well. Resuspend the cells in 1 mL media.

c) Transfer half of the cells to 1.5 mL tubes for screening, and the other half should be seeded in the same wells.

d) Spin the tubes at 600 rcf for 5 min. Discard the supernatant and remove the excess media from the tubes by inverting them on a tissue paper.

e) Resuspend the pellet in 300 μL of genomic DNA isolation buffer. Pipette thoroughly until the sample's viscosity is reduced. Incubate the samples overnight (14–16 h) at 50°C.

f) Add an equal volume of Phenol: Chloroform: Isoamyl (PCI) alcohol to the sample and vortex for 1 min.

g) Spin the tubes at 13500 rcf for 12 min at 4°C.

h) Collect the aqueous phase with care without disturbing the interphase, and transfer it to a new 1.5 mL tube.

i) To the sample, add 1/10th volume of 3M sodium acetate (pH 5.5) and 2.5 volumes of absolute ethanol. Vortex the tubes and place them in a −20°C freezer for an hour.

j) Pellet the precipitated DNA by centrifuging the tubes for 12 min at 4°C at 13500 rcf.

k) To remove residual salt, wash the pellet twice with 70% ethanol. Decant the ethanol and allow the pellet to air dry for 15–20 min.

l) Dissolve the DNA pellet in 100 μL TE buffer.

2. Validation of knockout clones

a) Prepare the PCR mix for the validation of clones.

b) Add 2 μL of genomic DNA obtained from Step 10l to the PCR mix.

c) Adjust the PCR to the following parameters:

d) Run all of the samples on an agarose gel

e) Complement the above PCR results with another round of PCRs with PCRin oligo set to confirm the outcome of the clones.