Optimized protocol for CRISPR knockout of human iPSC-derived macrophages

Summary

Here, we present a protocol for lentiviral delivery of CRISPR-Cas9 to human induced pluripotent stem cell (iPSC)-derived macrophages using co-incubation with VPX virus-like particles (VPX-VLPs). We describe steps for producing polybrene and puromycin kill curves, VPX viral production, and VPX-VLP titration by western blotting. We then detail procedures for iPSC macrophage precursor lentiviral transduction and lentiviral CRISPR-Cas9-based knockout in iPSC-derived macrophages. This protocol uses efficient genome-editing techniques to explore macrophage involvement in immune response, chronic inflammation, neurodegenerative disease, and cancer progression.

For complete details on the use and execution of this protocol, please refer to Navarro-Guerrero et al.¹

Graphical abstract

Highlights

• •Protocol to perform CRISPR-Cas9 screening in human iPSC-derived macrophages
• •Production and titration of VPX virus-like particles to improve transduction
• •CRISPR-Cas9-based knockout in human iPSC-derived macrophages
• •Steps to identify key regulators of inflammation and innate immune response

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Here, we present a protocol for lentiviral delivery of CRISPR-Cas9 to human induced pluripotent stem cell (iPSC)-derived macrophages using co-incubation with VPX virus-like particles (VPX-VLPs). We describe steps for producing polybrene and puromycin kill curves, VPX viral production, and VPX-VLP titration by western blotting. We then detail procedures for iPSC macrophage precursor lentiviral transduction and lentiviral CRISPR-Cas9-based knockout in iPSC-derived macrophages. This protocol uses efficient genome editing techniques to explore macrophage involvement in immune response, chronic inflammation, neurodegenerative disease, and cancer progression.

Before you begin

Monocytes and macrophages play a key role in innate immunity and multiple diseases, including but not limited to atherosclerosis, sepsis, cancer, tuberculosis, and virus infection. There are several protocols for generating human primary monocytes and macrophages for in vitro studies, but these differ in cell yield, purity, and activation status of cells. The cells used in this work are produced by a simple and efficient process based on homogeneous monocytes from hES cells that are differentiated into functional macrophages, and these cells do not require additional purification.

CRITICAL: The most critical step prior to screening is to differentiate IPSC into a homogeneous population of iPSC-derived macrophages CD11b+ CD14+ CD80low CD163low and to confirm the innate immune activation capacity by intracellular TNFa cytokine staining using flow cytometry upon LPS. For complete details on the use and execution of this protocol, please refer to Navarro-Guerrero et al.¹

The adaptation of CRISPR/SpCas9 technology to mammalian cells is transforming the study of human functional genomics. Pooled viral vectors libraries targeting human protein-coding genes can systematically create gene knockouts in a variety of systems. The TKOv3 library (gRNA pooled library in lentiCRISPRv2 - Original TKOv3 - from Addgene, #90294) targets 18,053 protein-coding genes and it has 71,090 guides (four sgRNAs per gene and 142 non-target sgRNAs). Due to its relatively small size, the TKOv3 library is ideal for genome wide CRISPR screens in models where cell numbers are limiting factor.

CRITICAL: Perform amplification, sequencing and viral production of TKOv3 library strictly following author’s protocol.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Mouse polyclonal SAMHD1 antibody (1:500)	Abcam	AB67820
Alexa Fluor 750 goat anti-mouse (1:15,000)	Thermo Fisher Scientific	A-21037
Bacterial and virus strains
pSIV3+	Laboratory of J. Rehwinkel	Nègre et al. 20002
psPAX2	Addgene	12260
pCMV-VSV-G	Addgene	8454
TKOv3 library	Addgene	90294
Chemicals, peptides, and recombinant proteins
Resazurin sodium salt	Sigma	R7017
Polybrene	Sigma	H9268
Puromycin	GE Life Sciences HyClone	SV30075.01
jetPRIME reagent and buffer	Polyplus transfection	114-15
DMEM	Sigma-Aldrich	21969035
X-VIVO 15	Lonza	02-060Q
Fetal bovine serum, heat inactivated	Merck	F9665
Tween 20	Sigma	P1379
1× PBS	Life Technologies Ltd.	10100056
24-well plates	Greiner	662160
6-well plates	Corning	3516
RIPA buffer	Thermo Fisher Scientific	89900
Halt protease inhibitor cocktail	Thermo Fisher Scientific	78429
Bolt 4%–12% Bis-Tris Plus gel	Thermo Fisher Scientific	NW04120BOX
Whatman Protran nitrocellulose membrane	MilliporeSigma	Z670766
Powdered skimmed non-fat dry milk	Cell Signaling Technology	99999
Experimental models: cell lines
Human: HEK293T cells	ATCC	CRL-3216

Materials and equipment

• •Resazurin sodium salt (Sigma-Aldrich #R7017) dye for redox assays to determine cellular metabolic reduction with minimal cell-toxicity. Prepare Resazurin sodium salt stock solution (1000×) by dissolving 100 mg Resazurin sodium salt into 10 mL of 1× PBS (Life Technologies Ltd, 10100056). Aliquot and storage Resazurin solution at −20°C up to five years. (Protocol steps in the sections relative to Resazurin have been partially modified from “Relative Viral Tittering with a Resazurin AlamarBlue Cell Viability Assay”, Broad Institute).
• •To prepare a Polybrene stock solution, dissolve PB (Sigma-Aldrich, #H9268) at a concentration of 8 mg/mL in water and sterilize the solution by passing it through a 0.22-μm filter.

CRITICAL: Store the Polybrene solution as small aliquots (0.25 mL) at −20°C. Discard after use.

• •Dissolve Puromycin (GE Life Sciences HyClone, # SV30075.01) in sterile 1× PBS at 1 mg/mL and store at −20°C for up to 1 year. iPSC-derived macrophages precursor, growth and differentiation medium X-VIVO 15 media (Lonza, #: 02-060Q).
• •Dissolve BSA (Sigma-Aldrich, #A9418) at a concentration of 15 g/L (=1.5%) in 1× PBS and sterilize the solution by passing it through a 0.22-μm filter.

CRITICAL: BSA solution should be made fresh at every use.

• •Tris-buffered saline with 0.1% Polysorbate 20 (also known as Tween 20) detergent (TBS-T) is an effective wash buffer for many immunoassays. To make 1 L of TBS-T wash buffer, add 100 mL of 10× TBS and 1 mL Tween 20 detergent to 900 mL of water. Store at room temperature in container protected from direct sunlight in a dry, cool and well-ventilated area. When properly stored, the reagent is stable for one year.

Step-by-step method details

Dose response curve for antibiotic selection of macrophages via resazurin assay

Timing: up to 2 weeks

To evaluate the lentivirus CRISPR library titer and consequentially utilizing the chosen MOI, it is important to determine the minimum concentration of antibiotic to kill 100% of non-transduced cells. The following protocol provides detailed instruction for determining the concentration of puromycin to select macrophages using TKOV3 library. In our knockout system, antibiotic selection begins 48 h after transduction.

Note: Before starting the screen ensure that cells adhere reasonably well to tissue culture vessels, and all-media requirements are met.

CRITICAL: Determine optimal cell plating density.

CRITICAL: Measure the Resazurin dye by fluorescence or absorbance mode.

Note: Fluorescence mode measurement offers greater assay linearity, reproducibility, robustness, and sensitivity.

• 1.
  Plate cells.

  • a.
    Plate iPSC-derived macrophage precursors at 0.25 million per well in 24 well plate.

Note: harvest the macrophage precursors from iPSC-monocytes factory and plate them 24 hours prior to the selection addition.

• 2.
  Add selection.

  • a.
    Replace medium with fresh medium supplemented with differentiation factors and a range of antibiotic concentrations 24 h after plating the cells.

CRITICAL: Remember to include untreated control cells, which have medium with 1× PBS and without selective antibiotic. Dilution range span 0 μg/mL to 10 μg/mL in 0.5 μg/mL increments.

• 3.
  Selection.

  • a.
    Observe the percentage of surviving cells under a microscope.

Note: Optimum effectiveness should be reached in 2–15 days, depending on the antibiotic used and differentiation medium. In our case, cells reach puromycin effectiveness after 10 days of selection.

CRITICAL: culture the cells in differentiation medium at this stage, so they do not proliferate. Since cells do not divide during the selection period, it would not be necessary to passage the cells.

Optional: If necessary, approximately every 2–4 days replace the medium with freshly prepared differentiation medium containing the range of antibiotic concentrations we test.

• 4.
  Resazurin assay.

  • a.
    Remove media and replace with 500 μL fresh growth media containing 10 μg/mL of Resazurin sodium salt by diluting 1:1000 the stock solution.

    CRITICAL: Make sure each well contains the same volume and remember to add it to an empty well as background.

  • b.
    Incubate for up to 6 h at 37°C in a cell culture incubator, protect from direct light.

    Note: Sensitivity of detection increases with longer incubation times. For samples with fewer cells or slow metabolism cells, use longer incubation times of up to 24 hours.

    Optional: Add 50 μL 15% SDS directly to the wells to stop the reaction.

  • c.
    Record results using fluorescence or absorbance at room temperature.

    • i.
      Read fluorescence using a fluorescence excitation wavelength of 540–570 nm. Peak excitation is 570 nm and peak emission is 585 nm.

      Note: When fluorescence instrumentation is unavailable, wrap assay plates or tubes in foil and store at 4°C. Then read within 1–3 days without affecting the fluorescence or absorbance values.

    • ii.
      Read absorbance of Resazurin at 570 nm, using 600 nm as a reference wavelength (normalize to the 600 nm value).
  • d.
    Plot the results.

    CRITICAL: The minimum antibiotic concentration to use is the lowest concentration that kills 100% of untreated control cells in 3–10 days from the start of antibiotic selection.

    Example: Use puromycin-supplemented media for 10 days to determine the optimal antibiotic concentration by a tolerance test of each macrophage harvest. Change media every 3–4 days.

    Note: We found most non-transduced macrophages died when exposed to 1 μg/mL puromycin.

Dose response curve for polybrene of macrophages via resazurin assay

Timing: 5 days

Polybrene is a positively charged polymer that can greatly enhance the efficiency of the retroviral or lentiviral infection into the mammalian cells.

• 5.
  Plate cells.

  • a.
    Plate iPSC-derived macrophages at 0.25 million per well in 24 well plate.

Note: Harvest the macrophage precursors from iPSC-monocytes factory and plate them 24 hours prior to polybrene addition.

• 6.
  Polybrene curve.

  • a.
    24 h after plating the cells, add polybrene at concentration ranging from 0 up to 16 μg/mL.

    CRITICAL: Remember to include untreated control cells with no polybrene.

  • b.
    After 18 h, remove the medium containing polybrene and replace with fresh medium.
• 7.
  Resazurin assay.

  • a.
    48 h after removing the polybrene, perform the Resazurin assay as described above.

Note: Read resazurin assay 72 hours after adding the polybrene.

CRITICAL: The optimal polybrene concentration is the highest tolerated by the cells without impacting viability.

Note: In our hands, the typical working concentration for polybrene in iPSC-derived macrophages precursors is 6 μg/mL.

Large scale production of VPX virus-like particles in 15-cm tissue culture plates

Timing: 5 days

CRITICAL: Biosafety precautions: Proper handling of lentivirus should be followed as outlined by your institution's Environmental Health and Safety Office.

• 8.
  Plate cells.

  • a.
    Plate HEK293T cells at 60% confluency in a T175 flask, about 9 million in each flask.
  • b.
    Repeat for as many T175 flasks as necessary.
• 9.
  DNA Transfection.

  • a.
    Replace the medium to 17 mL fresh Pen. /Strep. free medium 1 h before starting the transfection.
  • b.
    Allow the vial of jetPRIME Buffer to reach room temperature for around 1 h.
  • c.
    Prepare the transfection mixture;

    • i.
      Add 30 μg of DNA (psPAX2, pCMV-VSV-G, pSIV3 in equimolar ratio) per each flask.
    • ii.
      Add 2 mL of jetPRIME buffer.
    • iii.
      Add 60 μL of jetPRIME reagent per each flask to mixture DNA + buffer.
  • d.
    Mix by vortex 15 s and incubate for 15 min at room temperature.
  • e.
    Add 2 mL of the mixture to each flask drop-wise gently to not disturb or lift the cells.
• 10.
  Replace Media.

  • a.
    Very gently replace media with fresh media 18 h post transfection.
• 11.
  First Collection and Concentration.

  • a.
    Collect the media in 50 mL conical tubes and replace it with 17 mL fresh media without antibiotics.
  • b.
    Spin the media containing the virus at 2000 rpm for 5 min to remove cell debris. Keep the supernatant and discard the pellet.
  • c.
    Filter the supernatant with a 0.45 μm filter to remove cells completely.
  • d.
    Ultra-centrifuge the virus containing media to collect virus by spinning 29000 rpm (∼188000 g) for 2 h at 4°C.

    • i.
      Centrifuge Beckmann Optima XPN-100 Ultracentrifuge.
    • ii.
      Rotor SW32ti in sterile Ultra clear tubes (Beckmann, #344058).
    • iii.
      Rmax:152.5 (Maximum accelerating rate and maximum decelerating rate).
  • e.
    Aspirate supernatant and resuspend pellet in 1.5% sterile PBS/BSA. Use 0.5 mL/tube.
  • f.
    Keep virus at 4°C overnight in the tube, being careful to maintain sterility.
• 12.
  Second Collection.

  • a.
    Collect media, spin, filter and ultracentrifuge (Repeat Day 3 conditions).

    Note: Do two collections of virus separately to allow the first collection to be resuspended overnight at 4°C in BSA. Then we pool the first and the second virus collections.

    Note: This technique will increase the virus concentration in comparison with leaving the medium at 4°C overnight before the centrifugation.

  • b.
    Resuspend in 1.5% sterile PBS/BSA, use 0.25 mL per tube.
  • c.
    Mix both collections, aliquot in small aliquots and freeze-down at −80°C.

VPX virus-like particles titration by western blotting

Timing: 6 days

Note: Biosafety precautions: Proper handling of lentivirus should be followed as outlined by your institution's Environmental Health and Safety Office.

• 13.
  Plate cells.

  • a.
    Plate iPSC-derived macrophage precursors, 24 h before transduction, at 0.25 million per well in 24 well plate.
• 14.
  Vpx-VLPs transduction.

  • a.
    Thaw in ice a new aliquot of Vpx-VLPs if stored at −80°C.
  • b.
    Mix increasing amounts of Vpx-VLPs virus (1–11 μL) to 0.5 mL of cell medium containing Polybrene and add to each well.

    Note: Remember to have a sample without virus.

    • i.
      Add Polybrene to the media at the concentration that has been tested for each batch of cells.
    • ii.
      Add Vpx-VLPs 24 h after plating the cells.
  • c.
    Remove medium 18 h after addition of virus and Vpx-VLPs.

    Note: At this stage, cells should be infected and tightly adhered to plate.

    Optional: Gently wash plate with warm 1× PBS to remove any extraneous virus.

  • d.
    Add fresh medium and return plates to incubator.
• 15.
  Protein Extraction & western Blot.

  • a.
    72 h after transduction, gently remove medium and wash plates twice with cold 1× PBS to dislodge dead cells.

    CRITICAL: Place plates on ice during the PBS washes and RIPA buffer addition.

  • b.
    Add 50 μL/well cold 1× RIPA Buffer (Thermo Fisher, #89900) with added Halt Protease Inhibitor Cocktail (Thermo Fisher, #78429).

    • i.
      Keep on ice for 5 min.
  • c.
    Gather the cell lysate using a cell scraper and transfer it to a 1.5 mL-tube.
  • d.
    Spin at 14000 g for 15 min at 4°C.

    Optional: Increase yield by sonicating for 30 sec 50% pulse before centrifugation.

    • i.
      Transfer the supernatant, which contains the protein, to a tube.
    • ii.
      Discard the pellet.
  • e.
    Quantify protein content by BSA assay according to the manufacturer’s instructions.
  • f.
    Run 40 μg per sample by SDS-PAGE electrophoresis in Bolt gradient 4–12% Bis–Tris Plus acrylamide Gel (Thermo Fisher).
  • g.
    Transfer the gel to Whatman Protran nitrocellulose membrane (Millipore, #Z670766) using the Mini Trans-Blot Cell (Bio-Rad) according to the manufacturer’s instructions.
  • h.
    Block the membrane in 4% skim milk in TBST.
  • i.
    Incubate in the membrane with SAMHD1 antibody Abcam ab67820 (dilution 1:500) in 1% skimmed milk in TBST overnight.
• 16.
  Western Blot results.

  • a.
    Wash the membrane 3 times with TBST for 10 min on mild agitation.
  • b.
    Incubate the membrane in 1% skimmed milk in TBST with secondary antibody anti-animal (dilution 1:15000) for 1 h at RT.

Note: In our hands, the minimum volume of VPX virus-like particles (produced and stored as described above) that eliminates SAMHD1 is 1.3–5 μL per 1 million macrophages (Figure 1).

Figure 1.

Example of VPX virus-like particles titration by western blotting of SAMHD1

Top panel: Western blot analysis of iPSC-derived macrophages treated with increasing concentration of VPX virus like particles to knock-out the target gene SAMHD1. Bottom panel: Western blot analysis of GAPDH as loading control. Figure reprinted with permission from Navarro-Guerrero et al., 2021.

Lentiviral CRISPR library MOI determination in iPSC-derived macrophages

Timing: up to 17 days

This protocol describes a 24 well format relative tittering for lentiviral stocks, based on transduction at low multiplicity of infection (MOI), selection for transduced cells (with puromycin, or other antibiotics varying by vectors), and a viability assay to quantify cell survival.

CRITICAL: The multiplicity of infection must be determined under the same cell culture conditions used during the primary screen. This includes using the same tissue culture vessels, media constituents and volume, cell plating density, and pooled virus preparations that will be used in the screen.

Note: Measurements made in different formats cannot be reliably scaled to the screening format, but it can be useful to get estimate in smaller vessel and confirm it in the screening setting.

• 17.
  Plate cells.

  • a.
    Plate iPSC-derived macrophage precursors in a 24 well plate at 0.25 million per well in a 24 well plate.

Optional: To reduce well-to-well variability and edge effects, allow seeded plates to sit undisturbed on a flat surface at room temperature for at least 1 h before transferring to a tissue culture incubator overnight.

• 18.
  Transduction.

  • a.
    Thaw a new aliquot of Vpx-VLPs and a new aliquot of lentiviral CRISPR library at room temperature.

    Note: Keep aliquot in ice once thawed.

  • b.
    Prepare medium containing the correct amount of polybrene (PB) and Vpx-VLPs. Example:

    • i.
      PB = 4 μg/mL.
    • ii.
      Vpx-VLPs = 2.5 μL per 1 million macrophages.
    • iii.
      PB stock is 8000 μg/mL.
    • iv.
      Calculation of the volume we need (V):

      $$500\mu \mathrm{L}\times 4\mu \mathrm{g}/\text{mL}=8000\mu \mathrm{g}/\text{mL}\times \mathrm{V}\to \mathrm{V}=4\mu \mathrm{L}\text{PB}$$

      Note: These values can vary depending on the type of macrophages, cell density and passage.

  • c.
    Prepare a serial dilution series of the CRISPR library virus, with sufficient volumes for multiple replicate wells per dilution.
    Example: for a 1:100 dilution, transfer 495 μL fresh medium add 5 μL virus stock per well and mix well by pipetting.
  • d.
    Add polybrene to the media for a final concentration of 4 μg/mL.

    Note: Calculate first the correct concentration of polybrene.

    Note: Use an appropriate dilution such that the unknown samples fall within the linear range of the standard curve. Higher titer virus stocks will require a larger dilution factor.

    Example: Our serial dilution series starts at 1:100, the dilution factor is 2 and it has 11 points.
    Essential: Set a ‘no virus’ control.
  • e.
    Prepare cells for transduction.

    • i.
      Remove media.
    • ii.
      Add 500 μL of fresh medium supplemented with polybrene, Vpx-VLP and the diluted virus mixture.
  • f.
    Spinfection.

    • i.
      Spin plates at 800 × g for 2 h at 37°C.
    • ii.
      Replace with fresh medium.
    • iii.
      Return the plate(s) to the incubator.
• 19.
  Selection.

  • a.
    72 h post transduction, prepare fresh medium supplemented with differentiation factors, with and without puromycin (or specific antibiotic selection based on the CRISPR lentiviral library).

    Note: Calculate first the correct concentration of the specific selection.

  • b.
    Prepare at least one replicate in selective medium and one in medium without selection for each viral dilution.
• 20.
  Monitor cells.

  • a.
    Observe the percentage of surviving cells using a microscope.
  • b.
    Optimum effectiveness should be reached in 2–15 days, depending on the antibiotic we use and the differentiation medium.
  • c.
    The no virus control should be 100% dead cells, while the no selection control should be 100% viable.
• 21.
  Resazurin Assay and Library Titration.

  • a.
    Remove media and replace with 500 μL fresh growth media containing 10 μg/mL final of Resazurin sodium salt by diluting 1:1000 the previously prepared stock solution.
    Essential: Each well contains the same volume.

    Note: The ‘no virus’ control is the correct background.

  • b.
    Incubate for 6 h at 37°C in a cell culture incubator, protect from direct light.

    Note: Sensitivity of detection increases with longer incubation times.

    Note: For samples with fewer cells or slow metabolism cells, use longer incubation times of up to 24 hours.

    Optional: Add 50 μL 15% SDS directly to the wells to stop the reaction.

  • c.
    Record results using fluorescence or absorbance as described above.
  • d.
    Plot the results for the two series, plus and minus selection.

    • i.
      Determine virus volume that gives 30–40% survival with selection vs. without.
    • ii.
      This is the volume of library virus that gives MOI of 0.3 with the tissue culture conditions we use.
  • e.
    Back-calculate the amount of lentiviral CRISPR library necessary to transduce any given amount of target cells at any transduction efficiency using the formula:

    $$Viraltiter\left(TU/ml\right)=Numberofcellsattransduction\times MOI/\left(VolumeofLVprepusedattransduction\right)$$

Lentiviral CRISPR-Cas9 based knock out in iPSC-derived macrophages

Timing: up to 17 days

This protocol describes a 24 well knock out CRISPR-Cas9 technique, based on transduction at low multiplicity of infection (MOI).

CRITICAL: Perform amplification and viral production of lentiviral particles of the target gene(s) strictly following the author’s protocol. Determine each virus(es) MOI.

• 22.
  Plate cells.

  • a.
    Plate as many iPSC-derived macrophage precursors as necessary in 6 well plates at a density of 1 million per well.

    • i.
      Set up few controls, including non-target guides if necessary and no-virus.
    • ii.
      Allow seeded plates to sit undisturbed on a flat surface at room temperature for at least 1 h before transferring to a tissue culture incubator overnight, to reduce well-to-well variability and edge effects.
• 23.
  Transduction.

  • a.
    Thaw as many new aliquots of Vpx-VLPs and CRISPR virus as necessary at room temperature, then keep in ice.
  • b.
    Prepare the macrophages precursor medium and supplement with:

    • i.
      4 μL of polybrene.
    • ii.
      2.5 μL of Vpx-VLPs per million of macrophages.
    • iii.
      single-gene CRISPR-Cas9 lentivirus.
  • c.
    Remove media and replace with 500 μL of fresh medium supplemented with polybrene, Vpx-VPL and diluted virus mixture.

    Note: leave a well without virus.

  • d.
    Spin plates at 800 g for 2 h at 37°C.
  • e.
    Return the plate to the incubator.
• 24.Replace with fresh medium 18 h after transduction.
• 25.
  Differentiation.

  • a.
    72 h post transduction, replace 50% of the medium with fresh differentiation medium containing the differentiation factors.

    Note: For the antibiotic selection control, replace the medium with fresh medium containing the differentiation factors and the previously established concentration of selection.

    Replace 50% of the medium with fresh differentiation medium every 3–4 days.
  • b.
    Monitor the cells daily using a microscope.

    Note: The no-virus control should be 100% viable, while the selection control should have a viability similar to the one chosen based on the desirable MOI.

    Example: The desirable MOI of 0.3 correspond to 25% of cell viability (25% of infected cells).
• 26.
  Screening.

  • a.
    For the controls, gently remove the media and wash with 1× PBS.
  • b.
    Lift the cells by adding 10 mM EDTA and incubating for 10 min at 37°C.
  • c.
    Collect the cells in suspension by carefully pipetting.
  • d.
    Wash in 1× PBS, spin 5 min at 300 g and dispose of the supernatant.
  • e.
    The macrophages are now ready for a viability or phenotypic readout.

Note: Test the knockout effect (Figure 2) and the Cas9 expression (Figure 3) by western blot, and the library guides distribution by next-generation sequencing (Figure 3).

Figure 2.

Cas9 expression in iPSC-derived macrophage knock-out targeting HPRT1; PPIB and CDK4

Top panel: Western blot analysis of Cas9 protein in CRISPR-Cas9-transduced iPSC-derived macrophages knock-out targeting three macrophage non-essential genes: HPRT1; PPIB and CDK4 respectively. Bottom panel: Western blot analysis of GAPDH as loading control. Figure reprinted with permission from Navarro-Guerrero et al., 2021.

Figure 3.

Experimental characteristics of TKOv3 genome-wide knockout library in iPSC-derived macrophages

(A), Western blot analysis of Cas9 expression in TKOv3-transduced cells: in the top panel Cas9 is detected as ∼160 kDa band; in the bottom panel, GAPDH is loading control. (B), Histogram showing the total distribution of reads for the guides per each gene. The curve shows a maximum of 2233 reads per guide and a mean of 339 reads per guide. Figure reprinted with permission from Navarro-Guerrero et al., 2021.

Expected outcomes

Figures 2 and 3 show representative western-blot gels with the knockout effect (Figure 2) and the Cas9 expression (Figure 3) in iPSC-derived macrophages.

Limitations

Antibiotics selection post-transduction can activate certain types of immune cells.

We use puromycin to select macrophages transduced with TKOv3 library. In fact, using TNF expression as activation marker and quantifying it by flow cytometry, puromycin-treated transduced macrophages exhibited a decrease in TNF expression upon LPS stimulation from 71.9 ± 8.0% to 42.2 ± 7.0% (Figure 4). A potential explanation for the lower levels of increased TNF cells is that the transduced cells (puromycin-resistant) may have a suppressed production of pro-inflammatory mediators, most likely due to ingestion of dead cells, which is thought to be anti-inflammatory and immunosuppressive.

Figure 4.

Puromycin treatment decreases TNF expression upon LPS stimulation in CRISPR-Cas9 transduced iPSC-derived macrophages

We analyzed by flow cytometry the percentage of iPSC-derived macrophages able to produce TNF after TKOv3 infection in response to LPS treatment. (n = 3; p-value = 0.051). TNF expression upon LPS stimulation is 71.9 ± 8.0% without puromycin and 42.2 ± 7.0% with puromycin Figure reprinted with permission from Navarro-Guerrero et al., 2021.

Troubleshooting

Problem 1

Cells do not differentiate well into macrophage or have low/no marker expression. Related to step 2 at dose response curve for antibiotic selection of macrophages via Resazurin assay.

Potential solution

Prepare new differentiation media with freshly thawed aliquots or new reagents.

Problem 2

Poor or inefficient production of VPX virus-like particles. Related to steps 8–12 at large scale production of VPX virus-like particles in 15-cm tissue culture plates.

Potential solution

• •Plasmid DNA must be of high quality and concentration. Potentially check on agarose gel for DNA integrity. DNA resuspension solution must be endotoxin free and free from other transfection inhibitors.
• •Check for mycoplasma contamination in the 293T cells used for transfection.
• •Before transfection, the 293T cells must have been passaged more than twice and less than 20 times.
• •Do not overgrow the 293T cells (do not allow the cells to reach more than 90% confluency to keep the culture continuously in logarithmic growth phase).
• •Use HEPES pH 7.4, 20 mM final or an alternative to prevent cell medium to acidify.

Problem 3

Low transduction efficiency. Related to step 23 at lentiviral CRISPR-Cas9 based knock out in iPSC-derived macrophages.

Potential solution

Potential solution in this section have been partially modified from “Packaging, Tittering, and Transduction of Lentiviral Constructs” Cellecta.

• •A most trivial and easy to fix possibility is a potential loss of lentiviral titer during initial storage. In our hands, each freeze-thaw cycle typically causes reduction of the titer by ∼20%. The standard solution is to ensure storage of aliquoted packaged construct at –80°C. We always recommend using fresh stock for transduction.
• •Cells are typically infected in the presence of polybrene or protamine sulfate, to neutralize the charge repulsion between the virus and cell target surface and helps viral integration into the cell.
• •Spinfection is another technique that can be tested to achieve higher transduction for many cells. Overall, the ideal transduction conditions can be highly variable across samples and should be optimized. There are multiple factors: seeding density, polybrene concentration, MOI, Vpx-VLPs quality and concentration plus other specific variables (such as time, speed, temperature for spinfection). The correct balance will be determined to ensure successful transduction and minimal cost to cell viability.

Problem 4

Low cell viability after transduction. Related to step 13 at the VPX virus-like particles titration by western blotting or step 22 at the lentiviral CRISPR-Cas9 based knock out in iPSC-derived macrophages.

Potential solution

Increase cell density at plating step, before adding virus.

Problem 5

The amount of protein sample(s) is too low to perform traditional western blotting (i.e., less than 40 μg protein). Related to first step 15 at VPX virus-like particles titration by western blotting.

Potential solution

Use automated protein detection and analysis systems, for example WES or JESS Simple Western, these are capillary-based system to obtain higher sensitivity with a much lower amount of sample.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Elena Navarro-Guerrero (elena.navarroguerrero@ndm.ox.ac.uk).

Technical contact

Further information and requests for technical details should be directed to and will be fulfilled by Elena Navarro-Guerrero (elena.navarroguerrero@ndm.ox.ac.uk).

Materials availability

This study did not generate any unique materials or reagents.

Data and code availability

This study did not generate any unique datasets or code.