Cell Surface Receptor Identification Using Genome-Scale CRISPR/Cas9 Genetic Screens

1. Production and purification of biotinylated His-tagged proteins

1. Use a mammalian or insect cell-based protein expression system to produce soluble recombinant His-tagged biotinylated proteins (see plasmid constructs in Table 1).
   NOTE: A detailed protocol for the production of monomeric biotin and His-tagged proteins using the HEK293 cell expression system is described by Kerr et al.¹⁷. Protein ectodomains expressed using the HEK293 expression system are secreted into the culture medium.
2. Collect the soluble proteins by pelleting the cells by centrifugation at 3,000 x g for 20 min.
3. Filter the supernatant through a 0.22 µm filter and add the Ni²⁺-NTA agarose beads to the filtered protein supernatant in a 1:1,000 ratio (i.e., 50 µL of 50% agarose slurry into 50 mL of supernatant). Incubate overnight or at least 4-5 h at 4 °C on a rotating platform.
4. Wash the polypropylene column by adding 5 mL of His-purification wash buffer. Refer to Table 2 for all buffer compositions.
5. Pour the entire bead-protein supernatant mixture into the column. Beads will accumulate at the base.
6. Wash the beads 2x with 15 mL of wash buffer. To avoid protein dilution, carefully draw the residual wash buffer from the column with a 5 mL syringe and discard.
7. Carefully add 300-500 µL of His-purification elution buffer directly to the beads and incubate for at least 1 h. Collect the eluted protein by again carefully drawing out the liquid using a 1 mL syringe. Exchange the elution buffer to the desired buffer (e.g., normally PBS or HBS) using desalting columns. Store all proteins at 4 °C until further use.

2. Quantification and oligomerization of monomeric biotinylated protein

NOTE: To increase the binding avidity, oligomerize biotinylated monomeric proteins on tetrameric streptavidin-PE before using them in binding assays. Achieve optimal conjugation ratios of monomeric proteins and tetrameric streptavidin-PE by testing a dilution series of biotinylated monomers against a fixed concentration of streptavidin and by empirically establishing the minimum dilution at which no excess biotinylated monomers can be detected.

1. Make at least eight serial dilutions of biotinylated protein samples using an appropriate dilution buffer (either PBS or HBS with 1% bovine serum albumin [BSA]) in a 96 well plate. Ensure that the final volume of each dilution is at least 200 µL.
2. Make a duplicate plate of the samples by removing 100 µL from each well and transferring into a new 96 well plate. Always include a control. In this case controls are tag-only proteins (i.e., biotinylated His-tagged Cd4 domain 3+4 protein). This will be used as a control probe in all binding assays.
3. Dilute streptavidin-PE to 0.1 µg/mL in the dilution buffer.
4. On just one of the plates, add 100 µL of the diluted streptavidin-PE. The duplicate plate will serve as a control. Add 100 µL of dilution buffer in the control plate to equalize volumes.
5. Incubate for 20 min at room temperature (RT). In the meantime, block the wells of a streptavidin-coated plate with the dilution buffer for 15 min.
6. Transfer the total volume of the sample from both plates to individual wells of the streptavidin-coated plates and incubate for 1 h at RT.
7. Wash the plate 3x with 200 µL of wash buffer (i.e., either PBS or HBS with 0.1% Tween-20, 2% BSA). Add 100 µL of 2 µg/mL mouse anti-rat Cd4d3+4 IgG (OX68) and incubate for 1 h at RT.
8. Wash the plate 3x with the wash buffer. Add 100 µL of an anti-mouse alkaline phosphatase conjugate at 0.2 µg/mL for 1 h at RT.
9. Wash the plate 3x with wash buffer and 1x in dilution buffer.
10. Prepare p-nitrophenyl phosphate at 1 mg/mL in diethanolamine buffer. Add 100 µL in each well and incubate for 15 min.
11. Take absorbance readings at 405 nm. Use the minimum dilution at which there is no signal on the plate as the appropriate dilution factor to create tetramers (Figure 2).
12. Make a 10x tetramer staining solution for all samples and controls by incubating 4 µg/mL streptavidin-PE and the appropriate biotinylated protein dilution for 30 min at RT. Store conjugated proteins in a light-protected tube at 4 °C until further use.

3. Flow cytometry-based cell binding assays

1. For adherent cells, remove culture media and wash 1x with PBS without divalent cations. Then add cell detachment solutions (e.g., EDTA). Allow the cells to detach for 5-10 min. Gently tap the flask to release the cells.
   NOTE: Avoid using trypsin-based products as they can cleave cell surface proteins.
2. Collect detached cells into a tube. For cells growing in suspension (e.g., HEK293 cells), directly collect the cells from culture flasks into a tube.
3. Pellet cells at 200 x g for 5 min. Remove the supernatant and resuspend the pellet in wash buffer (i.e., PBS/1% BSA).
4. Count the cells using a hemocytometer and adjust the concentration to 2.5 x 10⁵-1 x 10⁶ cells/mL. Aliquot 100 µL of prepared cell mix on a 96 well U- or V- bottomed plate. Spin the plate for 5 min at 400 x g. Remove the supernatant with a multichannel pipette.
5. Add 100 µL of normalized fluorescently labeled highly avid protein probes and controls into the previously prepared plates with cells and incubate for 1 h at 4 °C. After binding for 1 h, spin the plate at 400 x g for 5 min.
6. Remove the supernatant and add 200 µL of wash buffer (i.e., PBS/1% BSA). Mix well by pipetting up and down.
7. Pellet the cells by centrifugation at 400 x g for 5 min. Repeat the wash step 1x. After two washes, completely remove the supernatant and resuspend the cell pellet in 100 µL of PBS.
8. Analyze the cells by flow cytometry. Use the yellow-green laser (i.e., 561 nm) to detect PE fluorescence.
   1. First analyze the cells that have been stained with the control probe. Based on the distribution of PE fluorescence, draw a gate for binding population such that no more than 1% of the control cell falls in this gate.
   2. Analyze the sample and determine the fraction of cells that falls in the binding gate.
      NOTE: Cell lines that display a higher binding population are desired for genetic screens, as they have a higher signal-to-noise ratio. Ideally over 80% of the cells should fall within this gate.

4. Determining binding contributions from heat labile epitopes and heparan sulphate sidechains

NOTE: The activity of many proteins is heat labile, so loss of binding activity following heat treatment is encouraging. It is advised to determine the contribution from negatively charged glycosaminoglycans, mainly heparan sulphate (HS), in mediating binding of the recombinant proteins. This is because the binding by HS in the cell binding assay described here can be additive rather than codependent on other receptors¹⁹. This means that the observed binding could be entirely mediated by HS side chains of cell surface proteoglycans and not by a specific receptor. Binding to HS on the cell surface is not necessarily nonspecific, but rather a property of a protein, which is useful to know before performing a full genetic screen.

1. Prepare heat-treated protein samples to use in binding assays.
   1. Heat the normalized but unconjugated monomeric protein at 80 °C for 10 min.
   2. Conjugate the heat-treated protein to streptavidin-PE assuming the same conjugation ratio as its untreated counterpart as determined by ELISA (refer to section 2).
2. Prepare heparin-blocked protein samples.
   1. Prepare eight 1:3 dilutions of soluble heparin in PBS with a starting concentration of 2 mg/mL and final volume of 100 µL.
   2. Incubate 100 µL of prepared binding probes in the heparin dilutions for at least 30 min.
3. Use heat-treated protein and the full 200 µL of heparin/protein mixture in the binding assays described in section 3. Representative results are shown in Figure 3A,B.

5. Selection of cell lines stably expressing Cas9

NOTE: Before the cell line that binds the probe of interest can be used in CRISPR screening, it must first be engineered to express the Cas9 nuclease and a highly active clone selected¹⁹.

1. Use the following general lentivirus production protocol to produce lentivirus using the lentiviral construct for Cas9 expression (refer to Table 1).
   1. Culture HEK293-FT cells in DMEM/10% FBS media at 37 °C and 5% CO₂. Seed HEK293-FT cells 1 day prior to transfection so that they are ~80% confluent on the day of transfection.
      NOTE: HEK293FT cells are loosely adherent; therefore, when they are used for production of lentiviruses, consider plating them on culture flasks coated with 0.1% (w/v) gelatin to increase adherence.
   2. Perform transfections in the morning. Add transfer vector, packaging mix, and transfection reagent into prewarmed transfection compatible media (e.g., Opti-MEM). Mix by inverting the tube 10-15x. Incubate for 5 min at RT. Refer to Table 3 for exact volumes.
   3. Add the transfection reagent as suggested by the manufacturer. Mix by quick vortexing. Incubate for 30 min at RT.
   4. Very carefully aspirate the spent medium. Add transfection compatible media to plate.
   5. Add the transfection reagent/DNA complexes dropwise onto the side of the plate and slowly spread through the plate by swirling very gently.
   6. Incubate at 37 °C for 3-5 h and replace the medium with D10 medium. Incubate overnight.
   7. Next day in the morning, replace the medium with fresh D10 medium. Incubate overnight.
   8. Next day late in the afternoon, collect the viral supernatant. Filter with a 0.45 µm filter with low protein binding. Optionally, add fresh D10 medium, incubate overnight and recollect the supernatant the next day.
   9. Virus supernatants are stable at 4 °C for only for a few days. Store at -80 °C for long-term storage.
      NOTE: To generate a highly concentrated lentiviral preparation, which could be desirable for the transduction of difficult to transduce cells, supernatants can also be concentrated by centrifugation at 6,000 x g overnight at 4 °C. Mark the translucent viral pellet with an ethanol-resistant pen and discard the supernatant. Resuspend the pellet in 1/100th of the original volume for a 100x increase in the concentration.
2. Transduce the cells with lentiviruses.
   1. Plate 1 x 10⁶ cells per well in a 6 well plate with 3 mL of appropriate culture media. Some cells are more readily transduced than others. For easy to transduce cells (e.g., HEK cells), directly add the lentivirus to the cells. For difficult to transduce cells, it might be necessary to follow a spinoculation protocol as described below.
      1. Aliquot 2 mL of 2-5 x 10⁶ cells/mL in a 15 mL conical tube.
      2. Add lentivirus together with 8 µg/mL hexadimethrine bromide and incubate at RT for 30 min.
      3. Centrifuge for 100 min at 800 x g at 32 °C. Then resuspended the cells in the same media and add the cell suspension into appropriate culture flasks with appropriate media.
   2. Allow transductions for at least 24 h. Afterwards remove the media containing the virus and add fresh medium.
   3. After another 24 h, change the media to one that is supplemented with the appropriate antibiotics. The Cas9 construct contains a blasticidin resistance cassette for selection.
      NOTE: The amount of blasticidin must be optimized for each cell line by performing a dose response kill curve. A blasticidin concentration between 2.5-50 µg/mL should kill most untransduced cell lines within 10 days of transduction.
3. Perform selection until all cells in the control plate (i.e., nontransduced cells that have been treated with the same concentration of selection antibiotics) are killed.

6. Selecting high Cas9-activity clones

NOTE: Polyclonal Cas9 can be used to successfully perform genetic screens; however, selecting a clone with high Cas9 activity improves the screening results¹⁸.

1. Use limiting dilution or single-cell sort individual blasticidin-resistant cells into wells of three 96 well plates containing culture media supplemented with blasticidin. Clones will start to emerge between 2-4 weeks. Select 10-20 clones and expand in 6 well plates.
2. Assay the clones for Cas9 activity using the quick-to-assess GFP-BFP (green fluorescent protein-blue fluorescent protein) system, which uses an exogenous gene knockout system in which cells are transduced either with a construct expressing GFP with a gRNA-targeting GFP or an empty gRNA as a control¹⁸.
   1. Order reporter plasmids: GFP-BFP plasmid, Control-BFP plasmid (Table 1).
   2. Produce lentivirus for both the GFP-BFP plasmid and Control-BFP plasmid using the lentivirus production protocol described in section 5.1.
   3. Transduce each Cas9-expressing cell line clone with the lentivirus encoding the GFP-BFP system and Control-BFP separately. Follow the protocol in section 5.2.
   4. After 3 days of transduction, examine the GFP-BFP fluorescence of each clone using flow cytometry. Use 488 nm laser and 405 nm laser to detect GFP and BFP respectively.
   5. Quantitate the Cas9 activity in each clone by examining the ratio of BFP only to GFP-BFP-double positive cells. High activity Cas9 cells should ideally have >95% GFP knockout efficiency (Figure 4).

7. Generation of genome-wide CRISPR-Cas9 screening knockout library

1. For the genome-wide screening using the Human V1 library¹⁸, order the genome-wide library (refer to Table 1) and prepare the plasmid library from the bacterial stab using the protocol provided under "Protocols for Library Replication" in the manufacturer's manual.
2. Use the genome-wide library plasmid preparation to produce a lentiviral library encoding gRNAs for targeted disruption of human genes using the lentivirus production protocol described in section 5.1.
   NOTE: A good practice is to produce a single batch of lentiviral preparation that is optimized for transduction to improve experimental consistency.
3. Use the transduction protocol in section 5.2 to perform small scale test transductions to determine the required amount of virus for each cell line to achieve 30% transduction. Use flow cytometry to assess BFP fluorescence as a proxy for transduction efficiency.
4. To transduce HEK293 cells, simply add the predetermined lentiviral preparation to 30-50 x 10⁶ cells cultured in normal growth media for ~4 h. Then remove the media with lentivirus and replace with fresh growth media.
5. For other cell lines, use the spinoculation protocol in section 5.2.1 but at a larger scale, such that a total of 30-50 x 10⁶ cells are transduced. For this, aliquot 2 mL of 5 x 10⁶ cells/mL in a 15 mL conical tube and proceed as indicated.
6. For adherent cell lines, select transduced cells by adding puromycin 24 h after transduction.
   NOTE: Optimize puromycin concentrations by performing a dose response kill curve. Normally concentrations between 1-10 µg/mL should kill nontransduced cells within 3-5 days. Avoid using higher concentrations of puromycin because this may increase the chances of selecting cells that have been transduced by more than onesingle guide RNA (sgRNA).
7. For suspension cells, harvest transduced (i.e., BFP positive) cells 3 days posttransduction using a cell sorter and generate libraries that contain at least 10 x 10⁶ cells. Once selected using BFP, grow the cells in media supplemented with appropriate amount of puromycin.
   NOTE: Avoid selections only with puromycin for suspension cell lines, because it is difficult to remove dead cells and debris from suspension cell cultures that can interfere with cell sorting.
8. Culture mutant library for 9-16 days posttransduction with regular passage every 2-3 days.

8. Genetic screening for cell surface binding

1. Pellet the mutant cell library at 200 x g for 5 min and resuspend the cells in PBS.
2. Divide the cells into two 15 mL conical tubes with at least 50 x 10⁶ cells in each tube.
3. Spin one conical tube at 200 x g for 5 min, remove the supernatant, and freeze the cell pellet at -20 °C. This is the control population and will be processed later.
4. Resuspend the pellet in the other tube in 10 mL of PBS/1% BSA. Set aside 100 µL of cells as a negative control on a 96 well plate.
5. Add the appropriate preconjugated recombinant protein to the cell suspension in the conical tube and negative control proteins to the 96 well plate.
6. Perform cell staining for at least 1 h at 4 °C on a benchtop rotor with gentle rotation (6 rpm).
7. Pellet the cells at 200 x g for 5 min, remove the supernatant. Perform two wash steps, then resuspend the cells in 5 mL of PBS.
8. Strain the cells though a 30 µm cell strainer to remove cell clusters. Analyze using a flow sorter.
9. Use the negative control sample to gate for BFP+/PE- cells.
10. Sort the sample and collect the BFP+/PE- cells. The sort gates will depend on the binding of the cells to the protein but is normally 1-5% of the PE negative samples are collected. An example of a sorting gate is provided in Supplementary Figure 1.
11. Collect 500,000-1,000,000 cells from the selected gate. Given the low number of cells, consider collecting the samples in a 1.5 mL centrifugation tube to minimize losses.
12. Pellet the sorted cells by centrifuging at 500 x g for 5 min. Carefully remove the supernatant and discard. It is possible to store the pellet at -20 °C for up to 6 months.

9. Genomic DNA extraction and first PCR for gRNA enrichment

1. Extract genomic DNA from high-complexity control population.
   1. If the control population was frozen at -20 °C, take out the conical tube and add PBS. Keep on ice to thaw the pellet.
   2. Use a commercial kit (see Table of Materials) using the manufacturer's recommendations to extract genomic DNA from 50 x 10⁶ cells. Adjust the DNA concentration to 1 mg/mL.
   3. For each sample, set up a master mix for PCR corresponding to 72 µg of DNA. Aliquot 50 µL per well in 36 wells of a 96 well PCR plate. The necessary primer sequences are listed in Table 4. Use the guide in Table 5 and 6.
   4. Resolve 5 µL of the PCR from 6-12 representative samples on a 2% (w/v) agarose gel. A single clear band at ~250 bp should be observed. If the bands are faint, repeat the PCR for an additional 2-3 cycles.
   5. Use a multichannel pipette to collect 5 µL of PCR products from each well (180 µL in total) and pool them in a reservoir with 900 µL of binding buffer from a commercial kit (see Table of Materials).
   6. Purify the PCR products using a commercial PCR purification kit. Elute DNA into 50 µL of elution buffer from a commercial kit (see Table of Materials) and measure the DNA concentration.
2. Samples that have been sorted for the loss of binding phenotype are unlikely to be composed of a large number of independent clones. Therefore, it is not necessary to perform PCR with 72 µg of DNA. Isolate the DNA using an appropriate commercial kit (see Table of Materials). Set up 3-4 PCR reactions using the protocol described before (section 9.1.3) with 100 ng/µL DNA. If the sorted cell number is less than 100,000 use cell lysates instead of genomic DNA preparations.
   1. Aliquot approximately 10,000 cells/well in a 96 well PCR plate.
   2. Pellet the cells in the plate and carefully remove most of the supernatant. The pellet will not be visible.
   3. Add 25 µL of water in each well and heat the samples at 95 °C for 10 min.
   4. Add 5 µL of 2 mg/mL freshly diluted proteinase K to each well for 1 h and incubate at 56 °C. Then heat the sample for 10 min at 95 °C to inactivate the proteinase K.
   5. Use 10 µL of cell lysate mixture per PCR reaction. Lysates should be used within 24 h.

10. Second round of PCR for index barcoding and sequencing

1. Dilute the products from the first round PCR to 40 pg/µL.
2. Set up one PCR per sample (use the guide provided in Tables 7 and 8). The use of a high-fidelity polymerase is important to minimize errors introduced by the polymerase during sgRNA amplification.
3. Resolve 5 µL of PCR products on a 2% (w/v) agarose gel. A single clear band at ~330 bps should be observed.
4. Purify PCR products using paramagnetic beads by adding 31.5 µL of (0.7x total volume) of resuspended beads to the PCR products, mixing well, and incubating for 5 min at RT.
5. Place the tube on a magnetic rack for 3 min. The beads should be captured on the side of the plate and the solution should be clear. Carefully remove and discard the supernatant.
6. Add 150 µl of 80% freshly prepared ethanol to the tube. Incubate for 30 s, and then carefully remove and discard the supernatant.
7. Repeat step 13.6, this time with 180 µL. Then air dry the beads for 5 min.
8. Remove the tube from the magnet. Elute DNA target from beads into 35 µL of sterile EB buffer. Incubate for 3 min, then put the tube back in the magnetic rack for 3 min.
9. Transfer approximately 30 µL of the supernatant containing the eluted PCR products to a clean tube.
10. Sequence the samples on a next-generation sequencing platform. For the HumanV1 gRNA library, use the custom primer listed in Table 4 to sequence 19 bp.

11. Bioinformatics analysis to identify the receptor and related pathways

1. Map sequences from sorted and unsorted population to the reference library using the count function of MAGeCK. The function will yield a raw count file (Supplementary Table 1).
   NOTE: Detailed instructions on the installation of MAGeCK and the usage of different functions within MAGeCK are described in a previously published protocol by Wang et al.²⁰.
2. Check the technical standard of the control library used in the screen.
   1. Median normalize the raw counts and use the ggplot2 package in R²¹ or equivalent software to plot an empirical cumulative density function plot of the counts in plasmid and control unsorted samples (Figure 5A).
   2. Run the -test function of MAGeCK using counts from plasmid population as "control" and the counts from unsorted control samples as the "test" sample. The function is will yield a gene summary file (Supplementary Table 1).
   3. Open the gene summary file and draw the distribution of log-fold-changes (neg|lfc column) for previously categorized essential and nonessential genes²² (Figure 5B).
   4. Select significantly depleted genes (neg|fdr < 0.05) and perform pathway enrichment analysis using the enrichr²³ package or any equivalent pathway enrichment packages in R (Figure 5C).
3. Run the -test function of MAGeCK with default setting. Use raw counts from unsorted control sample as "control" and counts from sorted sample as "treatment" when performing the analysis.
4. Open the gene summary file generated by MAGeCK and rank the pos|rank column in ascending order. Use FDR (pos|fdr column) < 0.05 as a cutoff for identification of hits. The receptor is usually ranked highly, often in the first position.
5. Plot the Robust-Ranking-Algorithm (RRA) scores for positive selection (pos|score) in R or an equivalent software (Figure 5D).
6. Select the gene hits and perform pathway enrichment analysis using the enrichr package or any equivalent pathway enrichment packages in R to identify the enriched pathways.