Apr 02, 2026
dSpCRISPRi tiling screen targeting the TOX locus in primary human CD8+ T cells
- Christian McRoberts Amador1,
- Aretha Gao1,
- Charles Gersbach1
- 1Duke University
- Gersbach Lab
Protocol Citation: Christian McRoberts Amador, Aretha Gao, Charles Gersbach 2026. dSpCRISPRi tiling screen targeting the TOX locus in primary human CD8+ T cells. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vz4e72lx1/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 31, 2026
Last Modified: April 02, 2026
Protocol Integer ID: 314198
Funders Acknowledgements:
NIH
Grant ID: HG012053
Disclaimer
This protocol was adapted from the work of Christian McRoberts Amador and colleagues in the Gersbach lab at Duke University.
Abstract
Lentiviral dSpCRISPRi tiling screen targeting TOX locus +- 100 kB from transcript start and end site in primary human CD8+ T cells (n = 3 donors). In brief, CD8+ human T cells are activated, transduced 24 hours later with lentiviral library, and expanded for a total of 10 days. T cells are restimulated with anti CD3/CD28 at a 3:1 ratio every two days post-transduction for a total of three restimulations. Top and bottom 10% TOX expressing cells are sorted via flow cytometry for subsequent genomic DNA extraction, PCR for next-gen library construction, and next-gen sequencing.
Materials
| A | |
| Complete DMEM* | |
| DMEM GlutaMAX | |
| 10% FBS | |
| 1% P/S | |
| 1 mM Sodium Pyruvate | |
| 1x MEM NEAA | |
| 10 mM HEPES solution |
| A | |
| Complete Opti-MEM | |
| Opti-MEM‱ I Reduced Serum Medium (OPTI-MEM) | |
| 1x Glutmax | |
| 5% FBS | |
| 1 mM Sodium Pyruvate | |
| 1x MEM NEAA |
| A | |
| Complete primary human T cell expansion media (cPRIME) | |
| 1 L of PRIME media | |
| 50 mL hPlatelet lysate solution (5%) | |
| 10 mL P/S (1%) |
Dynabeads (ThermoFisher 11132D)
FOXP3 TF staining kit (ThermoFisher 00-5523-00)
TOX:APC antibody (Miltenyi 130-118-335)
Troubleshooting
Library design
Generate SpCas9 library using guidescan tiling - chr8:58705412-59219147. Filter guides by a cutting specificity score of >0.37. Additionally 325 NT guides are designed with similar nucleotide contents to the library. Total library size - 7377 guides.
Generating viral pool
| A | |
| Plate 7M 293Ts in the afternoon in 12 mL of cOpti-MEM | |
| The following morning, prepare L3000mix: Lipofectamine 3000 reagent (ThermoFisher) dilution in Opti-MEM media (no supplements), mix well | |
| Prepare DNA mix in Opti-MEM (no supplements) and then add P3000 (vortex) | |
| Combine L3000 mix and DNA mix 1:1 | |
| Incubate for 15 minutes at RT | |
| Remove 6 mL of media from well | |
| Add 3 mL per 10cm dropwise | |
| Incubate for 6 hours before media change | |
| Discard media and replace with 12 mL of Complete Opti-MEM (cOpti-MEM) | |
| Collect supernatant 24 hrs after transfection and keep at 4C (first harvest), replace with 12 mL cOpti-MEM | |
| Collect 48 hrs after transfection and pool with first collection (second harvest) | |
| Spin down at 600xg for 10 minutes to remove cellular debris | |
| Transfer supernatant to conical and add 4x LentiX concentrator | |
| Incubate for 2-24 hours at 4C | |
| Spin down at 1500xg for 45 minutes | |
| Aspirate and resuspend in 1/100th original volume of cOpti-MEM |
Screen
The library consists of 7377 guides, for 100x coverage in PBMCs, the required amount of cells is: 7377 / 0.20 (fraction transduced from titer experiment) x 100 = 3.7 million cells per rep.
| A | B | |
| Day | Activity | |
| -1 | Thaw 5 M CD8 T cells per biorep (Stem cell tech), activate all in cPRIME and 100 U/mL of IL-2. | |
| -1 | Plate three wells of a 6 well dish for each biorep, each with 5 M activated cells. | |
| 0 | Add 260 uL of TOX sp library lentivirus to each well. | |
| 2 | Count and expand cells into 10 cm plates with 8 mL of fresh media. Restimulate with dynabeads by removing old dynabeads with magnet and adding 3:1 dynabead:cell. | |
| 4 | Count and feed with 5 mL of fresh media. Restimulate with dynabeads by removing old dynabeads with magnet and adding 3:1 dynabead:cell. | |
| 6 | Count and expand into 15 cm plates with 10 mL of fresh media. Restimulate with dynabeads by removing old dynabeads with magnet and adding 3:1 dynabead:cell. | |
| 8 | Feed with 10 mL of fresh media. | |
| 10 | Harvest and count cells before staining for Thy1.1, fix/perm stain for TOX, and flow for Thy1.1+ and top/bottom 10% TOX CD8 Tex. |
Cell staining
Note: For each FACS+ab/ Perm+ab staining step, resuspend in a volume to achieve 10x10^6 cells/mL based on D10 cell count.
Ab dilutions:
- Thy1.1:PE - 1:300
- TOX:APC - 1:100
Surface Marker Staining
- Transfer full volume of each T cell condition into labeled 50 mL conical tubes. Make sure to also transfer a condition that will not be stained and will be used as your negative control.
- Place on magnet for 2-5 minutes to remove beads and transfer supernatant to another tube.
- Count cells using countess.
- Centrifuge at 300 g for 5 minutes.
- Aspirate supernatant.
- Resuspend in 3500 uL (this is an example volume, try to aim for resuspension concentration of 10x10^6 cells/mL) of FACS+Antibodie(s) solution (typically Thy1.1:FITC is used for transduction efficiency, and then other antibodies for whatever desired protein that is being overexpressed/repressed). For the negative control, simply resuspend in FACS buffer and it is ready to use.
- Place samples in shaker in cold room, cover with aluminum foil, and wait 30 minutes for staining.
- Add 17500 uL (again, use the calculated volume not the example one and add 5x as much of FACS buffer) of FACS buffer to samples.
- Centrifuge at 300 g for 5 minutes.
- Aspirate supernatant.
- Resuspend in 1000 uL of FACS buffer (again, example volume).
Intracellular Staining
Buffers and solution preparation
- Prepare fresh Foxp3 Fixation/Permeabilization (FOXP3 TF staining kit, ThermoFisher) working solution by mixing 1 part of Foxp3 Fixation/Permeabilization Concentrate with 3 parts of Foxp3 Fixation/Permeabilization Diluent. One mL of the working solution is required for each sample, if staining in tubes.
- Prepare a 1X working solution of Permeabilization Buffer by mixing 1 part of 10X Permeabilization Buffer with 9 parts of distilled water. 8.5 mL of the working solution is required for each sample, if staining in tubes.
Experimental Procedure in 12 x 75 mm Tubes
- Prepare a single-cell suspension. Refer to ‘Cell Preparation Protocols for Flow Cytometry’
- [Optional] Stain cells with a Fixable Viability Dye. Refer to ‘Viabilty Dye Staining, Protocol C’ for more details
- After the final wash, discard the supernatant and pulse vortex the sample to completely dissociate the pellet. Typically about 100 µL of residual volume remains.
- Add 1 mL of Foxp3 Fixation/Permeabilization working solution to each tube and pulse vortex
- Incubate for 30-60 minutes at 2-8°C or room temperature. Protect from light. (Mouse samples can be incubated for up to 18 hours at 2-8°C in the dark).
- Add 2 mL of 1X Permeabilization Buffer to each tube and centrifuge samples at 400-600 x g for 5 minutes at room temperature. Discard the supernatant.
- [Optional] Repeat Step 7.
- Resuspend pellet in residual volume of 1X Permeabilization Buffer. This is typically 100 µL after decanting.
- [Optional] Block with 2% normal mouse/rat serum by adding 2 µL directly to the cells. Incubate for 15 minutes at room temperature.
- Add 3.5 mL of 1x Perm buffer (again, this depends on the concentration).
- Without washing, add the recommended amount of directly conjugated antibody for detection of intracellular antigen(s) to cells and incubate for at least 30 minutes at room temperature. Protect from light. (1:100 dilution for TOX:APC ab, Miltenyi)
- Add 2 mL of 1X Permeabilization Buffer to each tube and centrifuge samples at 400-600 x g for 5 minutes at room temperature. Discard the supernatant.
- Repeat Step 13.
- Resuspend stained cells in an appropriate volume of Flow Cytometry Staining Buffer (based on concentration).
Proceed to flow cytometric analysis.