Jun 15, 2024
Immune Saturation Genome Editing (Immune SGE): Variant Analysis in T cells
- 1Seattle Children's Research Institute
- IGVF
Protocol Citation: Nathan Camp, Sam Dawson 2024. Immune Saturation Genome Editing (Immune SGE): Variant Analysis in T cells. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqn7e3gk5/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: April 02, 2024
Last Modified: June 15, 2024
Protocol Integer ID: 99390
Keywords: Saturation Genome Editing, Immune Cells, T cells, Primary cells, T cell receptor, CD3, CD28, CARD11, BCL10, IRF4, ssODN, CFSE
Funders Acknowledgements:
NHGRI (IGVF)
Grant ID: HG011969
Abstract
This protocol details immune saturation genome editing (Immune SGE) and variant analysis in T cells. In brief, primary T cells are isolated from donor PBMCs, edited with a variant library centered around a guide cut site, and variants are scored based on how they affect T cell proliferation in response to T cell receptor (TCR) stimulation. This method works well for genes that regulate the response to TCR activation.
Guidelines
Overview of repair template design of ssODN libraries. A) Example of an intron/exon border. We typically go 4 bp into the introns on either side of the exon. Bold characters indicate the original sgRNA sequence locations. Red characters indicate the original PAM sequence location. B) Our ssDNA repair templates are typically ~140 bp, including 30-50 bp of region to be edited and homology arms on both ends to make up the rest of the 140 bp. Underlined bases indicate synonymous mutations within the guide and PAM sites to discourage recutting of gDNA after repair. We typically go with 3 synonymous mutations with the thinking that some of our variants will revert one of them back to the original. In that case we will still have 2 synonymous mutations to limit recutting of the gDNA after repair. N indicates variant with any of the 4 bases.
Of note, using N as a designation for different variants when ordering oPool libraries results in ~50% of the edits containing the WT sequence with synonymous edits, thus reducing the fraction of your variants of interest. We are currently looking into how variant coverage changes when we use the other 3 bases in each location and completely limit the possibility of the WT base at that position:
Important considerations:
- We identify and test guides using ICE analysis before ordering oPools. We typically test guides alone and any guide that cuts >10% on its own should suffice. Although we prefer guides to cut with efficiency levels >50%, inclusion of repair templates and increasing guide to Cas9 ratios should significantly increase cutting efficiency.
- It is best to keep editing regions centered around the cut site and within 20bp of the cut site. We see significant dropout of variants that are >20 bp from the cut site. If possible, we will keep our editing regions no larger than 40 bp with 20 bp on either side of the cut site. Sometimes we don’t have a choice, so we transfect and sample more cells to ensure reasonable sequencing coverage.
- Occasionally we will have guides and/or PAM sites that cross into the intron. We recommend that the 3 synonymous variants within the “WT sequence with synonymous edits” do not alter the bases involved in splicing as these will most likely result in loss of variants within your analysis.
Materials
Reagents:
- T cell culture media
- RPMI
- 20% FBS
- 1% Glutamax
- 10 mM HEPES
- 0.1% BME
- Add fresh IL-2 on passage days (Peprotech AF200-02, 1x = 50 ng/mL)
- CD4+ T cell isolation kit (stemcell)
- Dynabeads™ Human T-Expander CD3/CD28Thermo FisherCatalog #11141D
- ImmunoCult Human CD2/3/28 STEMCELL Technologies Inc.Catalog #10970
- CellTrace™ CFSE Cell Proliferation Kit, for flow cytometryThermo FisherCatalog #C34570
- TRIzol ReagentThermo Fisher ScientificCatalog #15596026 )
- Quick-DNA miniprep plus kit Zymo ResearchCatalog #D4069
OVERVIEW: ssODN repair template and oPool design notes
OVERVIEW: ssODN repair template and oPool design notes
Determine your editing region of interest, trying to stay within 20bp on either side of the cut site.
Add respective homology arms to both the 5’ and 3’ ends of the editing region such that ssODN is at least 140 bp long.
Introduce 3 synonymous mutations in the editing region within the guide homology region and the PAM. Ideally you have one mutation in the PAM site.
Use custom scripts to create your list of variants within the editing region.
Determine number of stop codons in library. Add up to 10 stop codons total if there aren’t enough.
Take all variants and add the homology arms (we do this in excel).
Add asterisks after the first 2 bases and before the last 2 bases if incorporating phosphorothioate bonds (this greatly improves stability as most cells contain nucleases that will degrade nonmodified ssDNA).
Get quote from IDT. Phosphorothiate and 3bp codes are not currently supported on IDT’s website when ordering oPools.
OVERVIEW: T Cell Culture method
OVERVIEW: T Cell Culture method
Step 1 - Isolate CD4+ T cells from PBMCs
Step 2 - Activate CD4+ T cells with CD3/28 beads for 72 hours in Complete media + IL-2
Step 3 - Remove CD3/28 beads and allow cells to proliferate o/n
Step 4 - Transfect cells with Maxcyte
Step 5 - Collect day 2/3 samples for gDNA and RNA
Step 6 - Replace media with IL-2 every 2-3 days (M/W/F)
Step 7 - Take off IL-2 on day 9
Step 8 - Take 2-4e6 cells for gDNA and RNA
Step 9 - Stain cells with CFSE and stim with CD2/3/28 (no IL-2) for 72 hours
Step 10 - Sort cells on flow cytometer
Step 1: Isolate CD4+ T cells from PBMCs (starting from ~3.5 mL)
Step 1: Isolate CD4+ T cells from PBMCs (starting from ~3.5 mL)
22m 33s
22m 33s
Pipette5 mL of filtered FBS into a 50mL conical tube.
Thaw a vial of frozen cells in a 37 °C water bath until cells are completely thawed.
Use a 5ml serological pipette to take cells from vial and slowly add the thawed cells dropwise into the FBS in the 50mL conical while swirling.
Add PBS 2% FBS (or EasySep buffer) dropwise to the 50 mL conical until the volume reaches 20 mL .
Spin down cells and aspirate sup.
Optional step: If cell pellets appear red, add in 5 mL of 1x ACK buffer. Let sit for 00:02:00 Room temperature . Dilute out ACK with 20 mL PBS before centrifuge.
2m
Resuspend cells in 20 mL CD4+ T cell isolation buffer.
At this point you can take 10 µL of the cells for counting. While counting, spin down cells.
If you use hemocytometer to count total PBMC, make 1:10 dilution with Trypan blue before counting: If you use cell counter, follow manufacturer’s protocol. Record cell concentration (cells/ml) and calculate total PBMC counts.
Aspirate the supernatant and resuspend the cells to 5.0E+07cells/mL in EasySep buffer (PBS containing 2% FBS and 1 millimolar (mM) EDTA).
Transfer cells to a 14 mL Falcon round bottom polystyrene tube.
Add the EasySep Human CD4+ T Cell enrichment cocktail at 50 uL/mL cells (e.g. add 100 µL of cocktail to 2 mL of cells). Mix well and incubate at Room temperature (15 °C - 25 °C ) for 00:10:00 .
10m
Vortex the EasySep™ D Magnetic Particles for 00:00:30 . Ensure that the particles are in a uniform suspension with no visible aggregates. (Solution should be uniformly brown).
30s
Add the EasySep™ D Magnetic Particles at 100 µL/mL cells (e.g. for 2 mL of cells, add 200 µL of magnetic particles). Mix well and incubate at Room temperature (15 °C - 25 °C ) for 00:05:00 .
5m
Bring the cell suspension up to a total volume of 2.5 mL (for 5ml tube), 5 mL (for samples 2 mL ), 10 mL (for samples 2 mL ) by adding EasySep buffer. Mix the cells in the tube by gently pipetting up and down 2 - 3 times. Place the tube (without cap) into the magnet. Set aside for 00:05:00 .
5m
Pick up the EasySep™ Magnet, and in one continuous motion invert the magnet and tube, pouring off the desired fraction into a new 14 mL polystyrene tube.
- The magnetically labeled unwanted cells will remain bound inside the original tube, held by the magnetic field of the EasySep™ Magnet.
- Leave the magnet and tube in inverted position for 00:00:02 - 00:00:03 , and then return to an upright position.
3s
Take an aliquot for counting, and spin down.
Count and resuspend in cell culture media + IL-2 at 0.5e6 cells/mL.
Record the PBMC count and T cells count in the PBMC tracking file at the proper place.
Step 2: Activate CD4+ T cells with CD3/28 beads
Step 2: Activate CD4+ T cells with CD3/28 beads
3d 0h 0m 45s
3d 0h 0m 45s
Calculate volume of beads needed. We use 30 µL beads/1e6 cells.
Vortex CD3/CD28 beads for 00:00:10 -00:00:15 to fully resuspend beads. Add desired volume of beads to 1.5 mL Eppendorf.
15s
Add 500 µL media into each Eppendorf tube and place in DynaMag™-2 magnet for 00:00:30 .
30s
Remove media avoiding beads (should be visible held to the side of the tube).
Resuspend beads with media (same volume of beads suspension) and add to each well containing T cells.
Gently mix T-cells and CD3/CD28 beads with a P1000 pipette or 5ml serological pipette. Place cells in 37 °C incubator for 72:00:00 .
3d
Step 3: Remove CD3/28 beads
Step 3: Remove CD3/28 beads
8h 2m
8h 2m
Observe cells under microscope before removing beads. Cells should be clumping in the well and most of cells should be visibly larger prior to removing beads.
Use a P1000 pipette or 5ml serological pipettes to gently pipet up and down 10 times to separate cells from beads.
Collect cells in 15 mL tube, at no more than 10 mL per tube (multiple tubes if culture volume is greater than 10 mL).
Place on magnet for at least 00:01:00 .
1m
Carefully transfer cells in culture to a new tube without touching the beads.
Wash culture wells with 0.5 mL -1 mL fresh media and add to tube containing only the beads. Take the tube off the magnet to re-suspend the beads well.
Place on magnet for at least 00:01:00 to separate the beads and media, and then collect media to the tube containing cells.
1m
Discard tube containing only beads.
Repeat steps 36 – 37 at least once more to ensure all beads are removed.
Count cells and record cell concentration as well as total cell numbers. Resuspend cells at a concentration to 0.5x106 cells/mL in fresh media + IL-2.
Culture at 37 °C for ∼16 hours (Overnight ) before transfection.
Note
Cells should double or triple overnight. Transfection will most likely fail if cells do not expand.
8h
Step 4: Transfect cells with Maxcyte
Step 4: Transfect cells with Maxcyte
1h 20m
1h 20m
Note
We typically make a125 µL transfection mix containing 5e6 cells, sgRNA, Cas9, and oPool library and pipette 100 µL into a single well of a 100x2 maxcyte assembly or 50 µL each into 2 wells of a 50x8 assembly.
If using crRNA, complex with trRNA. crRNA and trRNA are resuspended at 100 micromolar (µM) and mixed 1:1. Add 15 µL of crRNA + 15 µL trRNA in sterile PCR tube, mix well. Heat to 95 °C 5 minutes and allow to cool slowly to Room temperature ,00:30:00 -01:00:00 .
1h
Add 7.5 µL of recombinant Cas9 (Cas9 stock is 40 micromolar (µM) ). You now have a guide:Cas9 ratio of 4:1. Allow mix to rest at Room temperature for 00:15:00 -00:20:00 .
20m
While Cas9 guide complex is forming, spin down cells, resuspend in PBS and count. Spin down enough cells for transfection, then wash 1x with Maxcyte buffer.
Resuspend 5e6 cells in 82.5 µL of Maxcyte buffer.
Add 5 µL of oPool HDR library (oPool library is at 40 pmol/uL) to the Cas9 guide complex, then add cells.
Add cells to Maxcyte assembly and transfect with protocol “Expanded T Cell-1”.
Transfer cells to 4 mL -5 mL media + IL-2.
Step 5: Collect day 2/3 samples for gDNA and RNA
Step 5: Collect day 2/3 samples for gDNA and RNA
If there are enough cells (>10e6), collect 2 samples, each containing 2-3e6 cells. Spin down and aspirate supernatant.
For sample 1, isolate gDNA with favorite kit. We use the Quick-DNA miniprep plus kit from Zymo, cat. No. D4069.
For sample 2, resuspend in 600 µL of Trizol reagent and store at -20 °C or -80 °C until processing.
Step 6: Replace media with IL-2 every 2-3 days (M/W/F)
Step 6: Replace media with IL-2 every 2-3 days (M/W/F)
Replace media with IL-2 every 2-3 days. If cell counts are <40e6, we plate cells at 1e6/mL. If cell counts are >40e6/mL (typically after 7 days post transfection) we plate at 2e6/mL.
Step 7: Take off IL-2 on day 9
Step 7: Take off IL-2 on day 9
1 day prior to CFSE labeling and restimulation with CD3/28, spin down cells and resuspend at 1-2e6/mL of media without IL-2.
Step 8: Collect day 10 samples for gDNA and RNA
Step 8: Collect day 10 samples for gDNA and RNA
Wash day 10 cells 1x with PBS. Resuspend in 10 mL PBS and count cells.
Like section "Collect day 2/3 samples for gDNA and RNA" above, collect 2 samples, each containing 2-3e6 cells. Spin down and aspirate supernatant.
For sample 1, isolate gDNA with favorite kit.
For sample 2, resuspend in 600 µL of Trizol reagent and store at -20 °C or -80 °C until processing.
Stain 80e6 cells with CFSE and stim with CD2/3/28 (below).
Step 9: Stain cells with CFSE and stimulate with CD2/3/28 (no IL-2) for 72 hours
Step 9: Stain cells with CFSE and stimulate with CD2/3/28 (no IL-2) for 72 hours
1h 46m
1h 46m
Resuspend each vial of CFSE (Thermo, cat. No. C34570) in 18 µL DMSO.
Resuspend 80e6 cells in 40 mL PBS. Add 20 µL of CFSE, mix well and incubate at Room temperature for 00:05:00 .
5m
Add 5 mL of FBS, mix well and incubate at Room temperature for 00:05:00 .
5m
Spin down cells, wash 1x in10 mL media without IL-2.
Resuspend cells in 40 mL media without IL-2 and add 125 µL of ImmunoCult Human CD2/3/28 (Stemcell, cat. No. 10970). Place in incubator and wait 72:00:00 -96:00:00 before sorting.
1h 36m
Step 10: Sort cells on flow cytometer
Step 10: Sort cells on flow cytometer
4d
4d
72-96:00:00 post stimulation, spin down cells and resuspend in PBS at 5-10e6 cells/mL.
4d
Pipette through strain cap into FACS tube and sort 2-4e6 cells based on the lowest and highest CFSE staining:
Isolate gDNA from both CFSE Lo and CFSE Hi fractions.