TITLE: Immune Saturation Genome Editing (Immune SGE): Variant Analysis in T cells
AUTHORS: Nathan Camp, Sam Dawson

[DESCRIPTION]
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]

 

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.

 

[STEPS]
SECTION: OVERVIEW: ssODN repair template and oPool design notes
1. Determine your editing region of interest, trying to stay within 20bp on either side of the cut site.
SECTION: OVERVIEW: ssODN repair template and oPool design notes
2. Add respective homology arms to both the 5’ and 3’ ends of the editing region such that ssODN is at least 140 bp long.
SECTION: OVERVIEW: ssODN repair template and oPool design notes
3. 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.
SECTION: OVERVIEW: ssODN repair template and oPool design notes
4. Use custom scripts to create your list of variants within the editing region.
SECTION: OVERVIEW: ssODN repair template and oPool design notes
5. Determine number of stop codons in library. Add up to 10 stop codons total if there aren’t enough.
SECTION: OVERVIEW: ssODN repair template and oPool design notes
6. Take all variants and add the homology arms (we do this in excel).
SECTION: OVERVIEW: ssODN repair template and oPool design notes
7. 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).
SECTION: OVERVIEW: ssODN repair template and oPool design notes
8. Get quote from IDT. Phosphorothiate and 3bp codes are not currently supported on IDT’s website when ordering oPools.
SECTION: Step 1: Isolate CD4+ T cells from PBMCs (starting from ~3.5 mL)
19. Pipette5 mL of filtered FBS into a 50mL conical tube.
SECTION: Step 1: Isolate CD4+ T cells from PBMCs (starting from ~3.5 mL)
20. Thaw a vial of frozen cells in a 37 °C water bath until cells are completely thawed. 
SECTION: Step 1: Isolate CD4+ T cells from PBMCs (starting from ~3.5 mL)
21. 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.
SECTION: Step 1: Isolate CD4+ T cells from PBMCs (starting from ~3.5 mL)
22. Add PBS 2% FBS (or EasySep buffer) dropwise to the 50 mL conical until the volume reaches 20 mL. 
SECTION: Step 1: Isolate CD4+ T cells from PBMCs (starting from ~3.5 mL)
23. Spin down cells and aspirate sup.
SECTION: Step 1: Isolate CD4+ T cells from PBMCs (starting from ~3.5 mL)
24. Optional step: If cell pellets appear red, add in 5 mL of 1x ACK buffer. Let sit for 2 min. Dilute out ACK with 20 mL PBS before centrifuge.

SECTION: Step 1: Isolate CD4+ T cells from PBMCs (starting from ~3.5 mL)
25. Resuspend cells in 20 mL CD4+ T cell isolation buffer.
SECTION: Step 1: Isolate CD4+ T cells from PBMCs (starting from ~3.5 mL)
26. At this point you can take 10 µL of the cells for counting. While counting, spin down cells.
SECTION: Step 1: Isolate CD4+ T cells from PBMCs (starting from ~3.5 mL)
27. 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.
SECTION: Step 1: Isolate CD4+ T cells from PBMCs (starting from ~3.5 mL)
28. Aspirate the supernatant and resuspend the cells to 5.0E+07cells/mL in EasySep buffer (PBS containing 2% FBS and 1 millimolar (mM) EDTA).
SECTION: Step 1: Isolate CD4+ T cells from PBMCs (starting from ~3.5 mL)
29. Transfer cells to a 14 mL Falcon round bottom polystyrene tube.
SECTION: Step 1: Isolate CD4+ T cells from PBMCs (starting from ~3.5 mL)
30. Add the EasySep Human CD4+ T Cell enrichment cocktail at 50 uL/mL cells (e.g. add 100 µLof cocktail to 2 mL of cells). Mix well and incubate at Room temperature (15 °C - 25 °C) for 10 min.
SECTION: Step 1: Isolate CD4+ T cells from PBMCs (starting from ~3.5 mL)
31. Vortex the EasySep™ D Magnetic Particles for 30 s. Ensure that the particles are in a uniform suspension with no visible aggregates. (Solution should be uniformly brown).
SECTION: Step 1: Isolate CD4+ T cells from PBMCs (starting from ~3.5 mL)
32. 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 5 min.
SECTION: Step 1: Isolate CD4+ T cells from PBMCs (starting from ~3.5 mL)
33. 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 5 min.
SECTION: Step 1: Isolate CD4+ T cells from PBMCs (starting from ~3.5 mL)
34. 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 2 s - 3 s, and then return to an upright position. 
SECTION: Step 1: Isolate CD4+ T cells from PBMCs (starting from ~3.5 mL)
35. Take an aliquot for counting, and spin down.
SECTION: Step 1: Isolate CD4+ T cells from PBMCs (starting from ~3.5 mL)
36. Count and resuspend in cell culture media + IL-2 at 0.5e6 cells/mL. 
SECTION: Step 1: Isolate CD4+ T cells from PBMCs (starting from ~3.5 mL)
37. Record the PBMC count and T cells count in the PBMC tracking file at the proper place.
SECTION: Step 2: Activate CD4+ T cells with CD3/28 beads
38. Calculate volume of beads needed. We use 30 µL beads/1e6 cells.
SECTION: Step 2: Activate CD4+ T cells with CD3/28 beads
39. Vortex CD3/CD28 beads for 10 s-15 s to fully resuspend beads. Add desired volume of beads to 1.5 mL Eppendorf.
SECTION: Step 2: Activate CD4+ T cells with CD3/28 beads
40. Add 500 µL media into each Eppendorf tube and place in DynaMag™-2 magnet for 30 s.
SECTION: Step 2: Activate CD4+ T cells with CD3/28 beads
41. Remove media avoiding beads (should be visible held to the side of the tube).
SECTION: Step 2: Activate CD4+ T cells with CD3/28 beads
42. Resuspend beads with media (same volume of beads suspension) and add to each well containing T cells.  
SECTION: Step 2: Activate CD4+ T cells with CD3/28 beads
43. Gently mix T-cells and CD3/CD28 beads with a P1000 pipette or 5ml serological pipette.  Place cells in 37 °C incubator for 4320 min. 

SECTION: Step 3: Remove CD3/28 beads
44. 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. 
SECTION: Step 3: Remove CD3/28 beads
45. Use a P1000 pipette or 5ml serological pipettes to gently pipet up and down 10 times to separate cells from beads.
SECTION: Step 3: Remove CD3/28 beads
46. Collect cells in 15 mL tube, at no more than 10 mL per tube (multiple tubes if culture volume is greater than 10 mL).  
SECTION: Step 3: Remove CD3/28 beads
47. Place on magnet for at least 1 min. 
SECTION: Step 3: Remove CD3/28 beads
48. Carefully transfer cells in culture to a new tube without touching the beads. 
SECTION: Step 3: Remove CD3/28 beads
49. Wash culture wells with 0.5 mL-1 mLfresh media and add to tube containing only the beads. Take the tube off the magnet to re-suspend the beads well. 

SECTION: Step 3: Remove CD3/28 beads
50. Place on magnet for at least 1 min to separate the beads and media, and then collect media to the tube containing cells. 

SECTION: Step 3: Remove CD3/28 beads
51. Discard tube containing only beads.
SECTION: Step 3: Remove CD3/28 beads
52. Repeat steps 36 – 37 at least once more to ensure all beads are removed.
SECTION: Step 3: Remove CD3/28 beads
53. 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.
SECTION: Step 3: Remove CD3/28 beads
54. Culture at 37 °C for ∼16 hours (960 min) before transfection.
 

SECTION: Step 4: Transfect cells with Maxcyte
55. 
 

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 and allow to cool slowly to Room temperature,30 min-60 min.
SECTION: Step 4: Transfect cells with Maxcyte
56. Add 7.5 µLof recombinant Cas9 (Cas9 stock is 40 micromolar (µM)). You now have a guide:Cas9 ratio of 4:1.  Allow mix to rest at Room temperaturefor 15 min-20 min.
SECTION: Step 4: Transfect cells with Maxcyte
57. 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.
SECTION: Step 4: Transfect cells with Maxcyte
58. Resuspend 5e6 cells in 82.5 µL of Maxcyte buffer.
SECTION: Step 4: Transfect cells with Maxcyte
59. Add 5 µL of oPool HDR library (oPool library is at 40 pmol/uL) to the Cas9 guide complex, then add cells.
SECTION: Step 4: Transfect cells with Maxcyte
60. Add cells to Maxcyte assembly and transfect with protocol “Expanded T Cell-1”.
SECTION: Step 4: Transfect cells with Maxcyte
61. Transfer cells to 4 mL-5 mL media + IL-2.
SECTION: Step 5: Collect day 2/3 samples for gDNA and RNA
62. If there are enough cells (>10e6), collect 2 samples, each containing 2-3e6 cells. Spin down and aspirate supernatant. 
SECTION: Step 5: Collect day 2/3 samples for gDNA and RNA
63. For sample 1, isolate gDNA with favorite kit. We use the Quick-DNA miniprep plus kit from Zymo, cat. No. D4069.
SECTION: Step 5: Collect day 2/3 samples for gDNA and RNA
64. For sample 2, resuspend in 600 µL of Trizol reagent and store at -20 °C or -80 °C until processing.

SECTION: Step 6: Replace media with IL-2 every 2-3 days (M/W/F)
65. 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.
SECTION: Step 7: Take off IL-2 on day 9
66. 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.
SECTION: Step 8: Collect day 10 samples for gDNA and RNA
67. Wash day 10 cells 1x with PBS. Resuspend in 10 mL PBS and count cells. 
SECTION: Step 8: Collect day 10 samples for gDNA and RNA
68. 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. 
SECTION: Step 8: Collect day 10 samples for gDNA and RNA
69. For sample 1, isolate gDNA with favorite kit. 
SECTION: Step 8: Collect day 10 samples for gDNA and RNA
70. For sample 2, resuspend in 600 µL of Trizol reagent and store at -20 °Cor -80 °Cuntil processing.
SECTION: Step 8: Collect day 10 samples for gDNA and RNA
71. Stain 80e6 cells with CFSE and stim with CD2/3/28 (below).
SECTION: Step 9: Stain cells with CFSE and stimulate with CD2/3/28 (no IL-2) for 72 hours
72. Resuspend each vial of CFSE (Thermo, cat. No. C34570) in 18 µL DMSO.
SECTION: Step 9: Stain cells with CFSE and stimulate with CD2/3/28 (no IL-2) for 72 hours
73. Resuspend 80e6 cells in 40 mL PBS. Add 20 µL of CFSE, mix well and incubate at Room temperature for 5 min.
SECTION: Step 9: Stain cells with CFSE and stimulate with CD2/3/28 (no IL-2) for 72 hours
74. Add 5 mL of FBS, mix well and incubate at Room temperature for 5 min.
SECTION: Step 9: Stain cells with CFSE and stimulate with CD2/3/28 (no IL-2) for 72 hours
75. Spin down cells, wash 1x in10 mL media without IL-2.
SECTION: Step 9: Stain cells with CFSE and stimulate with CD2/3/28 (no IL-2) for 72 hours
76. 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 4320 min-5760 min before sorting.

SECTION: Step 10: Sort cells on flow cytometer
77. 72-5760 min post stimulation, spin down cells and resuspend in PBS at 5-10e6 cells/mL.

SECTION: Step 10: Sort cells on flow cytometer
78. Pipette through strain cap into FACS tube and sort 2-4e6 cells based on the lowest and highest CFSE staining:

 

SECTION: Step 10: Sort cells on flow cytometer
79. Isolate gDNA from both CFSE Lo and CFSE Hi fractions.
SECTION: OVERVIEW: T Cell Culture method
9. Step 1 - Isolate CD4+ T cells from PBMCs
SECTION: OVERVIEW: T Cell Culture method
10. Step 2 - Activate CD4+ T cells with CD3/28 beads for 72 hours in Complete media + IL-2
SECTION: OVERVIEW: T Cell Culture method
11. Step 3 - Remove CD3/28 beads and allow cells to proliferate o/n
SECTION: OVERVIEW: T Cell Culture method
12. Step 4 - Transfect cells with Maxcyte
SECTION: OVERVIEW: T Cell Culture method
13. Step 5 - Collect day 2/3 samples for gDNA and RNA
SECTION: OVERVIEW: T Cell Culture method
14. Step 6 - Replace media with IL-2 every 2-3 days (M/W/F)
SECTION: OVERVIEW: T Cell Culture method
15. Step 7 - Take off IL-2 on day 9
SECTION: OVERVIEW: T Cell Culture method
16. Step 8 - Take 2-4e6 cells for gDNA and RNA
SECTION: OVERVIEW: T Cell Culture method
17. Step 9 - Stain cells with CFSE and stim with CD2/3/28 (no IL-2) for 72 hours
SECTION: OVERVIEW: T Cell Culture method
18. Step 10 - Sort cells on flow cytometer