Dec 09, 2023
TFome CRISPRko screens
- Sean R. McCutcheon1,
- Adam M. Swartz1,
- Michael C. Brown1,
- Alejandro Barrera1,
- Christian McRoberts Amador1,
- Keith Siklenka1,
- Lucas Humayun1,
- Maria A. ter Weele1,
- James M. Isaacs1,
- Andrea R Daniel1,
- Timothy E. Reddy1,
- Andrew S. Allen1,
- Smita K. Nair1,
- Scott J. Antonia1,
- Charles A. Gersbach1
- 1Duke University
- Andrea R Daniel: This protocol was adapted from work of Sean McCutcheon and colleagues in the Gersbach lab at Duke University.
- Gersbach Lab
Protocol Citation: Sean R. McCutcheon, Adam M. Swartz, Michael C. Brown, Alejandro Barrera, Christian McRoberts Amador, Keith Siklenka, Lucas Humayun, Maria A. ter Weele, James M. Isaacs, Andrea R Daniel, Timothy E. Reddy, Andrew S. Allen, Smita K. Nair, Scott J. Antonia, Charles A. Gersbach 2023. TFome CRISPRko screens. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbxk8olpk/v1
Manuscript citation:
McCutcheon, S.R., Swartz, A.M., Brown, M.C. et al. Transcriptional and epigenetic regulators of human CD8+ T cell function identified through orthogonal CRISPR screens. Nat Genet (2023). https://doi.org/10.1038/s41588-023-01554-0
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: December 08, 2023
Last Modified: December 09, 2023
Protocol Integer ID: 92033
Keywords: CRISPR, screen, knockout, BATF3, T cells
Funders Acknowledgements:
NIH
Grant ID: HG012053
Abstract
This protocols describes methods for transcription factor CRISPR knockout screens to reveal cofactors of BATF3 in T cells.
gRNA library construction
gRNA library construction
A total of 550 NT gRNAs were included in the library for a total of 7,000 gRNAs (available in Supplementary Table 6 of McCutcheon et al. Nature Genetics, 2023. https://doi.org/10.1038/s41588-023-01554-0).
This gRNA library was cloned into SpCas9 gRNA lentiviral plasmids with either mCherry or BATF3.
gRNA library cloning
gRNA library cloning
Oligonucleotide pools containing variable gRNA sequences and constant regions for polymerase chain reaction (PCR) amplification were synthesized by Twist Bioscience.
gRNA amplicons were gel extracted, PCR purified and input into 20 μl Gibson reactions (5:1 molar ratio of insert to backbone) with 200 ng of Esp3I digested and 1 × solid-phase reversible immobilization (SPRI)-selected (Beckman Coulter) plasmid backbone.
Gibson reactions were purified using ethanol precipitation and transformed into Lucigen’s Endura ElectroCompetent Cells.
Transformed cells were cultured overnight and plasmids were isolated using Qiagen Midi Kits.
TFome CRISPRko screens
TFome CRISPRko screens
A total of 20 × 106 CD8+ T cells from two donors were activated with CD3/CD28 dynabeads at a 1:1 ratio.
At 24 h post-activation, CD8+ T cells were split evenly and transduced in parallel with TFome CRISPRko gRNA libraries with mCherry or BATF3.
At 48 h post-activation, cells were electroporated with Cas9 protein. Briefly, the cells were collected, spun down at 90g for 10 min, resuspended in 100 μl of Lonza P3 Primary Cell buffer with 3.2 μg Cas9 (Thermo) per 106 cells, and electroporated with the pulse code EH115.
After electroporation, warm medium was immediately added to each cuvette and cells were recovered at 37 °C for 20 min before being transferred into a six-well plate.
On day 3 post transduction, cells were selected with 2 μg ml−1 of puromycin for 3 days. On day 9 post transduction, cells were stained for CD8, IL7R and a viability dye.
| Antibody Target | Fluorophore/Sequence | Clone | Isotype | Dilution | Application | Manufacturer | Catalog # | |
| IL7RA | PE-Cy5 | eBioRDR5 | Mouse / IgG1, kappa | 1:100 | Flow cytometry | Thermo | 15-1278-42 | |
| CD8 | bv-421 | HIT8a | Mouse IgG1, κ | 1:50 | Flow cytometry | BD Biosciences | 740078 |
An SH800 FACS Cell Sorter (Sony Biotechnology) was used for cell sorting and analysis.
Cells were then washed with flow buffer, spun down at 300g for 5 min and resuspended in flow buffer for cell sorting.
Viable CD8+ T cells in the lower and upper 10% tails of IL7R expression were sorted for subsequent gRNA library construction and sequencing.
All replicates were maintained and sorted at a minimum of 75× coverage.
Protocol references
45. Lambert, S. A. et al. The human transcription factors. Cell 172, 650–665 (2018).
83. Doench, J. G. et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat. Biotechnol. 34, 184–191 (2016).