TITLE: TF and epigenetic modifier CRISPRi/a screens in human T cells
AUTHORS: Andrea R Daniel

[DESCRIPTION]
This protocol describes methods for CRISPR interference or activation screens identifying transcriptional and epigenetic regulators of human CD8+ T cell state. 

[STEPS]
SECTION: TF and epi-modifier CRISPRi/a gRNA library construction
1. The TSSs for each TF and epi-modifier were extracted using CRISPick and 1,000-bp windows were constructed around each TSS (−500 to +500 bp).
SECTION: TF and epi-modifier CRISPRi/a gRNA library construction
2. After establishing an SaCas9 gRNA database with the strict PAM variant (NNGRRT) using guideScan66, the genomic windows were input into the guidescan_guidequery function to generate the gRNA library.
SECTION: TF and epi-modifier CRISPRi/a gRNA library construction
3. Any gRNA that aligned to another genomic site with fewer than four mismatches was removed from the library. The final gRNA library contained at least seven gRNAs targeting 120/121 target gene (there were no PBX2-targeting gRNAs) with an average of 16 gRNAs per gene. 
SECTION: TF and epi-modifier CRISPRi/a gRNA library construction
4. A total of 120 NT gRNAs were included in the library for a total of 2,099 gRNAs (available in Supplementary Table 2, McCutcheon et al. Nature Genetics, 2023. https://doi.org/10.1038/s41588-023-01554-0)
SECTION: gRNA library cloning
5. Oligonucleotide pools containing variable gRNA sequences and constant regions for polymerase chain reaction (PCR) amplification were synthesized by Twist Bioscience.
SECTION: gRNA library cloning
6. 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. 
SECTION: gRNA library cloning
7. Gibson reactions were purified using ethanol precipitation and transformed into Lucigen’s Endura ElectroCompetent Cells. 
SECTION: gRNA library cloning
8. Transformed cells were cultured overnight and plasmids were isolated using Qiagen Midi Kits.
SECTION: gRNA library cloning
6.1. Addgene: pLV hU6-gRNA hUbC-dSaCas9-KRAB-T2A-Thy1.1 (Addgene 194278) and pLV hU6-gRNA hUbC-VP64-dSaCas9-VP64-T2A-Thy1.1 (Addgene 194279).
SECTION: Transfections for high-titer lentiviral production
9. Plate 1.2 x 106 or 7 x 106 HEK293T cells in a 6 well plate or 10 cm dish in the afternoon with 2 mL or 12 mL of complete opti-MEM (Opti-MEM‱ I Reduced Serum Medium supplemented with 1x  Glutamax, 5% FBS, 1 mM Sodium Pyruvate, and 1x MEM Non-Essential Amino Acids).
SECTION: Transfections for high-titer lentiviral production
10. The next morning, transfect HEK293T cells with 0.5 μg pMD2.G, 1.5 μg psPAX2, and 0.5 μg transgene for 6 well plates or 3.25 μg pMD2.G, 9.75 μg psPAX2, and 4.3 μg transgene for 10 cm dishes using Lipofectamine 3000.
SECTION: Transfections for high-titer lentiviral production
11. Exchanged media 6 hours after transfection and collect and pool lentiviral supernatant at 24 hours and 48 hours after transfection.
SECTION: Transduction of primary human T cells
12. Centrifuged lentiviral supernatant at 600g for 10 min to remove cellular debris. 
SECTION: Transduction of primary human T cells
13. Concentrate lentivirus to 50–100× the initial concentration using Lenti-X Concentrator (Takara Bio). 
SECTION: Transduction of primary human T cells
14. Transduce T cells at 5–10% v/v of concentrated lentivirus at 24 h post-activation. For dual transduction experiments, T cells were serially transduced at 24 h and 48 h.
SECTION: TF and epi-modifier CRISPRi/a gRNA screens
15. CD8+CCR7+ T cells were sorted and transduced with either CRISPRi  or CRIPRa TF + epi-modifier gRNA libraries at a low MOI.
SECTION: TF and epi-modifier CRISPRi/a gRNA screens
16. Cells were expanded for 10 days and then stained for Thy1.1 (a marker to identify transduced cells) and CCR7 (a marker associated with T cell state). 
SECTION: TF and epi-modifier CRISPRi/a gRNA screens
17. An SH800 FACS Cell Sorter (Sony Biotechnology) was used for cell sorting and analysis.
SECTION: TF and epi-modifier CRISPRi/a gRNA screens
16.1.  
    
  Antibody
  Target
  Fluorophore/Sequence
  Clone
  Isotype
  Dilution
  Application
  Manufacturer
  Catalog #
  Notes
  
  CCR7
  FITC
  150503
  Mouse IgG2a
  1:100
  Flow cytometry
  BD Biosciences
  561271
  Stain at 37C
  
  Thy1.1
  PE
  OX-7
  Mouse IgG1, κ
  1:300
  Flow cytometry
  StemCell Technologies
  60024PE
  -
  
 
SECTION: TF and epi-modifier CRISPRi/a gRNA screens
18. For antibody staining of Thy1.1 cells were collected, spun down at 300g for 5 min, resuspended in flow buffer (1× phosphate-buffered saline (PBS), 2 mM ethylenediaminetetraacetic acid and 0.5% bovine serum albumin) with the appropriate antibody dilutions and incubated for 30 min at 4 °C on a rocker. Antibody staining of CCR7 was carried out for 30 min at 37 °C. 
SECTION: TF and epi-modifier CRISPRi/a gRNA screens
19. Cells were then washed with flow buffer, spun down at 300g for 5 min and resuspended in flow buffer for cell sorting.
SECTION: TF and epi-modifier CRISPRi/a gRNA screens
20. Transduced cells in the lower and upper 10% tails of CCR7 expression were sorted for subsequent gRNA library construction and sequencing. All replicates were maintained and sorted at a minimum of 300× coverage.
SECTION: gRNA sequencing
21. Genomic DNA was isolated using Qiagen’s DNeasy Blood and Tissue Kit. Genomic DNA was split across 100 μl PCR reactions (25 cycles at 98 °C for 10 s, 60 °C for 30 s, and 72 °C for 20 s) with Q5 2× Master Mix and up to 1 μg of genomic DNA per reaction.

SECTION: gRNA sequencing
22. PCRs were pooled together for each sample and purified using double-sided (SPRI)bead selection at 0.6× and 1.8×. 
SECTION: gRNA sequencing
23. Libraries were run on a High Sensitivity D1000 tape (Agilent) to confirm amplicon size and quantified using Qubit’s dsDNA High Sensitivity assay.

SECTION: gRNA sequencing
24. Libraries were diluted to 2 nM, pooled together at equal volumes, and sequenced using Illumina’s MiSeq Reagent Kit v2 (50 cycles).
SECTION: gRNA sequencing
25. Primers are available in Supplementary Table 5 of McCutcheon et al. Nature Genetics, 2023. https://doi.org/10.1038/s41588-023-01554-0
SECTION: Processing gRNA sequencing and gRNA analysis
26. FASTQ files were aligned to custom indexes for each gRNA library (generated from the bowtie2-build function) using Bowtie 2 (ref. 67). 
SECTION: Processing gRNA sequencing and gRNA analysis
27. Counts for each gRNA were extracted and used for further analysis in R.
SECTION: Processing gRNA sequencing and gRNA analysis
28. Individual gRNA enrichment was determined using the DESeq2 (ref. 68) package to compare gRNA abundance between groups for each screen.
SECTION: Gene-level analysis for CRISPRi/a screens
29. DESeq2 P values were empirically transformed to cumulative probabilities using a midpoint linear interpolation of the 120 NT gRNA P values between 0 and 1. This transformation aligns the data with the null hypothesis that NT gRNA P values have a uniform distribution between 0 and 1. 
SECTION: Gene-level analysis for CRISPRi/a screens
30. Within each gene, transformed P values were aggregated using a modified robust rank aggregation method to detect genes with nonuniform (non-null) gRNA P values. 
SECTION: Gene-level analysis for CRISPRi/a screens
31. A gene-level P value was produced by comparison with 10 million gene-level null simulations of Pvalues randomly sampled from a uniform distribution. 
SECTION: Gene-level analysis for CRISPRi/a screens
32. NT gRNAs were randomly grouped into NT control ‘genes’ (NTCs) and analyzed in the same way.
SECTION: Gene-level analysis for CRISPRi/a screens
33. The number of gRNAs per NTC was sampled with replacement from the distribution of gRNAs per gene in the screen until all the NT gRNAs were used. 
SECTION: Gene-level analysis for CRISPRi/a screens
34. Genes were selected as hits if their Benjamini–Hochberg false discovery rate (FDR) was less than 0.05. Gene-level aggregation was done in Python. 
SECTION: Gene-level analysis for CRISPRi/a screens
35. Two effect sizes were computed for each gene by averaging gRNAs’ unshrunk DESeq2 log2FoldChange within the gene, weighted by each gRNA’s transformed one-sided P value. 
SECTION: Gene-level analysis for CRISPRi/a screens
36. The larger (absolute value) effect size was chosen for each gene. Effect sizes were estimated in R.