TITLE: CRISPRi tiling screens
AUTHORS: Andrea R Daniel

[DESCRIPTION]
This protocols describes methods for characterizing the activity of dSaCas9 as a repressor using promoter tiling guide RNA screens in primary human T cells. 

[STEPS]
SECTION: Primary human CD8+ T cell cultures
1. Isolated CD8+ T cells from individual donors were obtianed directly from vials purchased from StemCell Technologies. 
SECTION: Primary human CD8+ T cell cultures
2. Culture T cells were in PRIME-XV T cell Expansion XSFM (FujiFilm) supplemented with 5% human platelet lysate (Compass Biomed), 100 U ml−1 penicillin and 100 μg ml−1 streptomycin. All media were supplemented with 100 U ml−1 human IL-2 (Peprotech). 
SECTION: Designing CD2 and BM2 CRISPRi gRNA libraries
3. Saturation CD2 and B2M CRISPRi gRNA libraries were designed to tile a 1,050-bp window (−400 bp to 650 bp) around the transcription start site (TSS) of each target gene using CRISPick61. 
SECTION: Designing CD2 and BM2 CRISPRi gRNA libraries
4. Each gRNA library was designed to target dSaCas9’s relaxed protospacer adjacent motif (PAM) variant: 5′-NNGRRN-3′. NT gRNAs were generated for each library to match the nucleotide composition of the targeting gRNAs. 
SECTION: Designing CD2 and BM2 CRISPRi gRNA libraries
5. CD2 and B2M gRNA libraries are available in Supplementary Table 1 of McCutcheon et al. Nature Genetics, 2023. https://doi.org/10.1038/s41588-023-01554-0
SECTION: gRNA library cloning
6. Oligonucleotide pools containing variable gRNA sequences and constant regions for polymerase chain reaction (PCR) amplification were synthesized by Twist Bioscience. 
SECTION: gRNA library cloning
7. 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, pLV hU6-gRNA hUbC-dSaCas9-KRAB-T2A-Thy1.1 (Addgene 194278).
SECTION: gRNA library cloning
8. Gibson reactions were purified using ethanol precipitation and transformed into Lucigen’s Endura ElectroCompetent Cells. 
SECTION: gRNA library cloning
9. Transformed cells were cultured overnight and plasmids were isolated using Qiagen Midi Kits.
SECTION: Lentiviral transduction of T cells
10. CD8+ T cells from pooled peripheral blood mononuclear cell donors (see step 1) were transduced with all-in-one lentivirus encoding for dSaCas9–KRAB–2A–GFP and either CD2 or B2M gRNA libraries.
SECTION: Lentiviral transduction of T cells
12. Lentiviral supernatant was centrifuged at 600g for 10 min to remove cellular debris and concentrated to 50–100× the initial concentration using Lenti-X Concentrator (Takara Bio). 
SECTION: Lentiviral transduction of T cells
13. T cells were transduced at 5–10% v/v of concentrated lentivirus. 
SECTION: CRISPRi tiling screens
14. Following transduction, cells were expanded for 9 days and then stained for the target gene.
SECTION: CRISPRi tiling screens
17. An SH800 FACS Cell Sorter (Sony Biotechnology) was used for cell sorting and analysis. Fluorescent minus one (FMO) controls were used to set appropriate gates for all flow panels.
SECTION: CRISPRi tiling screens
15. For antibody staining of surface markers, 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. 
SECTION: CRISPRi tiling screens
15.1.  
                
  Antibody
  Target
  Fluorophore/Sequence
  Clone
  Isotype
  Dilution
  Application
  Manufacturer
  Catalog #
  
  CD2
  PE
  RPA-2.10
  Mouse / IgG1, kappa
  1:50
  Flow cytometry
  Thermo
  12-0029-42
  
  B2M
  PE
  A17082A
  Mouse IgG1, κ
  1:50
  Flow cytometry
  Biolegend
  395704
  
 
SECTION: CRISPRi tiling screens
16. Cells were then washed with flow buffer, spun down at 300g for 5 min and resuspended in flow buffer for cell sorting or analysis. 
SECTION: CRISPRi tiling screens
18. Transduced cells in the lower and upper 10% tails of target gene expression were sorted for subsequent gRNA library construction and sequencing. All replicates were maintained and sorted at a minimum of 350× coverage.
SECTION: gRNA sequencing
19. 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
20. PCRs were pooled together for each sample and purified using double-sided (SPRI)bead selection at 0.6× and 1.8×. 
SECTION: gRNA sequencing
21. 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
22. 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
23. 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
24. 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
25. Counts for each gRNA were extracted and used for further analysis in R.
SECTION: Processing gRNA sequencing and gRNA analysis
26. Individual gRNA enrichment was determined using the DESeq2 (ref. 68) package to compare gRNA abundance between groups for each screen.
SECTION: Lentiviral transduction of T cells
11. Lentivirus was produced as previously described60.