KEY RESOURCES TABLE
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
|
| ||
| anti-human TCR α/β Brilliant Violet 421 | BioLegend | Cat#306722; RRID: AB_2562805 |
| anti-human CD5 (UCHT2)-PE | Invitrogen | Cat#12-0059-42 |
| anti-human CD81 (5A6)-FITC | BioLegend | Cat#349504; RRID: AB_10642825 |
| anti-human CD3E (SK7)-APC | Invitrogen | Cat#17-0036-42 |
| anti-TCRα/β (BW242/412)-PE | Miltenyi | Cat#130-113-531; RRID: AB_2733903 |
| goat anti-mouse IgG (H+L) AlexaFluor 647 Fab | Jackson ImmunoResearch | Cat#115-607-003: RRID: AB_2338931 |
| Anti-HA (6E2)-AF647 | Cell Signaling Technology | Cat#3444S |
|
| ||
| Chemicals, peptides, and recombinant proteins | ||
|
| ||
| Dulbecco’s Modified Eagle Medium | Gibco | Cat#11965092 |
| Fetal Bovine Serum | VWR | Cat#1500-500GH |
| Penicillin/streptomycin | Gibco | Cat#15140122 |
| X-VIVO 15 Serum-free Hematopoietic Cell Medium | Lonza | Cat#04-418Q |
| 2-Mercaptoethanol | Gibco | 21985-023 |
| N-Acetyl-L-cysteine | Sigma-Aldrich | Cat#A7250 |
| DNase I Solution | STEMCELL Technologies | Cat#7900 |
| EasySep Buffer | STEMCELL Technologies | Cat#20144 |
| Dynabeads Human T-Activator CD3/CD28 for T cell Expansion and Activation | Gibco | Cat#11131D |
| Recombinant Human IL-2 | PeproTech | Cat#200-02 |
| Recombinant Human IL-7 | PeproTech | Cat#200-07 |
| Recombinant Human IL-7 | Miltenyi Biotec | Cat#130-095-367 |
| Recombinant Human IL-15 | R&D Systems | Cat#247-ILB-005/CF |
| Recombinant Human IL-15 | Miltenyi Biotec | Cat#130-095-760 |
| Alt-R S. pyogenes Cas9 Nuclease V3 | IDT | Cat#1081059 |
| Cell staining buffer | BioLegend | Cat#420201 |
| Ghost Dye Violet 780 | Tonbo Bioscience | Fisher Scientific Cat#50-105-2988 |
| QuickExtract DNA Extraction Solution | Lucigen | Cat#QE09050 |
| Q5 High-Fidelity DNA Polymerase | NEB | Cat#M0491L |
| SPRIselect | Beckman Coulter | Cat#B23318 |
| KAPA HiFi Hotstart ReadyMix | Roche | Cat#KK2601 |
| ddPCR Supermix for Probes (No dUTP) | Bio-Rad | Cat#1863023 |
| Polyethylenimine | Polysciences Inc. | Cat#24314 |
| Opti-MEM Reduced Serum Medium | Gibco | Cat#31985070 |
| Lenti-X Concentrator | Takara | Cat#631231 |
| CellTrace Violet Cell Proliferation Kit | Invitrogen | Cat#C34571 |
| EvaGreen Supermix | Bio-Rad | Cat#186-4033 |
| 7-Aminoactinomycin D | Invitrogen | Cat#A1310 |
| TRIzol Reagent | Invitrogen | Cat#15596026 |
| TURBO DNase | Invitrogen | Cat#AM2238 |
| SUPERase-In RNase inhibitor | Invitrogen | Cat#AM2694 |
| SuperScript III Reverse Transcriptase | Invitrogen | Cat#18080044 |
| Random primers | Promega | Cat#C1181 |
| iTaq Universal SYBR Green Supermix | Bio-Rad | Cat#1725124 |
|
| ||
| Critical commercial assays | ||
|
| ||
| EasySep Human T Cell Isolation Kit | STEMCELL Technologies | Cat#17951 |
| P3 Primary Cell 96-well Nucleofector Kit | Lonza | Cat#V4SP-3096 |
| iSeq 100 i1 Reagent v2 | Illumina | Cat#20031371 |
| NovaSeq 600 S1 v1.5 | Illumina | Cat#20028317 |
| NovaSeq 600 S4 v1.5 | Illumina | Cat#20028312 |
| Chromium Next GEM Single Cell 3’ GEM Kit v3.1 | 10x Genomics | Cat#1000123 |
| Chromium Next GEM Single Cell 3’ Library Kit v3.1 | 10x Genomics | Cat#1000157 |
| Chromium Next GEM Single Cell 3’ Gel Bead Kit v3.1 | 10x Genomics | Cat#1000122 |
| TotalSeq-A0251-1 anti-human Hashtag Reagents | BioLegend | Cat#394601 |
|
| ||
| Deposited data | ||
|
| ||
| Raw and processed single cell RNA sequencing data | This paper | GEO: GSE241838, GSE241839, and GSE241882 |
| Clinical single cell RNA sequencing data | Stadtmauer et al.6 | dbGaP: PHS001707.v2 |
|
| ||
| Experimental models: Cell lines | ||
|
| ||
| Human embryonic kidney 293T cells | UC Berkeley Cell Culture Facility | N/A |
| Primary adult peripheral blood mononuclear cells | Allcells Inc. | Fisher Scientific Cat#50-156-841 or 50-156-574. |
| Primary adult peripheral blood mononuclear cells | StemCell Technologies | Cat#70500 |
|
| ||
| Oligonucleotides | ||
|
| ||
| Cas9 gRNAs | IDT/Synthego | Tables S1, S7, S8, and S11 |
| MULTI-seq barcodes | IDT | Table S3 and S6 |
| CROP-seq library | IDT | Table S5 |
| ddPCR primers/probes | IDT | Tables S4, S7, and S8 |
| RT-qPCR primers | IDT | Table S12 |
| NGS primers | IDT | Tables S2 and S7 |
|
| ||
| Recombinant DNA | ||
|
| ||
| CROP-seq-opti-EGFP (modified from CROP-seq-opti) | This paper, modified from Addgene | Addgene#106280 |
| psPax2 (Gag-pol) | Addgene | Addgene#12260 |
| pCMV-VSV-G | Addgene | Addgene #8454 |
| pAAV-TRAC-1928z | Eyquem et al.22 | N/A |
| pHelper | Eyquem et al.22 | N/A |
| pAAV Rep-Cap | Eyquem et al.22 | N/A |
|
| ||
| Software and algorithms | ||
|
| ||
| FlowJo v10.5.3 | FlowJo | https://www.flowjo.com/solutions/flowjo/downloads |
| CRISPResso2 | Clement et al.60 | https://github.com/pinellolab/CRISPResso2 |
| 10x Cell Ranger | 10x Genomics | https://support.10xgenomics.com/single-cell-gene-expression/software/overview/welcome |
| Scanpy v1.9.3 | Virshup et al. 2023 | https://github.com/scverse/scanpy |
| inferCNV | inferCNV of the Trinity CTAT Project | https://github.com/broadinstitute/infercnv |
| MAGeCK | Li et al.61 | https://sourceforge.net/p/mageck/wiki |
| inDelphi | Shen et al.62 | https://indelphi.giffordlab.mit.edu/about |
| KEGG | Kanehisa and Goto63 | https://www.genome.jp/kegg/ |
| GSEApy v1.0.3 | Fang et al.64 | https://github.com/zqfang/GSEApy |
| Prism v9.4.1 | GraphPad | https://www.graphpad.com/ |
| Memento | Kim et al.65 | https://doi.org/10.1101/2022.11.09.515836 |
| R v4.1 | The Comprehensive R Archive Network | https://www.R-project.org/ |
| Python v3.9.12 | Python Software Foundation | https://www.python.org/ |
| ENCODE Portal: Encyclopedia of DNA Elements | Sloan et al.66 | www.enccodeproject.org |
| Scripts for chromosome loss estimation (gene-expression matrix generation, sgRNA assignments, demultiplexing, inferCNV analysis, breakpoint estimation) | This paper | www.github.com/yelabucsf/Cas9_chromosome_loss_scRNA-seq |