KEY RESOURCES TABLE
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| Mouse monoclonal anti-Vinculin (H-10) | Santa Cruz Biotechnology | Cat# sc-25336; RRID:AB_628438 |
| Rabbit monoclonal anti-Actinin (D6F6) | Cell Signaling Technology | Cat# 6487; RRID: AB_11179206 |
| Mouse monoclonal anti-GAPDH (D16H11) | Cell Signaling Technology | Cat# 5174; RRID: AB_10622025 |
| Rabbit monoclonal anti-Ku70 (D10A7) | Cell Signaling Technology | Cat# 4588; RRID: AB_11179211 |
| Rabbit polyclonal anti-CCAR1 | Bethyl | Cat# A300-270A; RRID:AB_155903 |
| Rabbit monoclonal anti-FANCA (D1L2Z) | Cell Signaling Technology | Cat# 14657; RRID:AB_2798558 |
| Rabbit polyclonal anti-FANCD2 | Novus | Cat# NB100-182; RRID AB_10002867 |
| Mouse monoclonal anti-FANCD2 (FI17) | Santa Cruz Biotechnology | Cat# sc-20022; RRID:AB_2278211 |
| Rabbit monoclonal anti-Histone H2A.X, phospho (Ser139) (20E3) | Cell Signaling Technology | Cat# 9718; RRID: AB_2118009 |
| Rabbit polyclonal anti-FANCE | Bethyl | Cat# 302-125A; RRID:AB_1720357 |
| Sheep polyclonal anti-FANCG | This study | N/A |
| Goat polyclonal anti-FANCL | GeneTex | Cat# GTX88033; RRID:AB_10720542 |
| Mouse monoclonal anti-FLAG (M2) | Sigma-Aldrich | Cat# F3165; RRID: AB_259529 |
| Rabbit polyclonal anti-U2AF35/U2AF1 | Abcam | Cat# ab86305; RRID:AB_1925546 |
| Rabbit polyclonal anti-U2AF2 | Cell Signaling Technology | Cat# 70471; RRID: N/A |
| Goat anti-rabbit IgG, HRP-linked | Cell Signaling Technology | Cat# 7074; RRID: AB_2099233 |
| Horse anti-mouse IgG, HRP-linked | Cell Signaling Technology | Cat# 7076; RRID: AB_330924 |
| Donkey anti-goat IgG (H+L), Secondary antibody, HRP | Thermo Fisher Scientific | Cat# A15999; RRID:AB_2534673 |
| Rabbit anti-sheep IgG-H&L HRP Conjugated | Abcam | Cat# ab6747; RRID: AB_955453 |
| Goat anti-mouse IgG (H+L) Highly adsorbed, Secondary antibody, Alexa Flour 488 | Thermo Fisher Scientific | Cat# A11029; RRID:AB_2534088 |
| Goat anti-rabbit IgG (H+L) Highly adsorbed, Secondary antibody, Alexa Flour 568 | Thermo Fisher Scientific | Cat# A11036; RRID:AB_10563566 |
| Chemicals, peptides, and recombinant proteins | ||
| Olaparib | Selleckchem | Cat# S1060 |
| Mitomycin C | Sigma-Aldrich | Cat# M0503 |
| Topotecan HCl | Selleckchem | Cat# S1231 |
| Cycloheximide | Sigma-Aldrich | Cat# 01810 |
| MG-132 | Selleckchem | Cat# S2619 |
| Lipofectamine CRISPRMAX Cas9 Transfection Reagent | Thermo Fisher Scientific | Cat# CMAX00003 |
| Lipofectamine LTX Reagent with PLUS Reagent | Thermo Fisher Scientific | Cat# 15338030 |
| TrueCut Cas9 Protein v2 | Thermo Fisher Scientific | Cat# A36499 |
| Alt-R™ S.p. HiFi Cas9 Nuclease V3 | Integrated DNA Technologies | Cat# 1081060 |
| Alt-R™ Cas9 Electroporation Enhancer | Integrated DNA Technologies | Cat# 1075915 |
| SF Cell Line 4D-Nucleofector™ X Kit | Lonza | Cat# V4XC-2012 |
| CalPhos Mammalian Transfection Kit | Takara Bio | Cat# 631312 |
| Polybrene | Sigma-Aldrich | Cat# TR-1003-G |
| Puromycin dihydrochloride from Streptomyces alboniger | Sigma-Aldrich | Cat# P7255 |
| Blasticidin S HCl | Thermo Fisher Scientific | Cat# A1113903 |
| GENETICIN (G418 Sulfate) | Thermo Fisher Scientific | Cat# 10131027 |
| Glycine | Sigma-Aldrich | Cat# G7126 |
| Urea | Sigma-Aldrich | Cat# U5378 |
| Dithiothreitol | Cell Signaling Technology | Cat# 7016L |
| Ultrapure 1M Tris-HCl pH 7.5 | Thermo Fisher Scientific | Cat# 15567027 |
| 5M NaCl | Thermo Fisher Scientific | Cat# AM9760G |
| IGEPAL CA-630 | Sigma-Aldrich | Cat# I8896 |
| Sodium deoxycholate | Sigma-Aldrich | Cat# D6750 |
| UltraPure SDS Solution, 10% | Thermo Fisher Scientific | Cat# 15553027 |
| 1M MgCl2 | Thermo Fisher Scientific | Cat# AM9530G |
| Crystal Violet certified by the Biological Stain Commission | Sigma-Aldrich | Cat# C0775 |
| ProLong Diamond Antifade Mountant with DAPI | Thermo Fisher Scientific | Cat# P36966 |
| ProLong Gold Antifade Mountant with DNA Stain DAPI | Thermo Fisher Scientific | Cat# P36941 |
| Triton X-100 | Sigma-Aldrich | Cat# T8787 |
| TWEEN-20 | Sigma-Aldrich | Cat# P9416 |
| RIPA Buffer (10X) | Cell Signaling Technology | Cat# 9806S |
| Protease/Phosphatase Inhibitor Cocktail (100X) | Cell Signaling Technology | Cat# 5872S |
| 2x Laemmli Sample Buffer | Bio-Rad Laboratories | Cat# 1610737 |
| 2-Mercaptoethanol | Sigma-Aldrich | Cat# M3148 |
| NuPAGE 4X LDS Sample Buffer | Thermo Fisher Scientific | Cat# NP0008 |
| NuPAGE Sample Reducing Agent (10X) | Thermo Fisher Scientific | Cat# NP0009 |
| Fish Serum Blocking Buffer | Thermo Fisher Scientific | Cat# 37527 |
| RNeasy Mini Kit | QIAGEN | Cat# 74104 |
| Direct-zol RNA MiniPrep | Zymo Research | Cat# R2050 |
| SuperScript IV First-Strand cDNA Synthesis Reaction | Thermo Fisher Scientific | Cat# 18091050 |
| High-Capacity RNA-to-cDNA kit | Thermo Fisher Scientific | Cat# 4387406 |
| RNase Inhibitor | Thermo Fisher Scientific | Cat# N8080119 |
| Q5 High-Fidelity 2X Master Mix | New England Biolabs | Cat# M0494S |
| SYBR Safe DNA Gel Stain | Thermo Fisher Scientific | Cat# S33102 |
| Cell Lysis Buffer (10X) | Cell Signaling | Cat# 9803S |
| cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail | Roche | Cat# 11836170001 |
| Dynabeads Protein G for Immunoprecipitation | Thermo Fisher Scientific | Cat# 10004D |
| Dynabeads Protein A for Immunoprecipitation | Thermo Fisher Scientific | Cat# 10002D |
| Micrococcal Nuclease Solution | Thermo Fisher Scientific | Cat# 88216 |
| Sequencing Grade Modified Trypsin | Promega | Cat# V5113 |
| 16% Formaldehyde, Methanol-Free | Cell Signaling Technology | Cat# 12606S |
| Paraformaldehyde 16% solution EM Grade | Electron Microscopy Sciences | Cat#15710-S |
| Proteinase K | New England Biolabs | Cat# P8107S |
| TURBO DNase | Thermo Fisher Scientific | Cat# AM2238 |
| TRIzol LS reagent | Thermo Fisher Scientific | Cat# 10296028 |
| SUPERaseIn RNase Inhibitor (20U/ul) | Thermo Fisher Scientific | Cat# AM2694 |
| RNaseZap | Thermo Fisher Scientific | Cat# AM9780 |
| RNase-Free DNase Set | QIAGEN | Cat# 79254 |
| Critical commercial assays | ||
| CellTiter-Glo Luminescent Cell Viability Assay | Promega | Cat# G7573 |
| Pierce BCA Protein Assay Kit | Thermo Fisher Scientific | Cat# 23227 |
| Power SYBR Green PCR Master Mix | Thermo Fisher Scientific | Cat# 4367659 |
| NEBNext Ultra II RNA Library Prep Kit for Illumina | New England Biolabs | Cat# E7770 |
| Deposited data | ||
| Human Reference Genome GRCh38/hg38 | The Genome Reference Consortium | https://www.ncbi.nlm.nih.gov/grc/human |
| GENCODE Human Reference Gene Annotation Release 44 | GENCODE | https://www.gencodegenes.org/human/release_44.html |
| Ensembl GENCODE V43 Gene Annotation | UCSC Genome Browser | https://hgdownload.soe.ucsc.edu/goldenPath/hg38/database/knownGene.txt.gz |
| NCBI RefSeq Gene Annotation | UCSC Genome Browser | https://hgdownload.soe.ucsc.edu/goldenPath/hg38/database/ncbiRefSeq.txt.gz |
| UCSC RefSeq Gene Annotation | UCSC Genome Browser | https://hgdownload.soe.ucsc.edu/goldenPath/hg38/database/refGene.txt.gz |
| RNA-seq data | This study | GEO: GSE242781 |
| Raw data files and images | This study | Mendeley Data: https://doi.org/10.17632/drsrg8xtdm.2 |
| Experimental models: Cell lines | ||
| Human: RPE p53−/− | Lim et al.49 | N/A |
| Human: RPE p53−/−, CCAR1 knockout | This study | Table S2 |
| Human: RPE p53−/−, FANCA knockout | This study | N/A |
| Human: RPE p53−/−, CCAR1 knockout, FANCA poison exon-eliminated | This study | N/A |
| Human: RPE CCAR2 knockout | Iyer et al.28 | N/A |
| Human: HEK293T | ATCC | CRL-3216 |
| Human: HEK293T, CCAR1 knockout | This study | Table S2 |
| Human: K562 | ATCC | CCL-243 |
| Human: K562, CCAR1 knockout | This study | Table S2 |
| Human: K562, mini-gene reporter | This study | N/A |
| Human: K562, CCAR1 knockout, mini-gene reporter | This study | N/A |
| Oligonucleotides | ||
| Oligonucleotides | This study | Table S3 |
| Recombinant DNA | ||
| Plasmid: pMMP-puro (empty) | Kupfer et al.50 | N/A |
| Plasmid: pMMP-puro-FANCA | Kupfer et al.50 | N/A |
| Plasmid: pMMP-puro-FANCG | Garcia-Higuera et al.36 | N/A |
| Plasmid: pMMP-FLAG-FANCA | Kupfer et al.50 | N/A |
| Plasmid: pMMP-FLAG-FANCA-PE | This study | N/A |
| Plasmid: pLV-EF1a-IRES-Blast (pLV-Blast, EV) | Hayer et al.51 | Addgene# 85133 |
| Plasmid: pLV-EF1a-IRES-Neo | Hayer et al.51 | Addgene# 85139 |
| Plasmid: pLV-EF1a-IRES-Neo FANCA-PE mini-gene reporter | This study | N/A |
| Plasmid: CCAR1 (Myc-DDK-tagged) | Origene | Cat# RC224293 |
| Plasmid: pOZ-Flag-HA-CCAR1 | This study | N/A |
| Plasmid: pLV-Blast-CCAR1 | This study | N/A |
| Plasmid: pLV-Blast-CCAR1-3xFlag | This study | N/A |
| Plasmid: pLV-Blast-CCAR1 (ΔS1-like) | This study | N/A |
| Plasmid: pLV-Blast-CCAR1 (ΔSAP) | This study | N/A |
| Plasmid: pLV-Blast-CCAR1-3xFlag (Δ2–321) | This study | N/A |
| Plasmid: pLV-Blast-CCAR1-3xFlag (Δ2–380) | This study | N/A |
| Plasmid: pLV-Blast-CCAR1-3xFlag (Δ2–477) | This study | N/A |
| Plasmid: pLV-Blast-CCAR1 (Δ874–1150) | This study | N/A |
| Plasmid: pLV-Blast-CCAR1 (Δ924–1150) | This study | N/A |
| Plasmid: pLV-Blast-CCAR1 (Δ1033–1150) | This study | N/A |
| Plasmid: pLV-Blast-CCAR1-3xFlag (Δ2–146) | This study | N/A |
| Plasmid: pLV-Blast-CCAR1-3xFlag (Δ205–321) | This study | N/A |
| Plasmid: pLV-Blast-CCAR1-3xFlag (ΔEF) | This study | N/A |
| Plasmid: lentiCRISPR-v2 | Sanjana et al.52 | Addgene# 52961 |
| Plasmid: VsVg | Iyer et al.28 | N/A |
| Plasmid: Gag-pol | Iyer et al.28 | N/A |
| Plasmid: psPAX2 | Trono Lab | Addgene# 12260 |
| Plasmid: pMD2.G | Trono Lab | Addgene# 12259 |
| Software and algorithms | ||
| Sequest software | Eng et al.53 | https://www.thermofisher.com/order/catalog/product/OPTON-31014?SID=srch-srp-OPTON-31014 |
| Reactome Functional Interaction Plugin version 8.0.6 | Wu et al.54 | https://reactome.org/userguide/reactome-fiviz |
| Cytoscape version 3.10 | Shannon et al.55 | https://cytoscape.org/ |
| bcl2fastq version 2.17 | Illumina | https://support.illumina.com/sequencing/sequencing_software/bcl2fastq-conversion-software.html |
| DEXSeq version 1.46.0 | Anders et al.35 | https://bioconductor.org/packages/release/bioc/html/DEXSeq.html |
| dexseq_prepare_annotation.py program | Anders et al.35 | https://bioconductor.org/packages/release/bioc/html/DEXSeq.html |
| dexseq_count.py program | Anders et al.35 | https://bioconductor.org/packages/release/bioc/html/DEXSeq.html |
| STAR version 2.7.10b | Dobin et al56 | https://github.com/alexdobin/STAR |
| MISO version 0.5.4 | Katz et al.33 | https://miso.readthedocs.io/en/fastmiso/ |
| rMATS version v4.2.0 | Shen et al.34 | https://rnaseq-mats.sourceforge.io/ |
| rnaseqlib Python package | Katz et al.33 | https://miso.readthedocs.io/en/fastmiso/ |
| compare_miso program | Katz et al.33 | https://miso.readthedocs.io/en/fastmiso/ |
| filter_events program | Katz et al.33 | https://miso.readthedocs.io/en/fastmiso/ |
| sashimi_plot program | Katz et al.33 | https://miso.readthedocs.io/en/fastmiso/ |
| Kallisto version 0.48.0 | Bray et al.57 | https://pachterlab.github.io/kallisto/ |
| tximport Bioconductor R package | Soneson et al.58 | https://bioconductor.org/packages/release/bioc/html/tximport.html |
| edgeR version 3.40.2 | Robinson et al.59 | https://bioconductor.org/packages/release/bioc/html/edgeR.html |
| DESeq2 version 1.38.3 | Love et al.60 | https://bioconductor.org/packages/release/bioc/html/DESeq2.html |
| FlowJo™ Software version 10.9.0 | Becton, Dickinson and Company | https://www.flowjo.com/ |
| bedtools toolset version 2.31.0 | N/A | https://bedtools.readthedocs.io/ |
| limma version 3.54.2 | Ritchie et al.61 | https://bioconductor.org/packages/release/bioc/html/limma.html |
| Other | ||
| RS-2000 Irradiator | Rad Source Technologies | N/A |
| GE Amersham Imager 600 | GE HealthCare Technologies | N/A |
| CytoFLEX S | Beckman Coulter Life Sciences | Cat# B75442 |
| 4D-Nucleofector™ Core Unit | Lonza | Cat# AAF-1002B |
| 4D-Nucleofector™ X Unit | Lonza | Cat# AAF-1002X |
| ImageJ | U. S. National Institutes of Health | https://imagej.nih.gov/ij/ |
| CLARIOstar Plus Plate Reader | BMG Labtech | N/A |
| Zeiss Axio Observer | Zeiss | N/A |
| Cell Profiler | Broad Institute | https://cellprofiler.org/ |
| Auant Studio 7 Flex Real-Time PCR System | Thermo Fisher Scientific | N/A |
| Bioruptor UCD-200 | Diagenode | N/A |
| SpeedVac Vacuum Concentrators | Thermo Fisher Scientific | N/A |
| EASY n-LC | Thermo Fisher Scientific | N/A |
| Ortitrap Explors480 Mass Spectrometer | Thermo Fisher Scientific | N/A |
| Qubit 2.0 Fluorometer | Thermo Fisher Scientific | N/A |
| Illumina NovaSeq | Illumina | N/A |
| GraphPad Prism 10 | Graphpad | https://www.graphpad.com/ |