ARTICLE RESEARCH was determ tage BFP-positive cells (that is purchased from C arles Rive r Labora ned in hy cage cT16 (00 Ce 351 tin (Cell n our in viy 3500 and this l RD 2212) bbitIgG(H+L)(LI-COR. in-fixed, SW62 nd SWa troll r10min）i led in a afte which D n Liguid DAR Sub ing I ved as po ng p as calc edcel e sum of f WRN depe HCT116 parental cells Data availability included in the data 1.2 an re a e fr r( nich the c o d ty was assessed sin Code availability SPR- d co HC the 6f2 31. Cell growth Cyte-FLR 4> very6h usingan ders&Hube al expre 420182 ence count data 2010 35. 2014 et al by山 902555801 Article RESEARCH score was determined as the ratio of the percentage BFP-positive cells (that is, sgRNA-positive cells) on day 14 compared to day 4, as measured by flow cytometry. A co-competition score less than 1 indicates a relative reduction in BFP-positive cells, resulting from targeting of a loss-of-fitness gene. Clonogenic assay. Cell lines were transduced with lentivirus that encodes WRN sgRNA at around 100% efficiency as described above in six-well plates (2,000 cells per well), typically for 15–21 days. Cells were fixed using 100% ice-cold ethanol for 30 min followed by Giemsa staining overnight at room temperature. Western blot analysis. Cells were transduced at around 100% as described above in 10-cm dishes. Day 5 after transduction, cells were lysed with 200 μl RIPA buffer supplemented with protease and phosphatase inhibitors and lysates were used for SDS–PAGE and immunoblot analysis. Antibodies used were: WRN (Cell Signaling Technologies, 4666; dilution 1:2,000), WRN for domain rescue experiment (Thermo Fisher Scientific, PA5-27319); MLH1 (Cell Signaling Technologies, 3515; dilution 1:1,000); MSH3 (Santa Cruz Biotechnology, sc-271080; dilution 1:1,000); anti-Flag M2 (Sigma-Aldrich, F3165); β-actin (Cell Signaling Technologies, 4970); and anti-β-tubulin (Sigma-Aldrich, T4026: dilution 1:5,000). Secondary antibod￾ies included: IRDye 800CW donkey anti-mouse antibody (LI-COR, 926-32212); IRDye 680LT donkey anti-rabbit IgG (H+L) (LI-COR, 925-68023); anti-mouse IgG HRP-linked secondary antibody (GE Healthcare, NA931). Molecular weight markers included: SeeBlue Plus2 Pre-stained Protein Standard (Thermo Fisher Scientific, 5925) and Precision Plus Protein Standards (BioRad, 161-0373). WRN rescue experiment. SW620 and SW48 cells (2 × 105  cells) were transfected by nucleofection (Lonza 4D Nucleofector Unit X) with Cas9–sgRNA ribonucleo￾proteins (RNP) targeting human MAVS (used as a non-essential knockout control) or WRN, together with overexpression of 200 ng pmGFP control or 200 ng mouse Wrn cDNA (Origene, MR226496). From each sample after nucleofection, 5,000 cells were seeded in a 96-well plate and allowed to grow for 5 days, after which cells were collected for either CellTiter-Glo assay (Promega, G9241) or western blot analysis. CellTiter-Glo data were read on an Envision Multiplate Reader and data analysis was performed using GraphPad Prism 7 software. Student’s t-test was performed using the multiple t-test module in Prism 7. The sgRNA sequences that were used are listed in Supplementary Table 10. RNA interference. A pool of four siRNAs that target WRN were used (Dharmacon, L-010378-00-0005). HCT116 cells were grown and transfected with siRNA using the RNAiMAX (Invitrogen) transfection reagent following the manufacturer’s instructions. Each experiment included: mock control (transfection lipid only), ON-TARGETplus Non-targeting Control Pool (Dharmacon, D-001810-10-05) as a negative control, and polo‐like kinase 1 (PLK1) (Dharmacon, L-003290-00-0010), which served as a positive control. siRNA sequences are listed in Supplementary Table 10. Rescue of WRN dependency in HCT116 isogenic lines. HCT116 parental cells and derivatives carrying Chr.2, Chr.3, Chr.5 or Chr.3 + Chr.5 were transduced to express Cas9. After transduction, all lines displayed Cas9 activity >80%. To assess WRN dependency, cells were seeded at 1.5 × 103  cells per well in 100 μl complete growth medium in 96-well plastic cell culture plates. At day 0, cells were transduced with viral particles containing sgRNAs targeting essential or non-essential genes, or WRN sgRNA 1 and WRN sgRNA 4 in order to achieve a >90% transduction efficiency. The following day, the medium was replaced and 48 h after transduction puromycin was added at final concentration of 2 μg ml−1 . Plates were incubated at 37 °C in 5% CO2 for 7 days, after which the cell viability was assessed using CellTiter-Glo (Promega) by measuring luminescence on an Envision multiplate reader. Clonogenic assays were performed as described in the ‘WRN dependency in MSI cell lines’ section; and 48 h after transduction puromycin was added at a final concentration of 2 μg ml−1 . In vivo validation. WRN knockout using an inducible CRISPR–Cas9 system. To generate inducible WRN sgRNA-expressing HCT116 cells, we cloned WRN sgRNA 4 into the pRSGT16H-U6Tet-(sg)-CMV-TetRep-TagRFP-2A-Hygro vec￾tor (Cellecta). Cas9-expressing HCT116 cells were transduced and selected with 500 μg ml−1 of hygromycin (Thermo Fisher Scientific). To obtain cell popula￾tions that both uniformly express Cas9 and contain the inducible WRN-targeting sgRNA, we generated single-cell clones by serial dilution. To measure the growth rate of WRN sgRNA-expressing HCT116 cells after conditional induction of WRN knockout, cells were grown in flasks in the presence or absence of 2 μg ml−1 dox￾ycycline for 24 h and then seeded in 96-well plates, with or without the same concentration of doxycycline. Cell growth was monitored every 6 h using an automated IncuCyte-FLR 4X phase-contrast microscope (Essen Instruments). The average object-summed intensity was calculated using the IncuCyte software (Essen Instruments). Mouse xenograft studies. Female non-obese diabetic/severe combined immunode￾ficiency (NOD/SCID) mice (Charles River Laboratories) were used in all in vivo studies. All animal procedures were approved by the Ethical Committee of the Institute and by the Italian Ministry of Health (authorization 806/2016-PR). The methods were carried out in accordance with the approved guidelines. Mice were purchased from Charles River Laboratories, maintained in hyperventilated cages and manipulated under pathogen-free conditions. In particular, mice were housed in individually sterilized cages; each cage contained a maximum of seven mice and optimal amounts of sterilized food, water and bedding. HCT116 xenografts were established by subcutaneous inoculation of 2 × 106  cells into the right posterior flank of 5- to 6-week-old mice. Tumour size was evaluated by calliper measure￾ments, and the approximate volume of the mass was calculated using the formula 4/3π × (d/2)2 × (D/2), where d is the minor tumour axis and D is the major tumour axis. When tumours reached an average size of approximately 250–300 mm3 , ani￾mals with the most homogeneous size were selected and randomized by tumour size. Doxycycline (Sigma-Aldrich, D9891) was dissolved in water and administered daily at a 50 mg kg−1 concentration by oral gavage. For each experimental group, 8–10 mice were used to enable reliable estimation of within-group variability. Operators allocated mice to the different treatment groups during randomization but were blinded during measurements. The maximal tumour volume permitted in our in vivo experiments was 3,500 mm3 and this limit was never exceeded. In vivo procedures and related biobank data were managed using the Laboratory Assistant Suite, a web-based proprietary data management system for automated data tracking47. Immunohistochemistry. Formalin-fixed, paraffin-embedded tissues explanted from cell xenografts were partially sectioned (10-μm thick) using a microtome. Then, 4-μm paraffin tissue sections were dried in a 37 °C oven overnight. Slides were dep￾araffinized in xylene and rehydrated through graded alcohol to water. Endogenous peroxidase was blocked in 3% hydrogen peroxide for 30 min. Microwave anti￾gen retrieval was carried out using a microwave oven (750 W for 10 min) in 10 mmol l−1 citrate buffer, pH 6.0. Slides were incubated with monoclonal mouse anti-human KI-67 (1:100; DAKO) overnight at 4 °C inside a moist chamber. After washings in TBS, anti-mouse secondary antibody (DAKO Envision+System horse￾radish peroxidase-labelled polymer, DAKO) was added. Incubations were carried out for 1 h at room temperature. Immunoreactivities were revealed by incubation in DAB chromogen (DakoCytomation Liquid DAB Substrate Chromogen System, DAKO) for 10 min. Slides were counterstained in Mayer’s haematoxylin, dehy￾drated in graded alcohol, cleared in xylene and a coverslip was applied using DPX (Sigma-Aldrich). A negative control slide was processed with only the secondary antibody, omitting the primary antibody incubation. Immunohistochemically stained slides for KI-67 were scanned with a 40× objective. Ten representative images selected from three cases were then analysed using ImageJ (NIH), which segmented cells with positive and negative nuclei. The percentage of the area con￾taining positive cells was calculated as the brown area (positively stained cells) divided by the sum of brown and blue areas (negatively stained cells). The software interpretation was manually verified by visual inspection of the digital images to ensure accuracy. Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this paper. Data availability Data and analyses are included in the published article and supplementary data 1, 2 and 3 are available from FigShare (https://figshare.com/projects/ CRISPRtargetID/60146). The gene fitness scores of the cell lines, raw counts of the sgRNA data, and processed data and results are available from the project Score web portal: https://score.depmap.sanger.ac.uk. Code availability Software code are available through GitHub at https://github.com/francescojm/ CRISPRcleanR, https://github.com/francescojm/ADAM and https://github.com/ francescojm/BAGELR. 29. Ballouz, S. & Gillis, J. AuPairWise: a method to estimate RNA-seq replicability through co-expression. PLOS Comput. Biol. 12, e1004868 (2016). Home (25 Doggett St) 30. Hart, T. & Mofat, J. BAGEL: a computational framework for identifying essential genes from pooled library screens. 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