ARTICLE RESEARCH a prostate cance nded Data Fig 4 on-syonyomua s.promoter methylation and homozygous oulator ed co fitness of WRN-knockout co ed t ld-typ ancers (Fig 5b.Extended Data Fig &aand Supple e four ata Fig. -0-50 510152023 similar tocor yin MS cancer cell lines tended Data Fig.8d),and and WRN d the fo uto ellite stab with chr type.MS effect of WRN knoc not revert the 9) ial st y fo al res er plots s tion in th (E8A) case (R 105 ld-type (def)or he SW48 cells with MS nt Wrr and ex Data Fig Th td.Tum re se a WRNsequired line)P-0006 ty ay ANOVA.Data eva utar us en with do 50m4 wth supp on of alculated using a two-s ded Welch's t-test ded Data an Ve identified WRN thetic lethal ta ssarv to sust Ivo gro MSI by ssion DNA recomb and the yeast hon ape ormed CRISPR mbinationinregions otide mis the mic and tractability data tos minate new can to confer WRN dependency.this sug hich WRN i derpins the synthetic letha its to patients with ance mal rec teriz lutionar me engi ering and dise netics.Results ever.ta WrN could result in dat ge to nor ough the project Score se (https://score.depmap. NATUREIW Article RESEARCH rare (<1%) in many other tumour types, such as kidney, melanoma and prostate cancers17 and most (4 out of 5 tested) MSI cell lines from these tissues were not dependent on WRN (Extended Data Fig. 7c). Other tested RecQ family members (BLM, RECQL and RECQL5) were not associated as fitness genes in MSI cell lines. A focused analysis of non-synonymous mutations, promoter methylation and homozygous deletions of MMR pathway genes confirmed a significant association between WRN dependency and hypermethylation of the MLH1 pro￾moter (Student’s t-test, FDR = 7.72 × 10−3 ) or mutations in MSH6 (FDR = 3.85 × 10−2 ); as well as mutations in the epigenetic regulator MLL2 (also known as KMT2D) (FDR = 1.43 × 10−4 ) (Fig. 5a). To further validate WRN, we performed CRISPR-based co￾competition assays in which the relative fitness of WRN-knockout versus wild-type cells was compared. WRN knockout using four individual sgRNAs decreased fitness of WRN-knockout compared to wild-type cells in six MSI cell lines from colon, ovarian, endometrial and gastric cancers (Fig. 5b, Extended Data Fig. 8a and Supplementary Table 10). By contrast, there was no difference in all microsatellite stable cell lines from these four tissues. Consistently, WRN was selectively essen￾tial for MSI cells in clonogenic assays (Extended Data Fig. 8b, c). Of note, WRN knockout had a potent effect on cell fitness with an effect size similar to core fitness genes (Fig. 5a, b). Furthermore, we mined data from systematic RNA interference screens and confirmed WRN dependency in MSI cancer cell lines12 (Extended Data Fig. 8d), and confirmed that WRN downregulation by RNA interference robustly impaired growth in MSI HCT116 cells (Extended Data Fig. 8e, f), thus providing validation in an orthogonal experimental system. Despite the strong association between MMR deficiency and WRN dependency, knockout of MLH1 in microsatellite-stable SW620 cell line did not induce WRN dependency; conversely complementation of HCT116 cells with chromosomes that contain MLH1 and/or MSH3—to restore their expression and correct MMR deficiency18—did not revert the effect of WRN knockout (Extended Data Fig. 9). To determine whether the loss-of-fitness effect was selective to WRN and identify a potential strategy for drug targeting, we performed func￾tional rescue experiments using wild-type, or hypomorphic versions of mouse Wrn (resistant to the WRN sgRNAs that we used) with a mutation in the exonuclease (E78A) or helicase (R799C or T1052G) domain to impair protein function19–21. Expression of wild-type or exonuclease-deficient Wrn rescued knockout of WRN in MSI cells, whereas expression of helicase-deficient Wrn led to no (R799C) or weak (T1052G) rescue (Fig. 5c and Extended Data Fig. 10a, b). Thus, the helicase activity of WRN is required and is an important domain that can be used for therapeutic targeting. To evaluate in vivo sensitivity of MSI cells to WRN depletion, we developed a doxycycline-inducible WRN sgRNA system in HCT116 cells (Extended Data Fig. 10c, d). Following subcutaneous engraftment of WRN sgRNA-expressing HCT116 cells in mice, treatment with dox￾ycycline led to significant growth suppression of established tumours and a reduction in the number of proliferating cells (Fig. 5d–f and Extended Data Fig. 10e, f). These findings confirm that WRN is nec￾essary to sustain in vivo growth of colorectal cancer cells with MSI. Discussion New approaches are needed to effectively prioritize candidate ther￾apeutic targets for cancer treatments. We performed CRISPR–Cas9 screens in a diverse collection of cancer cells lines and combined this with genomic and tractability data to systematically nominate new can￾cer targets in an unbiased way. Confirmatory studies are necessary to further evaluate the priority targets that we identified. Even a modest improvement in drug-development success rates, and an expanded repertoire of targets, through approaches such as ours could provide benefits to patients with cancer. Our CRISPR–Cas9 screening results are also a resource with diverse applications in fundamental and evo￾lutionary biology, genome engineering and disease genetics. Results are available through the project Score database (https://score.depmap. sanger.ac.uk/). We identified WRN as a promising new synthetic lethal target in MSI tumours. This finding is corroborated by the accompanying study by Chan et al.22. WRN physically interacts with MMR proteins23, can resolve DNA recombination intermediates24, and the yeast homologue Sgs1 has a redundant function with MMR proteins to suppress home￾ologous recombination in regions of nucleotide mismatch25. Together with our finding that modulation of MMR proteins alone is insufficient to confer WRN dependency, this suggests a model in which WRN is required to resolve the genomic structures present in MMR-deficient cells, which are possibly homeologous recombination structures, and failure to efficiently resolve these underpins the synthetic lethal dependency. Mutation of WRN leads to Werner syndrome, an auto￾somal recessive disorder characterized by premature ageing and an increased risk of cancer16. Thus, loss of WRN is compatible with human development; however, targeting WRN could result in damage to nor￾mal cells. Consideration should be given to maximizing therapeutic benefits through patient selection and dose scheduling. A possible route 0.5 1.0 Normalised cell viability Control sgRNA Control vector WRN sgRNA WT Exonuclease def. Helicase def. Helicase def. NS Wrn E78A R799C T1052G Vehicle Dox Vehicle Dox 0 20 40 60 80 KI-67+ nuclei (% area) P = 4.8 × 10–16 MSI MSS Colorectal Ovarian Other MLH1 meth. MLL2 mut. MSH6 mut. –4 –2 0 10 100 WRN Core fitness genes Mutation rate (per Mb) a b 1,750 1,500 1,250 1,000 750 500 250 Doxycycline Days from treatment start –10 –5 0 5 10 15 20 25 Tumour volume (mm3 ) n = 9 n = 10 d f e c MSI MSS 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 Co-competition score sgEss P = 0.69 MSI MSS sgNon P = 0.62 MSI MSS WRN sgRNA Gastric Endometrial Colorectal Ovarian P = 2.8 × 10–19 P = 4.7 × 10–3 P = 2.3 × 10–2 Fig. 5 | WRN is a target in MSI cancer cells. a, Circle plot of cell lines. From the outer ring to inner ring the following are shown: the fitness effect of WRN knockout and mean effect of core fitness genes (red dashed line); cancer-type; MLH1 methylation (meth.) status; mutation (mut.) status of MLL2 and MSH6; and the DNA mutation rate. b, WRN dependency in a co-competition assay. sgRNAs that target essential (sgEss) and non-essential (sgNon) genes were used as controls. Each point represents the mean co-competition score for a cell line (seven MSI and seven microsatellite stable (MSS) lines in duplicate); four WRN sgRNA guides were used. A score less than 1 denotes selective depletion of sgRNA-expressing knockout cells. Box-and-whisker plots show 1.5× the interquartile range and the median. P values were determined using a two￾sided Welch’s t-test. c, WRN rescue using wild-type (WT), exonuclease￾deficient (def.) or helicase-deficient mouse Wrn in SW48 cells with MSI. Mean ± s.d. from 3 independent experiments. P values were calculated using a standard two-sided t-test assuming equal variance; comparison to wild-type Wrn. NS, not significant. d, Tumour volume of WRN sgRNA-expressing HCT116 (clone a) xenografts treated with doxycycline (yellow line) or vehicle (grey line). P = 0.006, two-way ANOVA. Data are mean ± s.e.m. Numbers of mice in each cohort are indicated. e, Representative KI-67 immunohistochemistry assessment of WRN sgRNA￾expressing HCT116 (clone a) tumours explanted after one week. Scale bar, 50 μm; 40× magnification. f, Quantification of KI-67 staining. Data are mean ± s.d. of 10 fields from three different samples. n = 30; P value were calculated using a two-sided Welch’s t-test. N A t U r e | www.nature.com/nature