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. 2025 Jun;45(6):709-713.
doi: 10.1002/cac2.70013. Epub 2025 Mar 17.

Whole-genome CRISPR-Cas9 knockout screens identify SHOC2 as a genetic dependency in NRAS-mutant melanoma

Andrea Y Gu  1   2 , Tet Woo Lee  1   2 , Aziza Khan  1 , Xuenan Zhang  1 , Francis W Hunter  1   3 , Dean C Singleton  1   2   4 , Stephen M F Jamieson  1   2   5
Affiliations

Whole-genome CRISPR-Cas9 knockout screens identify SHOC2 as a genetic dependency in NRAS-mutant melanoma

Andrea Y Gu et al. Cancer Commun (Lond). 2025 Jun.
No abstract available

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

FIGURE 1
FIGURE 1
Identification and validation of SHOC2 as a genetic vulnerability in NRAS‐mutant melanoma through CRISPR‐Cas9 whole‐genome knockout screens. (A) Percentage of all or NTC sgRNAs detected in NZM cell lines transduced with either the first virus batch of the Brunello library (Plasmid A) or the second virus batch (Plasmid B). The bar indicates the median. Cell lines with < 80% sgRNAs detected (dotted line) with at least one read count were excluded from further analyses. Concomitant dropout of NTC sgRNAs indicates that dropout is not solely due to knockout‐induced fitness defects. (B) The log2 fold‐change density plots of sgRNAs targeting either the expanded common essential reference set (red) or nonessential reference genes (blue) in NZM37 cells. (C) Distributions of the normalised BF values of genes from the common essential reference set (red), nonessential reference set (green), and candidate genes (blue) in NZM17. BF values determined by BAGEL2 analysis were normalised to the minimum and maximum BF values. (D‐E) Heatmap of normalised BF values for the top two candidate essential genes (NRAS and SHOC2), ranked by the Limma moderated t‐statistic method, comparing BF values between NRAS‐mutant and NRAS‐wildtype cell lines in (D) NZM screens and (E) DepMap Avana melanoma screens. BF values were normalised to the minimum and maximum values within each cell line, with red indicating increased essentiality and green indicating decreased essentiality. (F) Normalised essentiality scores (BF values) for NRAS and SHOC2 in NZM (left) and DepMap Avana melanoma cell line (right) screens. NRAS and SHOC2 had significantly higher BF values (indicating greater essentiality) in NRAS‐mutant compared to NRAS‐wildtype cell lines in both NZM lines and Avana screens, as determined by the Limma moderated t‐statistic method. (G‐H) Cell proliferation following SHOC2 knockout compared to a positive control (PCID2 KO, a common essential gene knockout) and negative controls (APOA4 KO, a nonessential gene knockout, and cells transduced with an NTC). Cells were grown in T25 flasks and passaged upon reaching 80% confluency. Cumulative cell growth was determined by cell counts at each passage. (G) SHOC2 KO reduced cell proliferation relative to APOA4 KO and NTC cells in NRAS‐mutant NZM17, NZM48, and NZM78 cell lines. In NZM48 and NZM78, SHOC2 KO cells exhibited similar growth to PCID2 KO cells. (H) SHOC2 KO cells grew similarly to APOA4 KO and NTC cells and proliferated faster than PCID2 KO cells in NRAS‐wildtype NZM37, NZM53, and NZM91 cell lines. The dashed line at 1 indicates the proliferation of NTC cells. (I) Western blot analysis of SHOC2, phosphorylated ERK1/2 (p‐ERK1/2), and total ERK1/2 expression in NZM17 (NRAS‐mutant) and NZM37 (NRAS‐wildtype) cultures collected 28 days after transduction. SHOC2 knockout was associated with reduced ERK phosphorylation in NRAS‐mutant NZM17 cells but not in NRAS‐wildtype NZM37 cells. R1, R2, and R3 indicate three independent transductions and screens. Abbreviations: BF, Bayes Factor; CEG, common essential gene; D28, 28 days; KO, knockout; NEG, nonessential gene; NTC, non‐targeting control; NZM, New Zealand Melanoma; p‐ERK1/2, phosphorylated ERK1/2; sgRNA, single guide RNA.
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