Supplementary Materials

The PDF file includes:

• Materials and Methods
• Fig. S1. Drug target ablation with CRISPR-Cas9.
• Fig. S2. Cell competition assays in multiple cancer types.
• Fig. S3. Verification of drug target-KOs.
• Fig. S4. Knocking out the verified genetic dependency MEK1 in A375 melanoma cells.
• Fig. S5. MDA-MB-231 clonal analysis.
• Fig. S6. Analysis of homolog gene expression in CRISPR-KO clones.
• Fig. S7. Analysis of homolog gene expression in published RNA-seq experiments.
• Fig. S8. Assessing putative cancer dependencies in whole-genome CRISPR and RNAi screens.
• Fig. S9. Targeting several putative cancer dependencies with CRISPRi.
• Fig. S10. Lack of sensitivity to several clinical chemotherapy agents in putative cancer dependency KOs.
• Fig. S11. Target-independent toxicity of RNAi reagents previously used to investigate several putative cancer dependencies.
• Fig. S12. Using CRISPR to validate the MOA of several anticancer drugs.
• Fig. S13. Off-target toxicity of two caspase-3–activating compounds in CASP3-KO clones.
• Fig. S14. Target-independent cancer cell killing in single-agent and combination therapy experiments.
• Fig. S15. Off-target toxicity of two putative HDAC6-inhibiting compounds in HDAC6-KO ovarian cancer clones.
• Fig. S16. A mutation in the xDFG residue of CDK11B in OTS964-resistant clones.
• Fig. S17. Requirement for CDK11 activity for progression through mitosis.
• References (70–257).

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Other Supplementary Material for this manuscript includes the following:

• Data file S1 (Microsoft Excel format). Literature supporting the designation of HDAC6, MAPK14, PAK4, PBK, and PIM1 as cancer genetic dependencies and CASP3 as a drug target.
• Data file S2 (Microsoft Excel format). Cell competition assay results.
• Data file S3 (Microsoft Excel format). Sources of the cell lines used in this manuscript.
• Data file S4 (Microsoft Excel format). CRISPR gRNA sequences.
• Data file S5 (Microsoft Excel format). CRISPRi gRNA sequences.
• Data file S6 (Microsoft Excel format). Quantitative polymerase chain reaction primers.
• Data file S7 (Microsoft Excel format). Antibody sources and concentrations.
• Data file S8 (Microsoft Excel format). Drugs and drug sources.
• Movie S1 (.mov format). A375 cells expressing H2B-mCherry released from a double-thymidine block into normal medium.
• Movie S2 (.mov format). A375 cells expressing H2B-mCherry released from a double-thymidine block into medium with 25 nM OTS964.
• Movie S3 (.mov format). A375 cells expressing H2B-mCherry released from a double-thymidine block into medium with 100 nM OTS964.
• Movie S4 (.mov format). A375^CDK11B-G579S cells expressing H2B-mCherry released from a double-thymidine block into medium with 100 nM OTS964.