(A) Domain structure of MELK and locations of the sequences targeted by 7 MELK gRNAs. (B) Genomic DNA was purified from the indicated population of MDA-MB-231 cells and the targeted loci were amplified by PCR. Percent indel formation was estimated using TIDE analysis. The highlighted region indicates 20 nucleotides or 15 nucleotides of the sequence recognized by the guide RNA. Other sequence traces are presented in Figure 1—figure supplements 1–3. (C) Western blot analysis of GFP+ MDA-MB-231 cells using the Abcam ab108529 MELK antibody. Alpha-tubulin levels were analyzed as a loading control. (D–F) Proliferation and doubling time analysis of A375, Cal51, and MDA-MB-231 cell lines transduced with 3 Rosa26 gRNAs or with 7 MELK gRNAs. (G) Images of colonies from the indicated Cal51 strains grown in soft agar. (H–I) Quantification of anchorage-independent growth in Cal51 or A375 cells transduced with the indicated gRNA. For each assay, colonies were counted in at least 15 fields under a 10x objective. Boxes represent the 25th, 50th, and 75th percentiles of colonies per field, while the whiskers represent the 10th and 90th percentiles.