assays, we also cloned and transduced three gRNA’s that target the non-essential and non-coding Rosa26 locus. To assess the efficacy of our CRISPR system, we purified GFP+ populations from cell lines harboring each individual guide RNA, and then we analyzed the targeted loci by Sanger sequencing. TIDE analysis, which decomposes raw sequencing traces into linear combinations of indel mutations, revealed high cutting efficiency at most targeted loci. Across the 21 samples, the median level of PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19826938 indel formation was 80%. These values likely underestimate the true mutation frequency, as TIDE analysis is not able to detect missense mutations and large indels will not be efficiently amplified by PCR. R-roscovitine Western blot analysis of MELK protein levels in A375, Cal51, and MDA-MB-231 sorted populations further confirmed that our CRISPR system effectively ablated MELK expression. We next set out to determine whether MELK was required for cancer cell fitness. To test this, we measured cell proliferation over 15 days in culture in the 30 independent lines of A375, Cal51, and MDA-MB-231 that we had generated. Surprisingly, we failed to detect any difference in proliferative capacity between the cell lines with wild-type or mutant MELK. For instance, in the A375 cell line, we calculated a mean doubling time of 16.9 hr among cells transduced with Rosa26 guide RNAs, while the cell lines transduced with MELK gRNAs exhibited a mean doubling time of 16.8 hr. MELK gRNA-transduced cell lines also exhibited wild-type levels of growth under anchorage-independent conditions. These results call into question the notion that MELK is a genetic dependency either across cancer types or in triple-negative breast cancers. MELK is not a common cancer cell dependency In order to assess whether a wider range of cancer cell lines were dependent on MELK for viability, we performed individual GFP dropout experiments in 13 Cas9-expressing cancer cell lines. In these assays, cancer cells are transduced with GFP-expressing guide RNA vectors at low MOI to create mixed populations of GFP+ and GFP- cells. A table summarizing the results presented in is displayed. DOI: 10.7554/eLife.24179.008 Lin et al. eLife 2017;6:e24179. DOI: 10.7554/eLife.24179 R R 6 of 17 Short report Cancer Biology Genes and Chromosomes 2014), 
and a genome-wide CRISPR screen in seven cell lines. Large-scale unbiased screens are prone to experimental artifacts, and variations in protocol, technology, or the method of analysis can cause different screens to yield different results. Nonetheless, each of these screens identified multiple mitotic kinases as essential in various cancer cell lines, including Aurora B, BubR1, CDK1, and Plk1. In contrast, MELK was not identified as essential in a single experiment. These negative results include 13 triplenegative breast cancer cell lines tested by Campbell et al. and 16 triple-negative breast cancer cell lines tested by the Moffatt lab. Several other published pan-cancer or breast cancer-focused screens have failed to identify MELK as either a general cancer dependency or a triple-negative breast cancer dependency. Thus, while we do not consider results from large-scale screens to be dispositive, we believe that these findings, coupled with our own experimental evidence, suggest that MELK expression is not required for cancer cell proliferation. OTS167 inhibits the growth of receptor-positive breast cancer cell lines and cells that harbor mutant MELK If MELK is not a cance