The assembly domain has a rigid structure that contributes to the capsid scaffold

TAK 438 free base web analysis of knockdown efficiency. While Cas9 mutagenesis is also susceptible to off-target editing, to the best of our knowledge, the off-target loci affected by CRISPR are unlikely to substantially overlap with those that are affected by RNAi. Moreover, sequencing the locus targeted by Cas9 can provide an unbiased readout of mutagenesis efficiency that is not sensitive to cell statedependent expression variability. Finally, unlike RNAi, CRISPR can be applied to generate clonal cell lines that harbor null mutations in a targeted gene. This technique bypasses the problems inherent in the analysis of mixed cell populations and partial loss-of-function phenotypes, and can provide significant insight into the genetic architecture of cancer. One limitation of CRISPR mutagenesis is that, over the time required to generate or select for a pure cell population, cells may engage compensatory mechanisms to buffer against the loss of a targeted protein. Thus, the analysis of knockout clones can be complemented with cell-cell competition assays, which allow less time for cells to adapt to gene loss and may reveal the presence of a transient or immediate fitness defect induced by CRISPR. We performed a total of 91 competition assays that failed to reveal an effect of MELK loss on cell fitness, further strengthening our conclusion that MELK is dispensable for cancer cell proliferation. CRISPR mutagenesis can also assist in the pharmacological study of potential drugs. Several lines of evidence indicate that OTS167 does indeed inhibit MELK: for PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19828152 instance, a crystal structure of OTS167 binding to the MELK kinase domain has been reported. However, these structural and biochemical studies are unable to conclusively demonstrate that a phenotype in a living cell is due to an on-target effect. We believe that CRISPR represents a useful tool to gain genetic insight into this question: if a CRISPR-induced null mutation of a putative drug target fails to confer resistance to that drug, then that drug must act through alternate targets or mechanisms. While the MELK-KO cell lines that we generated remain exquisitely sensitive to OTS167, at present, we do not know how OTS167 blocks cell division. One possibility, not ruled out by our studies, is that OTS167 exhibits polypharmacology, and kills cancer cells by inhibiting multiple kinases, potentially including MELK. The analysis of drug-resistant alleles of other mitotic kinases that OTS167 has been shown to inhibit may shed further light on the in vivo MOA of this compound. Our results leave open the question of what role, if any, MELK plays in mammalian biology and cell cycle progression. While MELK is up-regulated in diverse tumor types, it is also expressed in several normal cell lineages, including embryonic cells, hematopoietic cells, and neural progenitor cells. MELK may be required at a certain developmental stage, or for a specific cell type or organismal process. Similarly, we cannot currently rule out the possibility that MELK plays a role in tumorigenesis in vivo that was not assessed in our Lin et al. eLife 2017;6:e24179. DOI: 10.7554/eLife.24179 11 of 17 Short report Cancer Biology Genes and Chromosomes current work. At a minimum, our results suggest that MELK is dispensable for mitotic progression in most cancers. MELK may function in an overlapping or redundant pathway with other mitotic kinases, several of which are up-regulated along with MELK in tumor cells. Synthetic lethal screens and further i