The ability to engineer genomes in a specific, systematic, and costeffective way is critical for functional genomic studies. Recent advances using the CRISPR-associated single-guide RNA system (Cas9/sgRNA) illustrate the potential of this simple system for genome engineering in a number of organisms. Here we report an effective and inexpensive method for genome DNA editing in Drosophila melanogaster whereby plasmid DNAs encoding short sgRNAs under the control of the U6b promoter are injected into transgenic flies in which Cas9 is specifically expressed in the germ line via the nanos promoter. We evaluate the off-targets associated with the method and establish a Web-based resource, along with a searchable, genome-wide database of predicted sgRNAs appropriate for genome engineering in flies. Finally, we discuss the advantages of our method in comparison with other recently published approaches.nanos-Cas9 | HRMA M uch of our knowledge of the mechanisms underlying biological processes relies on genetic approaches, whereby gene activity is perturbed and the phenotypic consequences of perturbation are analyzed in detail. In recent years, several major advances have been made in the design of methods for specifically and efficiently perturbing genomes. Arguably, the most exciting advances rely on the ability to induce double-strand breaks (DSBs) by targeting a nuclease to a specific genomic sequence. Repair of DSBs by the error-prone nonhomologous endjoining (NHEJ) mechanism allows for the recovery of small deletions; moreover, repair of DSBs by homologous recombination (HR) in the presence of a donor template opens the door to a wide range of specifically engineered changes at the targeted site (1).Two nuclease-based systems, the zinc-finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN) systems, work effectively in a number of organisms (2-7). But because these approaches require the production of a construct encoding a unique DNA-binding protein fused to the nuclease domain, they can be both cumbersome and costly. In contrast, the recent approach based on the bacterial CRISPR-associated single-guide RNA (Cas9/sgRNA) system does not require production of specific fusion proteins for each targeted sequence (8-10).Cas9 was first identified in type II Streptococcus pyogenes as an RNA-guided defense system against invading viruses and plasmids (11-13). This adaptive immune-like system contains three components: CRISPR RNA (crRNA), trans-activating CRISPR RNA (tracrRNA), and Cas9. The tracrRNA triggers Cas9 nuclease activity and the crRNA guides Cas9 to cleave the specific foreign dsDNA sequence via base-pairing between the crRNA and the target DNA. Importantly, a single-guide RNA (sgRNA, also known as chiRNA), comprising the minimal crRNA and tracrRNA, can function similarly to the crRNA and tracrRNA, thereby providing a simplified method for genome editing (8)(9)(10)(14)(15)(16)(17)(18)(19)(20).Given the great promise of the Cas9/sgRNA method for genome engineering, we set out to test the sys...

The Hippo signaling pathway regulates cellular proliferation and survival, thus exerting profound effects on normal cell fate and tumorigenesis1-3. The pivotal effector of this pathway is YAP, a transcriptional co-activator amplified in mouse and human cancers, where it promotes epithelial to mesenchymal transition (EMT) and malignant transformation4-10. To date, studies of YAP target genes have focused on cell-autonomous mediators; here we show that YAP-expressing MCF10A breast epithelial cells enhance the proliferation of neighboring untransfected cells, implicating a noncell autonomous mechanism. We identify the epidermal growth factor receptor (EGFR) ligand, amphiregulin (AREG), as a transcriptional target of YAP, whose induction contributes to YAPmediated cell proliferation and migration, but not EMT. Knockdown of AREG or addition of an EGFR kinase inhibitor abrogates the proliferative effects of YAP expression. Suppression of the negative YAP regulators LATS1/2 is sufficient to induce AREG expression, consistent with physiological regulation of AREG by the Hippo pathway. Genetic interaction between the Drosophila YAP orthologue Yorkie and Egfr signaling components support the link between these two highly conserved signaling pathways. Thus, YAP-dependent secretion of AREG implicates activation of EGFR signaling as an important non-cell autonomous effector of the Hippo pathway, with implications for the regulation of both physiological and malignant cell proliferation.Normal cells require mitogenic growth signals to proliferate, whereas, tumor cells often generate their own proliferative signals through the secretion of growth factors or the activation of growth factor receptors 11 . We have shown that YAP transduced MCF10A cells proliferate in 3D acinar cultures in the absence of EGF 7 . MCF10A are immortalized, non transformed human mammary epithelial cells, which exhibit dependence on growth factors for proliferation and survival 12 , raising the possibility that YAP itself induces secretion of required growth factors or cytokines in these cells. To test this hypothesis, we performed mixing experiments with cells transduced with either GFP-labeled YAP or Red-Cherry tagged vector. MCF10A cells expressing GFP-YAP, but not Cherry-vector, formed acini in 3D cultures in the absence exogenous EGF. Remarkably, vector transduced cells did produce acini when co-cultured in a 1:1 ratio with YAP expressing cells (Fig. 1a) To identify mediators of this apparent YAP-induced non-cell-autonomous effect, we made use of a constitutively active YAP mutant, YAP-S127A. Mutation in the serine residue targeted for phosphorylation by the negative regulatory kinases LATS1/2 prevents cytoplasmic sequestration of YAP by 14-3-3 proteins 5 , 10 , thus resulting in its exclusively nuclear localization (Fig. S1a). Retroviral vectors containing wild type YAP or YAP-S127A induced similar levels of protein expression (Fig. 1b). Ectopic expression of YAP-S127A induced a strong EMT and cell migration phenotype ( Fig. S1b and S1c),...

Summary The CRISPR/Cas9 system has recently emerged as a powerful tool for functional genomic studies in Drosophila melanogaster. However, sgRNA parameters affecting the specificity and efficiency of the system in flies are still not clear. Here, we found that off-target effects did not occur in regions of genomic DNA with three or more nucleotide mismatches to sgRNAs. Importantly, we document for the first time a strong positive correlation between mutagenesis efficiency and sgRNA GC content of the six protospacer adjacent motif-proximal nucleotides (PAMPNs). Furthermore, by injecting well-designed sgRNA plasmids at the optimal concentration we determined, we could efficiently generate mutations in four genes in one step. Finally, we generated null alleles of HP1a using optimized parameters through homology-directed repair, and achieved an overall mutagenesis rate significantly higher than previously reported. Our work presents the most comprehensive optimization of sgRNA and promises to vastly simplify CRISPR/Cas9 experiments in Drosophila.