Citrus is a large genus that includes several major cultivated species, including C. sinensis (sweet orange), Citrus reticulata (tangerine and mandarin), Citrus limon (lemon), Citrus grandis (pummelo) and Citrus paradisi (grapefruit). In 2009, the global citrus acreage was 9 million hectares and citrus production was 122.3 million tons (FAO statistics, see URLs), which is the top ranked among all the fruit crops. Among the 10.9 million tons (valued at $9.3 billion) of citrus products traded in 2009, sweet orange accounted for approximately 60% of citrus production for both fresh fruit and processed juice consumption (FAO statistics, see URLs). Moreover, citrus fruits and juice are the prime human source of vitamin C, an important component of human nutrition.Citrus fruits also have some unique botanical features, such as nucellar embryony (nucellus cells can develop into apomictic embryos that are genetically identical to mother plant). Consequently, somatic embryos grow much more vigorously than the zygotic embryos in seeds such that seedlings are essentially clones of the maternal parent. Such citrus-unique characteristics have hindered the study of citrus genetics and breeding improvement 1,2 . Complete genome sequences would provide valuable genetic resources for improving citrus crops.Citrus is believed to be native to southeast Asia 3-5 , and cultivation of fruit crops occurred at least 4,000 years ago 3,6 . The genetic origin of the sweet orange is not clear, although there are some speculations that sweet orange might be derived from interspecific hybridization of some primitive citrus species 7,8 . Citrus is also in the order Sapindales, a sister order to the Brassicales in the Malvidae, making it valuable for comparative genomics studies with the model plant Arabidopsis.We aimed to sequence the genome of Valencia sweet orange (C. sinensis cv. Valencia), one of the most important sweet orange varieties cultivated worldwide and grown primarily for orange juice production. Normal sweet oranges are diploids, with nine pairs of chromosomes and an estimated genome size of ~367 Mb 9 . To reduce the complexity of the sequenced genome, we obtained a doublehaploid (dihaploid) line derived from the anther culture of Valencia sweet orange 10 . We first generated whole-genome shotgun pairedend-tag sequence reads from the dihaploid genomic DNA and built a de novo assembly as the citrus reference genome; we then produced shotgun sequencing reads from the parental diploid DNA and mapped the sequences to the haploid reference genome to obtain the complete genome information for Valencia sweet orange. In addition, we conducted comprehensive transcriptome sequencing analyses for four representative tissues using shotgun RNA sequencing (RNA-Seq) to capture all transcribed sequences and paired-end-tag RNA sequencing (RNA-PET) to demarcate the 5′ and 3′ ends of all transcripts. On the basis of the DNA and RNA sequencing data, we characterized the orange genome for its gene content, heterozygosity and evolutionary features. ...

Precise and efficient genome editing is very important for gene functional characterization. In recent years, sequence-specific DNA nucleases have been developed to increase the efficiency of gene targeting or genome editing in animals and plants. Among them, Zinc-Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs) are two most commonly used sequencespecific chimeric proteins (Gaj et al., 2013). Recently, a breakthrough gene-targeting tool based on RNA-guided Cas9 nuclease from type II prokaryotic Cluster Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated system has been developed (Jinek et al., 2012). CRISPR/Cas system is an adaptive defense system in prokaryotes to fight against alien nucleic acids (Horvath and Barrangou, 2010). The CRISPR loci are variable short spacers separated by short repeats, which are transcribed into synthetic single-guide RNA (sgRNA). The sgRNA forms a functional complex with CRISPR-associated nuclease (Cas9) and guide the nuclease to genomic loci matching a 20-bp complementary invading DNA, cleaving it immediately upstream of a required 5'-NGG Protospacer Adjacent Motif (PAM). A chimeric sgRNA that mimics the natural synthetic sgRNA can be used to target Cas9 for genome editing in eukaryotic cells.Until now, the CRISPR/Cas system has been successfully applied to efficient genome editing in bacteria, animals and plants (Jiang et al., 2013; Mali et al., 2013; Xie and Yang, 2013). To inform the selection of target sites and avoid off-target effects, Hsu and colleagues characterized Cas9 targeting specificity in human cells by a volume of experiments, and built a computational tool for optimized sgRNA selection in human and animal genomes (Hsu et al., 2013). However, with the wide application of CRISPR-system in plant genome editing, tools for optimized sgRNA selection in plants are highly needed. In the current analysis, we present a web application tool-CRISPR-P, for CRISPR sgRNA design in more than 20 plant species. CRISPR-P allows users to search for highly specific Cas9 target sites within DNA sequences of interest, which also provides off-target loci prediction for further analysis and marks restriction enzyme cutting sites for convenience. CRISPR-P is freely available at http://cbi.hzau.edu.cn/crispr/.Genomic data and annotation of the analyzed plant genomes are obtained from public databases. The genome and annotation of Arabidopsis lyrata (v.1.0), Arabidopsis thaliana (TAIR10), Brachypodium distachyon (v1.0), Brassica

Tumors with mutant BRAF and some with mutant RAS are dependent upon ERK signaling for proliferation, and their growth is suppressed by MAPK/ERK kinase (MEK) inhibitors. In contrast, tumor cells with human EGF receptor (HER) kinase activation proliferate in a MEK-independent manner. These findings have led to the development of RAF and MEK inhibitors as anticancer agents. Like MEK inhibitors, the RAF inhibitor PLX4032 inhibits the proliferation of BRAF V600E tumor cells but not that of HER kinase-dependent tumors. However, tumors with RAS mutation that are sensitive to MEK inhibition are insensitive to PLX4032. MEK inhibitors inhibit ERK phosphorylation in all normal and tumor cells, whereas PLX4032 inhibits ERK signaling only in tumor cells expressing BRAF V600E . In contrast, the drug activates MEK and ERK phosphorylation in cells with wildtype BRAF. In BRAF V600E tumor cells, MEK and RAF inhibitors affect the expression of a common set of genes. PLX4032 inhibits ERK signaling output in mutant BRAF cells, whereas it transiently activates the expression of these genes in tumor cells with wild-type RAF. Thus, PLX4032 inhibits ERK signaling output in a mutant BRAFselective manner. These data explain why the drug selectively inhibits the growth of mutant BRAF tumors and suggest that it will not cause toxicity resulting from the inhibition of ERK signaling in normal cells. This selectivity may lead to a broader therapeutic index and help explain the greater antitumor activity observed with this drug than with MEK inhibitors.

Selumetinib (AZD6244, ARRY-142886) is a selective, non–ATP-competitive inhibitor of mitogen-activated protein/extracellular signal–regulated kinase kinase (MEK)-1/2. The range of antitumor activity seen preclinically and in patients highlights the importance of identifying determinants of response to this drug. In large tumor cell panels of diverse lineage, we show that MEK inhibitor response does not have an absolute correlation with mutational or phospho-protein markers of BRAF/MEK, RAS, or phosphoinositide 3-kinase (PI3K) activity. We aimed to enhance predictivity by measuring pathway output through coregulated gene networks displaying differential mRNA expression exclusive to resistant cell subsets and correlated to mutational or dynamic pathway activity. We discovered an 18-gene signature enabling measurement of MEK functional output independent of tumor genotype. Where the MEK pathway is activated but the cells remain resistant to selumetinib, we identified a 13-gene signature that implicates the existence of compensatory signaling from RAS effectors other than PI3K. The ability of these signatures to stratify samples according to functional activation of MEK and/or selumetinib sensitivity was shown in multiple independent melanoma, colon, breast, and lung tumor cell lines and in xenograft models. Furthermore, we were able to measure these signatures in fixed archival melanoma tumor samples using a single RT-qPCR–based test and found intergene correlations and associations with genetic markers of pathway activity to be preserved. These signatures offer useful tools for the study of MEK biology and clinical application of MEK inhibitors, and the novel approaches taken may benefit other targeted therapies.