Identifying genes involved in biological processes is critical for understanding the molecular building blocks of life. The effectiveness of engineered CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) to efficiently mutate specific loci coupled with the accessibility of zebrafish (Danio rerio) provides an opportunity to screen for genes involved in vertebrate biological processes. Injection of Cas9-encoding mRNA and an engineered, single guide RNA (sgRNA) can cause biallelic mutations in injected embryos that phenocopy known mutant phenotypes. We found that increasing CRISPR efficiency and multiplexing sgRNAs allowed for phenocopy of known mutants across many phenotypes. We performed a proof-of-concept screen examining 48 loci by intersecting, multiplexed pool injections, and identified two new genes involved in electrical synapse formation. By deep-sequencing target loci we found that 90% of the genes were effectively screened. We conclude that CRISPR can be used as a powerful reverse genetic screening strategy in vivo in a vertebrate system.

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Summary Interactions between tumor cells and tumor-associated macrophages play critical roles in the initiation of tumor cell motility. To capture the cellular interactions of the tumor microenvironment with high-resolution imaging, we directly visualized tumor cells and their interactions with macrophages in zebrafish. Live-imaging in zebrafish revealed that macrophages are dynamic, yet maintain sustained contact with tumor cells. Additionally, the recruitment of macrophages to tumor cells promotes tumor cell dissemination. Using a Cre/LoxP strategy, we found that macrophages transfer cytoplasm to tumor cells in zebrafish and mouse models. Remarkably, macrophage cytoplasmic transfer correlated with melanoma cell dissemination. We further found that macrophages transfer cytoplasm to tumor cells upon cell contact in vitro. Thus, we present a model in which macrophage/tumor cell contact allows for the transfer of cytoplasmic molecules from macrophages to tumor cells corresponding to increased tumor cell motility and dissemination.

Forward genetic screens have elucidated molecular pathways required for innumerable aspects of life; however, identifying the causal mutations from such screens has long been the bottleneck in the process, particularly in vertebrates. We have developed an RNA-seq-based approach that identifies both the region of the genome linked to a mutation and candidate lesions that may be causal for the phenotype of interest. We show that our method successfully identifies zebrafish mutations that cause nonsense or missense changes to codons, alter transcript splicing, or alter gene expression levels. Furthermore, we develop an easily accessible bioinformatics pipeline allowing for implementation of all steps of the method. Overall, we show that RNA-seq is a fast, reliable, and cost-effective method to map and identify mutations that will greatly facilitate the power of forward genetics in vertebrate models.[Supplemental material is available for this article.]Forward genetic screens have illuminated how genes encode the information necessary for life (Crick et al. 1961;Brenner 1974;Nüsslein-Volhard and Wieschaus 1980;Meyerowitz and Pruitt 1985;Haffter et al. 1996;Nolan et al. 2000). However, the subsequent identification of the causal mutation has been the bottleneck in the forward genetics process. Indeed, only one-third of the mutants identified in the first large-scale forward screens undertaken in a vertebrate model (Haffter et al. 1996) have been cloned. This problem has been solved in invertebrate systems through the use of whole-genome sequencing (WGS) of mutant animals to identify candidate genes; the main advantage of both Caenorhabditis elegans and Drosophila is that the genomes are small, animals are isogenic, and chemically induced mutations are rare enough that the changes can be identified and quickly confirmed to be causative (Sarin et al. 2008;Blumenstiel et al. 2009). In the zebrafish, the genome is large (greater than 10 times larger than worm or fly), making it relatively expensive to use WGS. Additionally, the zebrafish genome is highly polymorphic, with each strain, and even each individual, carrying numerous polymorphisms. Thus sequencing a single animal is not sufficient to distinguish potential causative mutations from other polymorphisms. Here we describe an RNA-seq-based bulk segregant analysis (BSA) approach that allows for the inexpensive mapping and identification of candidate mutations from forward genetic screens. While we have used zebrafish as a model, this methodology is applicable to any model system with a sequenced genome.BSA identifies regions of the genome that are linked to a causative mutation in a group of phenotypically mutant animals. This is accomplished by identifying regions of homozygosity within mutants at genetic markers found throughout the genome (Supplemental Fig. 1). BSA using PCR-based testing of infrequent microsatellite markers has been the standard for the initial mapping of zebrafish mutations (Geisler et al. 2007; Zhou and Zon 2011). The approach is both laborious...

Hair cells possess a single primary cilium, called the kinocilium, early in development. While the kinocilium is lost in auditory hair cells of most species it is maintained in vestibular hair cells. It has generally been believed that the primary role of the kinocilium and cilia-associated genes in hair cells is in the establishment of the polarity of actin-based stereocilia, the hair cell mechanotransduction apparatus. Through genetic screening and testing of candidate genes in zebrafish (Danio rerio) we have found that mutations in multiple cilia genes implicated in intraflagellar transport (dync2h1, wdr35, ift88, and traf3ip), and the ciliary transition zone (cc2d2a, mks1, and cep290) lead to resistance to aminoglycoside-induced hair cell death. These genes appear to have differing roles in hair cells, as mutations in intraflagellar transport genes, but not transition zone genes, lead to defects in kinocilia formation and processes dependent upon hair cell mechanotransduction activity. These mutants highlight a novel role of cilia-associated genes in hair cells, and provide powerful tools for further study.