A Large-Scale Zebrafish Gene Knockout Resource for the Genome-Wide Study of Gene Function

With the completion of the zebrafish genome sequencing project, it becomes possible to analyze the function of zebrafish genes in a systematic way. The first step in such an analysis is to inactivate each protein-coding gene by targeted or random mutation. Here we describe a streamlined pipeline using proviral insertions coupled with high-throughput sequencing and mapping technologies to widely mutagenize genes in the zebrafish genome. We also report the first 6144 mutagenized and archived F1’s predicted to carry up to 3776 mutations in annotated genes. Using in vitro fertilization, we have rescued and characterized ~0.5% of the predicted mutations, showing mutation efficacy and a variety of phenotypes relevant to both developmental processes and human genetic diseases. Mutagenized fish lines are being made freely available to the public through the Zebrafish International Resource Center. These fish lines establish an important milestone for zebrafish genetics research and should greatly facilitate systematic functional studies of the vertebrate genome

The Vertebrate Codex Gene Breaking Protein Trap Library For Genomic Discovery and Disease Modeling Applications

The zebrafish is a powerful model to explore the molecular genetics and expression of the vertebrate genome. The gene break transposon (GBT) is a unique insertional mutagen that reports the expression of the tagged member of the proteome while generating Cre-revertible genetic alleles. This 1000+ locus collection represents novel codex expression data from the illuminated mRFP protein trap, with 36% and 87% of the cloned lines showcasing to our knowledge the first described expression of these genes at day 2 and day 4 of development, respectively. Analyses of 183 molecularly characterized loci indicate a rich mix of genes involved in diverse cellular processes from cell signaling to DNA repair. The mutagenicity of the GBT cassette is very high as assessed using both forward and reverse genetic approaches. Sampling over 150 lines for visible phenotypes after 5dpf shows a similar rate of discovery of embryonic phenotypes as ENU and retroviral mutagenesis. Furthermore, five cloned insertions were in loci with previously described phenotypes; embryos homozygous for each of the corresponding GBT alleles displayed strong loss of function phenotypes comparable to published mutants using other mutagenesis strategies (ryr1b, fras1, tnnt2a, edar and hmcn1). Using molecular assessment after positional cloning, to date nearly all alleles cause at least a 99+% knockdown of the tagged gene. Interestingly, over 35% of the cloned loci represent 68 mutants in zebrafish orthologs of human disease loci, including nervous, cardiovascular, endocrine, digestive, musculoskeletal, immune and integument systems. The GBT protein trapping system enabled the construction of a comprehensive protein codex including novel expression annotation, identifying new functional roles of the vertebrate genome and generating a diverse collection of potential models of human disease

Genome engineering using DNA-binding proteins: zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs)

Over the past two decades, research groups in both academia and private industry have developed key technologies, including viral delivery vectors and engineered transposon-based or zinc finger protein-based nucleases, towards achieving the long-sought goal of therapeutic genome editing in humans. To date, Zinc Finger Nucleases (ZFNs) have been the most promising reagents for potential therapeutic applications in humans, but the recently characterized Transcription Activator Like Effector (TALE) proteins may soon change this status quo. Although it remains to be seen whether nucleases based on these proteins (TALENs) will be as broadly applicable and effective as ZFNs, based on initial reports, TALENs look very promising. Currently, the primary advantage of TALENs is that the DNA binding code for TALENs appears to be simple and robust, making their synthesis relatively simple. In this dissertation, I summarize advances made in the field of genome editing over the past decade and compare and contrast the currently available tools, focusing on ZFNs and TALENs. Specifically, I describe our efforts to make ZFN technology more accessible by designing and implementing models to help researchers choose target sites that are most amenable to targeting using ZFNs. Also, to help explore the potential of TALENs as tools for genome editing, I describe the development of a simple protocol to aid in constructing TALENs. As ZFNs become easier to use, and TALENs become more robust, the use of genome editing techniques as therapeutics appears poised to become reality in the near future

Regulation of zebrafish sleep and arousal states: current and prospective approaches

Every day, we shift among various states of sleep and arousal to meet the many demands of our bodies and environment. A central puzzle in neurobiology is how the brain controls these behavioral states, which are essential to an animal's well-being and survival. Mammalian models have predominated sleep and arousal research, although in the past decade, invertebrate models have made significant contributions to our understanding of the genetic underpinnings of behavioral states. More recently, the zebrafish has emerged as a promising model system for sleep and arousal research. Here we review experimental evidence that the zebrafish, a diurnal vertebrate, exhibits fundamental behavioral and neurochemical characteristics of mammalian sleep and arousal. We also propose how specific advantages of the zebrafish can be harnessed to advance the field. These include tractable genetics to identify and manipulate molecular and cellular regulators of behavioral states, optical transparency to facilitate in vivo observation of neural structure and function, and amenability to high-throughput drug screens to discover novel therapies for neurological disorders

Building the vertebrate codex using the gene breaking protein trap library

One key bottleneck in understanding the human genome is the relative under-characterization of 90% of protein coding regions. We report a collection of 1200 transgenic zebrafish strains made with the gene-break transposon (GBT) protein trap to simultaneously report and reversibly knockdown the tagged genes. Protein trap-associated mRFP expression shows previously undocumented expression of 35% and 90% of cloned genes at 2 and 4 days post-fertilization, respectively. Further, investigated alleles regularly show 99% gene-specific mRNA knockdown. Homozygous GBT animals in ryr1b, fras1, tnnt2a, edar and hmcn1 phenocopied established mutants. 204 cloned lines trapped diverse proteins, including 64 orthologs of human disease-associated genes with 40 as potential new disease models. Severely reduced skeletal muscle Ca2+ transients in GBT ryr1b homozygous animals validated the ability to explore molecular mechanisms of genetic diseases. This GBT system facilitates novel functional genome annotation towards understanding cellular and molecular underpinnings of vertebrate biology and human disease

Defined Single-Gene and Multi-Gene Deletion Mutant Collections in Salmonella enterica sv Typhimurium

Artículo de publicación ISIWe constructed two collections of targeted single gene deletion (SGD) mutants and two collections of targeted multi-gene deletion (MGD) mutants in Salmonella enterica sv Typhimurium 14028s. The SGD mutant collections contain (1), 3517 mutants in which a single gene is replaced by a cassette containing a kanamycin resistance (KanR) gene oriented in the sense direction (SGD-K), and (2), 3376 mutants with a chloramphenicol resistance gene (CamR) oriented in the antisense direction (SGD-C). A combined total of 3773 individual genes were deleted across these SGD collections. The MGD collections contain mutants bearing deletions of contiguous regions of three or more genes and include (3), 198 mutants spanning 2543 genes replaced by a KanR cassette (MGD-K), and (4), 251 mutants spanning 2799 genes replaced by a CamR cassette (MGD-C). Overall, 3476 genes were deleted in at least one MGD collection. The collections with different antibiotic markers permit construction of all viable combinations of mutants in the same background. Together, the libraries allow hierarchical screening of MGDs for different phenotypic followed by screening of SGDs within the target MGD regions. The mutants of these collections are stored at BEI Resources (www.beiresources.org) and publicly available

The mouse genetics toolkit: revealing function and mechanism

Large-scale projects are providing rapid global access to a wealth of mouse genetic resources to help discover disease genes and to manipulate their function

Applications of machine learning to solve biological puzzles

The era of “big data” has led to the generation of more biological data than any human could hope to process. This flood of data has necessitated the development of computational methods to assist in analysis, and has made it possible to begin to model complex biological systems. Machine learning methods represent one avenue for modeling, and allow for the identification of intricate and often cryptic sequence signals underlying many biological processes. In this dissertation, I present two machine learning models, RPIDisorder and MEDJED, which were developed to predict RNA-protein interaction partners (RPIPs) and DNA double-strand break (DSB) repair by the microhomology-mediated end joining (MMEJ) pathway, respectively. I also present the Gene Sculpt Suite, a set of freely available web-based software tools for precision gene editing. RPIDisorder uses signals from protein and RNA sequences (some of which have been previously utilized in published RNA-protein partner prediction methods), but it additionally exploits signal from disordered protein regions to predict interactions with greater specificity than has been possible before. RPIDisorder allows for the prediction of biologically relevant RNA-protein interaction networks, which in turn can assist in the development of clinical interventions for the numerous cancers and neurological and metabolic disorders associated with disruptions in RNA-protein interactions. RPIDisorder is freely available at www.rpidisorder.org. MEDJED (Microhomology-Evoked Deletion Judication EluciDation) uses signal within and surrounding short stretches of homologous DNA sequence (microhomologies) on either side of an introduced DSB to predict the extent to which a targeted genomic site will be repaired using the MMEJ pathway. MEDJED is freely available at www.genesculpt.org/medjed/. The advent of gene editing nucleases including CRISPR/Cas systems, TALENs, and zinc finger nucleases has made it possible to insert, delete, and precisely edit DNA. A great deal of recent research has focused on improving the efficiency and precision of these nucleases by leveraging endogenous DSB repair pathways including non-homologous end joining (NHEJ) and homologous recombination (HR). However, homology-mediated end joining pathways (HMEJ), including MMEJ and single-strand annealing (SSA), provide many advantages over NHEJ and HR. The Gene Sculpt Suite is a set of three web-based tools (GTagHD, MEDJED, and MENTHU) that leverage HMEJ pathways to enhance exogenous DNA knock-in (GTagHD) and produce more efficient and precise gene knock-outs (MEDJED and MENTHU). The Gene Sculpt Suite is freely available at www.genesculpt.org. Taken together, the results of these studies demonstrate that machine learning models can be valuable for identifying sequence signals that regulate macromolecular recognition, with numerous potential applications in both basic and applied research