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CRISPR AND Zebrafish

Satoshi Ota, Kiyohito Taimatsu, Kanoko Yanagi, Tomohiro Namiki, Rie Ohga, Shin-Ichi Higashijima, Atsuo Kawahara
The CRISPR/Cas9 complex, which is composed of a guide RNA (gRNA) and the Cas9 nuclease, is useful for carrying out genome modifications in various organisms. Recently, the CRISPR/Cas9-mediated locus-specific integration of a reporter, which contains the Mbait sequence targeted using Mbait-gRNA, the hsp70 promoter and the eGFP gene, has allowed the visualization of the target gene expression. However, it has not been ascertained whether the reporter integrations at both targeted alleles cause loss-of-function phenotypes in zebrafish...
October 11, 2016: Scientific Reports
Charles E Vejnar, Miguel A Moreno-Mateos, Daniel Cifuentes, Ariel A Bazzini, Antonio J Giraldez
This protocol describes how to generate and genotype mutants using an optimized CRISPR-Cas9 genome-editing system in zebrafish (CRISPRscan). Because single guide RNAs (sgRNAs) have variable efficiency when targeting specific loci, our protocol starts by explaining how to use the web tool CRISPRscan to design highly efficient sgRNAs. The CRISPRscan algorithm is based on the results of an integrated analysis of more than 1000 sgRNAs in zebrafish, which uncovered highly predictive factors that influence Cas9 activity...
October 3, 2016: Cold Spring Harbor Protocols
Irina Sedykh, Jessica J TeSlaa, Rose L Tatarsky, Abigail N Keller, Kimberly A Toops, Aparna Lakkaraju, Molly K Nyholm, Marc A Wolman, Yevgenya Grinblat
Cilia are cell surface organelles with key roles in a range of cellular processes, including generation of fluid flow by motile cilia. The axonemes of motile cilia and immotile kinocilia contain 9 peripheral microtubule doublets, a central microtubule pair, and 9 connecting radial spokes. Aberrant radial spoke components RSPH1, 3, 4a and 9 have been linked with primary ciliary dyskinesia (PCD), a disorder characterized by ciliary dysmotility; yet, radial spoke functions remain unclear. Here we show that zebrafish Rsph9 is expressed in cells bearing motile cilia and kinocilia, and localizes to both 9 + 2 and 9 + 0 ciliary axonemes...
September 30, 2016: Scientific Reports
Shao-Lin Xie, Wan-Ping Bian, Chao Wang, Muhammad Junaid, Ji-Xing Zou, De-Sheng Pei
Contemporary improvements in the type II clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system offer a convenient way for genome editing in zebrafish. However, the low efficiencies of genome editing and germline transmission require a time-intensive and laborious screening work. Here, we reported a method based on in vitro oocyte storage by injecting oocytes in advance and incubating them in oocyte storage medium to significantly improve the efficiencies of genome editing and germline transmission by in vitro fertilization (IVF) in zebrafish...
September 29, 2016: Scientific Reports
Yuyan Wei, Kai Li, Shaohua Yao, Junping Gao, Jun Li, Yanna Shang, Jie Zhang, Le Zhang, Yanyan Li, Xianming Mo, Wentong Meng, Rong Xiang, Jiankun Hu, Ping Lin, Yuquan Wei
Human zinc finger protein 32 (ZNF32) is a Cys2-His2 zinc-finger transcription factor that plays an important role in cell fate, yet much of its function remains unknown. Here, we reveal that the zebrafish ZNF32 homologue zfZNF32 is expressed in the nervous system, particularly in the lateral line system. ZfZNF32 knock-out zebrafish (zfZNF-/-) were generated using the CRISPR-associated protein 9 system. We found that the regenerative capacity of the lateral line system was increased in zfZNF-/- upon hair cell damage compared with the wild type...
September 8, 2016: Oncotarget
Yubao Cui, Lili Yu
The clustered regularly-interspaced short palindromic repeats (CRISPR) structural family functions as an acquired immune system in prokaryotes. Gene editing techniques have co-opted CRISPR and the associated Cas nucleases to allow for the precise genetic modification of human cells, zebrafish, mice, and other eukaryotes. Indeed, this approach has been used to induce a variety of modifications including directed insertion/deletion (InDel) of bases, gene knock-in, introduction of mutations in both alleles of a target gene, and deletion of small DNA fragments...
December 2016: Parasitology International
Anand Narayanan, Guillermina Hill-Teran, Albertomaria Moro, Emma Ristori, Dionna M Kasper, Christine A Roden, Jun Lu, Stefania Nicoli
A large number of microRNAs (miRNAs) are grouped into families derived from the same phylogenetic ancestors. miRNAs within a family often share the same physiological functions despite differences in their primary sequences, secondary structures, or chromosomal locations. Consequently, the generation of animal models to analyze the activity of miRNA families is extremely challenging. Using zebrafish as a model system, we successfully provide experimental evidence that a large number of miRNAs can be simultaneously mutated to abrogate the activity of an entire miRNA family...
2016: Scientific Reports
Charles G Jennings, Rogier Landman, Yang Zhou, Jitendra Sharma, Julia Hyman, J Anthony Movshon, Zilong Qiu, Angela C Roberts, Anna Wang Roe, Xiaoqin Wang, Huihui Zhou, Liping Wang, Feng Zhang, Robert Desimone, Guoping Feng
Molecular genetic tools have had a profound impact on neuroscience, but until recently their application has largely been confined to a few model species, most notably mouse, zebrafish, Drosophila melanogaster and Caenorhabditis elegans. With the development of new genome engineering technologies such as CRISPR, it is becoming increasingly feasible to apply these molecular tools in a wider range of species, including nonhuman primates. This will lead to many opportunities for brain research, but it will also pose challenges...
August 26, 2016: Nature Neuroscience
Tetsuya Nakamura, Andrew R Gehrke, Justin Lemberg, Julie Szymaszek, Neil H Shubin
Understanding the evolutionary transformation of fish fins into tetrapod limbs is a fundamental problem in biology. The search for antecedents of tetrapod digits in fish has remained controversial because the distal skeletons of limbs and fins differ structurally, developmentally, and histologically. Moreover, comparisons of fins with limbs have been limited by a relative paucity of data on the cellular and molecular processes underlying the development of the fin skeleton. Here, we provide a functional analysis, using CRISPR/Cas9 and fate mapping, of 5' hox genes and enhancers in zebrafish that are indispensable for the development of the wrists and digits of tetrapods...
August 17, 2016: Nature
R Kreutz, A Schulz, A M Meyer, T Lorenzen, M Stoll, D Panakova
OBJECTIVE: The Munich Wistar Frömter (MWF) rat represents a rat model for chronic kidney disease (CKD) with the development of albuminuria, hypertension and structural kidney damage. By genomic analysis in MWF we have recently identified a novel candidate gene for albuminuria, i.e. transmembrane protein 63c (tmem63c). We set out to test the relevance of tmem63c by analysing its role for the functional integrity of the glomerular filtration barrier (GFB) in zebrafish. DESIGN AND METHOD: The transgenic zebrafish line Tg(fabp10a:gc-EGFP) expressing the vitamin D binding protein tagged with enhanced green fluorescent protein (EGFP) was used for analysis of GFB function...
September 2016: Journal of Hypertension
Éric Samarut, Alexandra Lissouba, Pierre Drapeau
BACKGROUND: The CRISPR/Cas9 system has become a regularly used tool for editing the genome of many model organisms at specific sites. However, two limiting steps arise in the process of validating guide RNA target sites in larvae and adults: the time required to identify indels and the cost associated with identifying potential mutant animals. RESULTS: Here we have combined and optimized the HotSHOT genomic DNA extraction technique with a two-steps Evagreen PCR, followed by a high-resolution melting (HRM) assay, which facilitates rapid identification of CRISPR-induced indels...
2016: BMC Genomics
A Willis, M Mazon-Moya, S Mostowy
The zebrafish (Danio rerio) is an important animal model to study cell biology in vivo. Benefits of the zebrafish include a fully annotated reference genome, an easily manipulable genome (for example, by morpholino oligonucleotide or CRISPR-Cas9), and transparent embryos for noninvasive, real-time microscopy using fluorescent transgenic lines. Zebrafish have orthologues of most human septins, and studies using larvae were used to investigate the role of septins in vertebrate development. The zebrafish larva is also an established model to study the cell biology of infection and has recently been used to visualize septin assembly during bacterial infection in vivo...
2016: Methods in Cell Biology
An Xiao, Bo Zhang
Using TALEN or CRISPR/Cas system to induce small indels into coding sequences has been implicated in broad applications for reverse genetic studies of many organisms including zebrafish. However, complete deletion of a large gene or noncoding gene(s) or removing a large genomic fragment spanning several genes or other chromosomal elements is preferred in various cases, as well as inducing chromosomal inversions. Here, we describe the detailed protocols for the generation of chromosomal deletion mutations mediated by Cas9 and a pair of gRNAs and the evaluation for the efficiencies in F0 founder fish and of germline transmission...
2016: Methods in Molecular Biology
Satoshi Ota, Atsuo Kawahara
The recent remarkable innovation of an RNA-guided nuclease system, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system, enables us the modification of specific genomic loci in various model animals including zebrafish. With this system, multiple guide RNAs simultaneously injected with the Cas9 nuclease into zebrafish embryos cause multiple genome modifications at different genomic loci with high efficiency; therefore, a simple method to detect individual mutations at distinct loci is desired...
2016: Methods in Molecular Biology
Thomas O Auer, Filippo Del Bene
Targeting nucleases like zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) system have revolutionized genome-editing possibilities in many model organisms. They allow the generation of loss-of-function alleles by the introduction of double-strand breaks at defined sites within genes, but also more sophisticated genome-editing approaches have become possible. These include the integration of donor plasmid DNA into the genome by homology-independent repair mechanisms after CRISPR/Cas9-mediated cleavage...
2016: Methods in Molecular Biology
Alvin C H Ma, Yi Chen, Patrick R Blackburn, Stephen C Ekker
Transcription activator-like effectors (TALEs) are important genomic tools with customizable DNA-binding motifs for locus-specific modifications. In particular, TALE nucleases or TALENs have been successfully used in the zebrafish model system to introduce targeted mutations via repair of double-stranded breaks (DSBs) either through nonhomologous end joining (NHEJ) or by homology-directed repair (HDR) and homology-independent repair in the presence of a donor template. Compared with other customizable nucleases, TALENs offer high binding specificity and fewer sequence constraints in targeting the genome, with comparable mutagenic activity...
2016: Methods in Molecular Biology
Annekatrien Boel, Woutert Steyaert, Nina De Rocker, Björn Menten, Bert Callewaert, Anne De Paepe, Paul Coucke, Andy Willaert
Targeted mutagenesis by the CRISPR/Cas9 system is currently revolutionizing genetics. The ease of this technique has enabled genome engineering in-vitro and in a range of model organisms and has pushed experimental dimensions to unprecedented proportions. Due to its tremendous progress in terms of speed, read length, throughput and cost, Next-Generation Sequencing (NGS) has been increasingly used for the analysis of CRISPR/Cas9 genome editing experiments. However, the current tools for genome editing assessment lack flexibility and fall short in the analysis of large amounts of NGS data...
2016: Scientific Reports
D G Howe, Y M Bradford, A Eagle, D Fashena, K Frazer, P Kalita, P Mani, R Martin, S T Moxon, H Paddock, C Pich, S Ramachandran, L Ruzicka, K Schaper, X Shao, A Singer, S Toro, C Van Slyke, M Westerfield
The Zebrafish Model Organism Database (ZFIN; serves as the central repository for genetic and genomic data produced using zebrafish (Danio rerio). Data in ZFIN are either manually curated from peer-reviewed publications or submitted directly to ZFIN from various data repositories. Data types currently supported include mutants, transgenic lines, DNA constructs, gene expression, phenotypes, antibodies, morpholinos, TALENs, CRISPRs, disease models, movies, and images. The rapidly changing methods of genomic science have increased the production of data that cannot readily be represented in standard journal publications...
2016: Methods in Cell Biology
A Felker, C Mosimann
Spatiotemporal transgene regulation by transgenic DNA recombinases is a central tool for reverse genetics in multicellular organisms, with excellent applications for misexpression and lineage tracing experiments. One of the most widespread technologies for this purpose is Cre recombinase-controlled lox site recombination that is attracting increasing interest in the zebrafish field. Tol2-mediated zebrafish transgenesis provides a stable platform to integrate lox cassette transgenes, while the amenability of the zebrafish embryo to drug treatments makes the model an ideal candidate for tamoxifen-inducible CreERT2 experiments...
2016: Methods in Cell Biology
J Ablain, L I Zon
The zebrafish has been a powerful model in forward genetic screens to identify genes essential for organogenesis and embryonic development. Conversely, using reverse genetics to investigate specific gene function requires phenotypic analysis of complete gene inactivation. Despite the availability and efficacy of morpholinos, the lack of tractable and efficient knockout technologies has impeded reverse genetic studies in the zebrafish, particularly in adult animals. The recent development of genome-editing technologies such as CRISPR/Cas9 greatly widened the scope of loss-of-function studies in the zebrafish, allowing for the rapid phenotypic assessment of gene silencing in embryos, the generation of knockout lines, and large-scale reverse genetic screens...
2016: Methods in Cell Biology
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