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CRISPR AND Zebrafish

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https://www.readbyqxmd.com/read/28416245/a-simple-and-efficient-method-for-crispr-cas9-induced-mutant-screening
#1
Yufeng Hua, Chun Wang, Jian Huang, Kejian Wang
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system provides a technological breakthrough in mutant generation. Several methods such as the polymerase chain reaction (PCR)/restriction enzyme (RE) assay, T7 endonuclease I (T7EI) assay, Surveyor nuclease assay, PAGE-based genotyping assay, and high-resolution melting (HRM) analysis-based assay have been developed for screening CRISPR/Cas9-induced mutants. However, these methods are time- and labour-intensive and may also be sequence-limited or require very expensive equipment...
April 4, 2017: Journal of Genetics and Genomics, Yi Chuan Xue Bao
https://www.readbyqxmd.com/read/28409342/zebrafish-as-a-model-of-kidney-disease
#2
Elvin E Morales, Rebecca A Wingert
Animal models have been an invaluable means to advance biomedical research as they provide experimental avenues for cellular and molecular investigations of disease pathology. The zebrafish (Danio rerio) is a good alternative to mammalian models that can be used to apply powerful genetic experimental methods normally used in invertebrates to answer questions about vertebrate development and disease. In the case of the kidney, the zebrafish has proven itself to be an applicable and versatile experimental system, mainly due to the simplicity of its pronephros, which contains two nephrons that possess conserved structural and physiological aspects with mammalian nephrons...
2017: Results and Problems in Cell Differentiation
https://www.readbyqxmd.com/read/28398638/a-functional-screening-of-the-kinome-identifies-the-polo-like-kinase-4-as-a-potential-therapeutic-target-for-malignant-rhabdoid-tumors-and-possibly-other-embryonal-tumors-of-the-brain
#3
Simone Treiger Sredni, Mario Suzuki, Jian-Ping Yang, Jacek Topczewski, Anders W Bailey, Tufan Gokirmak, Jeffrey N Gross, Alexandre de Andrade, Akihide Kondo, David R Piper, Tadanori Tomita
PURPOSE: Malignant rhabdoid tumors (MRTs) are deadly embryonal tumors of the infancy. With poor survival and modest response to available therapies, more effective and less toxic treatments are needed. We hypothesized that a systematic screening of the kinome will reveal kinases that drive rhabdoid tumors and can be targeted by specific inhibitors. METHODS: We individually mutated 160 kinases in a well-characterized rhabdoid tumor cell line (MON) using lentiviral clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)...
April 11, 2017: Pediatric Blood & Cancer
https://www.readbyqxmd.com/read/28398516/functional-analysis-of-nuclear-estrogen-receptors-ners-in-zebrafish-reproduction-by-genome-editing-approach
#4
Huijie Lu, Yong Cui, Liwen Jiang, Wei Ge
Estrogens signal through both nuclear and membrane receptors with most reported effects being mediated via the nuclear estrogen receptors (nERs). Although much work has been reported on nERs in the zebrafish, there is a lack of direct genetic evidence for their functional roles and importance in reproduction. To address this issue, we undertook this study to disrupt all three nERs in the zebrafish, namely esr1 (ERα), esr2a (ERβII) and esr2b (ERβI), by genome-editing technology CRISPR/Cas9. Using this loss-of-function genetic approach, we successfully created three mutant zebrafish lines with each nER knocked out...
April 10, 2017: Endocrinology
https://www.readbyqxmd.com/read/28390800/ca-2-release-via-two-pore-channel-type-2-tpc2-is-required-for-slow-muscle-cell-myofibrillogenesis-and-myotomal-patterning-in-intact-zebrafish-embryos
#5
Jeffrey J Kelu, Sarah E Webb, John Parrington, Antony Galione, Andrew L Miller
We recently demonstrated a critical role for two-pore channel type 2 (TPC2)-mediated Ca(2+) release during the differentiation of slow (skeletal) muscle cells (SMC) in intact zebrafish embryos, via the introduction of a translational-blocking morpholino antisense oligonucleotide (MO). Here, we extend our study and demonstrate that knockdown of TPC2 with a non-overlapping splice-blocking MO, knockout of TPC2 (via the generation of a tpcn2(dhkz1a) mutant line of zebrafish using CRISPR/Cas9 gene-editing), or the pharmacological inhibition of TPC2 action with bafilomycin A1 or trans-ned-19, also lead to a significant attenuation of SMC differentiation, characterized by a disruption of SMC myofibrillogenesis and gross morphological changes in the trunk musculature...
April 6, 2017: Developmental Biology
https://www.readbyqxmd.com/read/28364234/blocking-zebrafish-micrornas-with-morpholinos
#6
Alex Sutton Flynt, Mahesh Rao, James G Patton
Antisense morpholino oligonucleotides have been commonly used in zebrafish to inhibit mRNA function, either by inhibiting pre-mRNA splicing or by blocking translation initiation. Even with the advent of genome editing by CRISP/Cas9 technology, morpholinos provide a useful and rapid tool to knockdown gene expression. This is especially true when dealing with multiple alleles and large gene families where genetic redundancy can complicate knockout of all family members. miRNAs are small noncoding RNAs that are often encoded in gene families and can display extensive genetic redundancy...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28350298/oncogenic-braf-disrupts-thyroid-morphogenesis-and-function-via-twist-expression
#7
Viviana Anelli, Jacques A Villefranc, Sagar Chhangawala, Raul Martinez-McFaline, Eleonora Riva, Anvy Nguyen, Akanksha Verma, Rohan Bareja, Zhengming Chen, Theresa Scognamiglio, Olivier Elemento, Yariv Houvras
Thyroid cancer is common, yet the sequence of alterations that promote tumor formation are incompletely understood. Here, we describe a novel model of thyroid carcinoma in zebrafish that reveals temporal changes due to BRAF(V600E). Through the use of real-time in vivo imaging, we observe disruption in thyroid follicle structure that occurs early in thyroid development. Combinatorial treatment using BRAF and MEK inhibitors reversed the developmental effects induced by BRAF(V600E). Adult zebrafish expressing BRAF(V600E) in thyrocytes developed invasive carcinoma...
March 28, 2017: ELife
https://www.readbyqxmd.com/read/28341548/cos2-kif7-and-osm-3-kif17-regulate-onset-of-outer-segment-development-in-zebrafish-photoreceptors-through-distinct-mechanisms
#8
Tylor R Lewis, Sean R Kundinger, Amira L Pavlovich, Jonathan R Bostrom, Brian A Link, Joseph C Besharse
Zebrafish morphants of osm-3/kif17, a kinesin-2 family member and intraflagellar transport motor, have photoreceptor outer segments that are dramatically reduced in number and size. However, two genetic mutant lines, osm-3/kif17(sa0119) and osm-3/kif17(sa18340), reportedly lack any observable morphological outer segment defects. In this work, we use TALENs to generate an independent allele, osm-3/kif17(mw405), and show that both osm-3/kif17(sa0119) and osm-3/kif17(mw405) have an outer segment developmental delay in both size and density that is fully recovered by 6 days post-fertilization...
March 22, 2017: Developmental Biology
https://www.readbyqxmd.com/read/28331970/disease-modeling-in-genetic-kidney-diseases-zebrafish
#9
REVIEW
Heiko Schenk, Janina Müller-Deile, Mark Kinast, Mario Schiffer
Growing numbers of translational genomics studies are based on the highly efficient and versatile zebrafish (Danio rerio) vertebrate model. The increasing types of zebrafish models have improved our understanding of inherited kidney diseases, since they not only display pathophysiological changes but also give us the opportunity to develop and test novel treatment options in a high-throughput manner. New paradigms in inherited kidney diseases have been developed on the basis of the distinct genome conservation of approximately 70 % between zebrafish and humans in terms of existing gene orthologs...
March 22, 2017: Cell and Tissue Research
https://www.readbyqxmd.com/read/28318500/mutations-in-tmem260-cause-a-pediatric-neurodevelopmental-cardiac-and-renal-syndrome
#10
Asaf Ta-Shma, Tahir N Khan, Asaf Vivante, Jason R Willer, Pavle Matak, Chaim Jalas, Ben Pode-Shakked, Yishay Salem, Yair Anikster, Friedhelm Hildebrandt, Nicholas Katsanis, Orly Elpeleg, Erica E Davis
Despite the accelerated discovery of genes associated with syndromic traits, the majority of families affected by such conditions remain undiagnosed. Here, we employed whole-exome sequencing in two unrelated consanguineous kindreds with central nervous system (CNS), cardiac, renal, and digit abnormalities. We identified homozygous truncating mutations in TMEM260, a locus predicted to encode numerous splice isoforms. Systematic expression analyses across tissues and developmental stages validated two such isoforms, which differ in the utilization of an internal exon...
April 6, 2017: American Journal of Human Genetics
https://www.readbyqxmd.com/read/28300641/genome-editing-using-crispr-cas9-based-knock-in-approaches-in-zebrafish
#11
REVIEW
Shahad Albadri, Filippo Del Bene, Céline Revenu
With its variety of applications, the CRISPR/Cas9 genome editing technology has been rapidly evolving in the last few years. In the zebrafish community, knock-out reports are constantly increasing but insertion studies have been so far more challenging. With this review, we aim at giving an overview of the homologous directed repair (HDR)-based knock-in generation in zebrafish. We address the critical points and limitations of the procedure such as cutting efficiency of the chosen single guide RNA, use of cas9 mRNA or Cas9 protein, homology arm size etc...
March 11, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28285134/loss-of-gspt1l-disturbs-the-patterning-of-the-brain-central-arteries-in-zebrafish
#12
Hongcheng Wang, Lingfei Luo, Deqin Yang
The cranial vasculature is crucial for the survival and development of the central nervous system and is closely related to brain pathologies. Characterizations of the underlying mechanisms by which cranial vessels acquire their stereotypic patterning remain to be the key interest in the cerebrovascular research. In this report, we show an interesting zebrafish cq37 mutant displaying aberrant patterning of the central arteries. Genetic mapping results indicate that the gene responsible for cq37 encodes G1 to S phase transition 1, like (Gspt1l) with a nonsense mutation...
April 22, 2017: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/28282619/the-use-of-mrp1-deficient-danio-rerio-zebrafish-embryos-to-investigate-the-role-of-mrp1-in-the-toxicity-of-cadmium-chloride-and-benzo-a-pyrene
#13
Jingjing Tian, Jia Hu, Mingli Chen, Huancai Yin, Peng Miao, Pengli Bai, Jian Yin
Previous studies in our lab have revealed that both P-glycoprotein (Pgp) and multi-resistance associated protein (Mrp) 1 played important roles in the detoxification of heavy metals and polycyclic aromatic hydrocarbon (PAH) in zebrafish embryos. This paper aims to extend this research by using mrp1-deficient model to illustrate the individual function of Mrp1. In this respect, CRISPR/Cas9 system was employed to generate a frameshift mutation in zebrafish mrp1 causing premature translational stops in Mrp1. Significant reduction on the efflux function of Mrps was found in mutant zebrafish embryos, which correlated well with the significantly enhanced accumulation and toxicity of cadmium chloride (CdCl2) and benzo[a]pyrene (BαP), indicating the protective role of the corresponding protein...
March 2, 2017: Aquatic Toxicology
https://www.readbyqxmd.com/read/28281657/a-gcsfr-csf3r-zebrafish-mutant-models-the-persistent-basal-neutrophil-deficiency-of-severe-congenital-neutropenia
#14
Vahid Pazhakh, Sharon Clark, M Cristina Keightley, Graham J Lieschke
Granulocyte colony-stimulating factor (GCSF) and its receptor (GCSFR), also known as CSF3 and CSF3R, are required to maintain normal neutrophil numbers during basal and emergency granulopoiesis in humans, mice and zebrafish. Previous studies identified two zebrafish CSF3 ligands and a single CSF3 receptor. Transient antisense morpholino oligonucleotide knockdown of both these ligands and receptor reduces neutrophil numbers in zebrafish embryos, a technique widely used to evaluate neutrophil contributions to models of infection, inflammation and regeneration...
March 10, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28280001/a-rapid-and-effective-method-for-screening-sequencing-and-reporter-verification-of-engineered-frameshift-mutations-in-zebrafish
#15
Sergey V Prykhozhij, Shelby L Steele, Babak Razaghi, Jason N Berman
Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas9 adaptive immunity against pathogens in bacteria has been adapted for genome editing and applied in zebrafish (Danio rerio) to generate frameshift mutations in protein-coding genes. Although there are methods to detect, quantify and sequence CRISPR/Cas9-induced mutations, identifying mutations in F1 heterozygous fish remains challenging. Additionally, sequencing a mutation and assuming that it causes a frameshift does not prove causality because of possible alternative translation start sites and potential effects of mutations on splicing...
March 9, 2017: Disease Models & Mechanisms
https://www.readbyqxmd.com/read/28274275/whole-exome-sequencing-coupled-with-unbiased-functional-analysis-reveals-new-hirschsprung-disease-genes
#16
Hongsheng Gui, Duco Schriemer, William W Cheng, Rajendra K Chauhan, Guillermo Antiňolo, Courtney Berrios, Marta Bleda, Alice S Brooks, Rutger W W Brouwer, Alan J Burns, Stacey S Cherny, Joaquin Dopazo, Bart J L Eggen, Paola Griseri, Binta Jalloh, Thuy-Linh Le, Vincent C H Lui, Berta Luzón-Toro, Ivana Matera, Elly S W Ngan, Anna Pelet, Macarena Ruiz-Ferrer, Pak C Sham, Iain T Shepherd, Man-Ting So, Yunia Sribudiani, Clara S M Tang, Mirjam C G N van den Hout, Herma C van der Linde, Tjakko J van Ham, Wilfred F J van IJcken, Joke B G M Verheij, Jeanne Amiel, Salud Borrego, Isabella Ceccherini, Aravinda Chakravarti, Stanislas Lyonnet, Paul K H Tam, Maria-Mercè Garcia-Barceló, Robert M W Hofstra
BACKGROUND: Hirschsprung disease (HSCR), which is congenital obstruction of the bowel, results from a failure of enteric nervous system (ENS) progenitors to migrate, proliferate, differentiate, or survive within the distal intestine. Previous studies that have searched for genes underlying HSCR have focused on ENS-related pathways and genes not fitting the current knowledge have thus often been ignored. We identify and validate novel HSCR genes using whole exome sequencing (WES), burden tests, in silico prediction, unbiased in vivo analyses of the mutated genes in zebrafish, and expression analyses in zebrafish, mouse, and human...
March 8, 2017: Genome Biology
https://www.readbyqxmd.com/read/28272984/exogenous-gene-integration-mediated-by-genome-editing-technologies-in-zebrafish
#17
Hitoshi Morita, Kiyohito Taimatsu, Kanoko Yanagi, Atsuo Kawahara
Genome editing technologies, such as transcription activator-like effector nuclease (TALEN) and the clustered regularly interspaced short palindromic repeat (CRISPR)/ CRISPR-associated protein (Cas) systems, can induce DNA double-strand breaks (DSBs) at the targeted genomic locus, leading to frameshift-mediated gene disruption in the process of DSB repair. Recently, the technology-induced DSBs followed by DSB repairs are applied to integrate exogenous genes into the targeted genomic locus in various model organisms...
March 8, 2017: Bioengineered
https://www.readbyqxmd.com/read/28253259/scalable-design-of-paired-crispr-guide-rnas-for-genomic-deletion
#18
Carlos Pulido-Quetglas, Estel Aparicio-Prat, Carme Arnan, Taisia Polidori, Toni Hermoso, Emilio Palumbo, Julia Ponomarenko, Roderic Guigo, Rory Johnson
CRISPR-Cas9 technology can be used to engineer precise genomic deletions with pairs of single guide RNAs (sgRNAs). This approach has been widely adopted for diverse applications, from disease modelling of individual loci, to parallelized loss-of-function screens of thousands of regulatory elements. However, no solution has been presented for the unique bioinformatic design requirements of CRISPR deletion. We here present CRISPETa, a pipeline for flexible and scalable paired sgRNA design based on an empirical scoring model...
March 2017: PLoS Computational Biology
https://www.readbyqxmd.com/read/28217809/-generation-and-phenotype-analysis-of-zebrafish-mutations-of-obesity-related-genes-lepr-and-mc4r
#19
Fei Fei, Shao-Yang Sun, Yu-Xiao Yao, Xu Wang
Obesity has become a severe public health problem across the world, and seriously affects the health and life quality of human beings. Here we generated lepr and mc4r mutant zebrafish via the CRISPR/Cas9 technique, and performed morphological and functional characterizations of those mutants. We observed that there was no significant phenotypic difference between homozygous mutants and wild-type controls before 2.5 months post-fertilization (mpf). However, the adult lepr(-/-) and mc4r(-/-) individuals displayed increased food intake, heavier weight, and higher body fat percentage, the characteristics of obesity phenotypes...
February 25, 2017: Sheng Li Xue Bao: [Acta Physiologica Sinica]
https://www.readbyqxmd.com/read/28181494/microenvironment-derived-factors-driving-metastatic-plasticity-in-melanoma
#20
Isabella S Kim, Silja Heilmann, Emily R Kansler, Yan Zhang, Milena Zimmer, Kajan Ratnakumar, Robert L Bowman, Theresa Simon-Vermot, Myles Fennell, Ralph Garippa, Liang Lu, William Lee, Travis Hollmann, Joao B Xavier, Richard M White
Cellular plasticity is a state in which cancer cells exist along a reversible phenotypic spectrum, and underlies key traits such as drug resistance and metastasis. Melanoma plasticity is linked to phenotype switching, where the microenvironment induces switches between invasive/MITF(LO) versus proliferative/MITF(HI) states. Since MITF also induces pigmentation, we hypothesize that macrometastatic success should be favoured by microenvironments that induce a MITF(HI)/differentiated/proliferative state. Zebrafish imaging demonstrates that after extravasation, melanoma cells become pigmented and enact a gene expression program of melanocyte differentiation...
February 9, 2017: Nature Communications
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