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CRISPR AND Zebrafish

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https://www.readbyqxmd.com/read/29471431/making-waves-new-developments-in-toxicology-with-the-zebrafish
#1
Katharine A Horzmann, Jennifer L Freeman
The laboratory zebrafish (Danio rerio) is now an accepted model in toxicologic research. The zebrafish model fills a niche between in vitro models and mammalian biomedical models. The developmental characteristics of the small fish are strategically being used by scientists to study topics ranging from high-throughput toxicity screens to toxicity in multi- and transgenerational studies. High-throughput technology has increased the utility of zebrafish embryonic toxicity assays in screening of chemicals and drugs for toxicity or effect...
February 19, 2018: Toxicological Sciences: An Official Journal of the Society of Toxicology
https://www.readbyqxmd.com/read/29450543/mutation-in-the-zebrafish-cct2-gene-leads-to-abnormalities-of-cell-cycle-and-cell-death-in-the-retina-a-model-of-cct2-related-leber-congenital-amaurosis
#2
Yuriko Minegishi, Naoki Nakaya, Stanislav I Tomarev
Purpose: The compound heterozygous mutations in the β subunit of chaperonin containing TCP-1 (CCT), encoded by CCT2, lead to the Leber congenital amaurosis (LCA). In this study, a cct2 mutant line of zebrafish was established to investigate the role of CCT2 mutations in LCA in vertebrates. Methods: A cct2 mutant zebrafish line was produced using the CRISPR-Cas9 system. Changes in the eyes of developing wild-type and mutant larvae were monitored using microscopy, immunostaining, TUNEL, and EdU assays...
February 1, 2018: Investigative Ophthalmology & Visual Science
https://www.readbyqxmd.com/read/29450416/a-g-quadruplex-motif-at-the-3-end-of-sgrnas-improves-crispr-cas9-based-genome-editing-efficiency
#3
Smita Nahar, Paras Sehgal, Mohd Azhar, Manish Rai, Amrita Singh, Sridhar Sivasubbu, Debojyoti Chakraborty, Souvik Maiti
Originating as a component of prokaryotic adaptive immunity, the type II CRISPR/Cas9 system has been repurposed for targeted genome editing in various organisms. Although Cas9 can bind and cleave DNA efficiently under in vitro conditions, its activity inside a cell can vary dramatically between targets owing to the differences between genomic loci and the availability of enough Cas9/sgRNA (single guide RNA) complex molecules for cleavage. Most methods have so far relied on Cas9 protein engineering or base modifications in the sgRNA sequence to improve CRISPR/Cas9 activity...
February 16, 2018: Chemical Communications: Chem Comm
https://www.readbyqxmd.com/read/29449560/knockout-of-zebrafish-interleukin-7-receptor-il7r-by-the-crispr-cas9-system-delays-retinal-neurodevelopment
#4
Shijiao Cai, Yang Chen, Yue Shang, Jianlin Cui, Zongjin Li, Yuhao Li
Interleukin 7 receptor (il7r), a transmembrane receptor, belongs to the type I cytokine receptor family. Il7r is involved in the pathogenesis of neurodegenerative disorders, such as multiple sclerosis. Targeted knockdown of il7r leads to delayed myelination, highlighting the potential role of il7r in the development of the nervous system. Zebrafish is an ideal model for the study of neurogenesis; moreover, the il7r gene is highly conserved between zebrafish and human. The aim of the present study was to investigate the novel function of il7r in neurogenesis...
February 15, 2018: Cell Death & Disease
https://www.readbyqxmd.com/read/29445519/highly-efficient-generation-of-knock-in-transgenic-medaka-by-crispr-cas9-mediated-genome-engineering
#5
Ikuko Watakabe, Hisashi Hashimoto, Yukiko Kimura, Saori Yokoi, Kiyoshi Naruse, Shin-Ichi Higashijima
Background: Medaka ( Oryzias latipes ) is a popular animal model used in vertebrate genetic analysis. Recently, an efficient (~ 30%) knock-in system via non-homologous end joining (NHEJ) was established in zebrafish using the CRISPR/Cas9 system. If the same technique were applicable in medaka, it would greatly expand the usefulness of this model organism. The question of the applicability of CRISPR/Cas9 in medaka, however, has yet to be addressed. Results: We report the highly efficient generation of knock-in transgenic medaka via non-homologous end joining (NHEJ)...
2018: Zoological Letters
https://www.readbyqxmd.com/read/29438257/recent-insights-into-vascular-development-from-studies-in-zebrafish
#6
Ryota L Matsuoka, Didier Y R Stainier
PURPOSE OF REVIEW: Zebrafish has provided a powerful platform to study vascular biology over the past 25 years, owing to their distinct advantages for imaging and genetic manipulation. In this review, we summarize recent progress in vascular biology with particular emphasis on vascular development in zebrafish. RECENT FINDINGS: The advent of transcription activator-like effector nuclease and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 genome-editing technologies has dramatically facilitated reverse genetic approaches in zebrafish, as in other models...
February 12, 2018: Current Opinion in Hematology
https://www.readbyqxmd.com/read/29435650/targeted-knock-in-of-creer-t2-in-zebrafish-using-crispr-cas9
#7
Gokul Kesavan, Juliane Hammer, Stefan Hans, Michael Brand
New genome-editing approaches, such as the CRISPR/Cas system, have opened up great opportunities to insert or delete genes at targeted loci and have revolutionized genetics in model organisms like the zebrafish. The Cre-loxp recombination system is widely used to activate or inactivate genes with high spatial and temporal specificity. Using a CRISPR/Cas9-mediated knock-in strategy, we inserted a zebrafish codon-optimized CreER T2 transgene at the otx2 gene locus to generate a conditional Cre-driver line. We chose otx2 as it is a patterning gene of the anterior neural plate that is expressed during early development...
February 12, 2018: Cell and Tissue Research
https://www.readbyqxmd.com/read/29374849/zebrafish-embryonic-slow-muscle-is-a-rapid-system-for-genetic-analysis-of-sarcomere-organization-by-crispr-cas9-but-not-ngago
#8
Mengxin Cai, Yufeng Si, Jianshe Zhang, Zhenjun Tian, Shaojun Du
Zebrafish embryonic slow muscle cells, with their superficial localization and clear sarcomere organization, provide a useful model system for genetic analysis of muscle cell differentiation and sarcomere assembly. To develop a quick assay for testing CRISPR-mediated gene editing in slow muscles of zebrafish embryos, we targeted a red fluorescence protein (RFP) reporter gene specifically expressed in slow muscles of myomesin-3-RFP (Myom3-RFP) zebrafish embryos. We demonstrated that microinjection of RFP-sgRNA with Cas9 protein or Cas9 mRNA resulted in a mosaic pattern in loss of RFP expression in slow muscle fibers of the injected zebrafish embryos...
January 27, 2018: Marine Biotechnology
https://www.readbyqxmd.com/read/29346415/acute-multi-sgrna-knockdown-of-keops-complex-genes-reproduces-the-microcephaly-phenotype-of-the-stable-knockout-zebrafish-model
#9
Tilman Jobst-Schwan, Johanna Magdalena Schmidt, Ronen Schneider, Charlotte A Hoogstraten, Jeremy F P Ullmann, David Schapiro, Amar J Majmundar, Amy Kolb, Kaitlyn Eddy, Shirlee Shril, Daniela A Braun, Annapurna Poduri, Friedhelm Hildebrandt
Until recently, morpholino oligonucleotides have been widely employed in zebrafish as an acute and efficient loss-of-function assay. However, off-target effects and reproducibility issues when compared to stable knockout lines have compromised their further use. Here we employed an acute CRISPR/Cas approach using multiple single guide RNAs targeting simultaneously different positions in two exemplar genes (osgep or tprkb) to increase the likelihood of generating mutations on both alleles in the injected F0 generation and to achieve a similar effect as morpholinos but with the reproducibility of stable lines...
2018: PloS One
https://www.readbyqxmd.com/read/29331378/loss-of-zebrafish-smyd1a-interferes-with-myofibrillar-integrity-without-triggering-the-misfolded-myosin-response
#10
Christoph Paone, Steven Rudeck, Christelle Etard, Uwe Strähle, Wolfgang Rottbauer, Steffen Just
Sarcomeric protein turnover needs to be tightly balanced to assure proper assembly and renewal of sarcomeric units within muscle tissues. The mechanisms regulating these fundamental processes are only poorly understood, but of great clinical importance since many cardiac and skeletal muscle diseases are associated with defective sarcomeric organization. The SET- and MYND domain containing protein 1b (Smyd1b) is known to play a crucial role in myofibrillogenesis by functionally interacting with the myosin chaperones Unc45b and Hsp90α1...
January 10, 2018: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/29330802/microinjection-of-antisense-morpholinos-crispr-cas9-rnp-and-rna-dna-into-zebrafish-embryos
#11
Yi Xin, Cunming Duan
In this chapter, we describe a stepwise protocol of microinjection. Using this method, antisense morpholinos, CRISPR-Cas9 ribonucleoprotein complexes, capped mRNA, and DNA can be delivered into fertilized zebrafish eggs to manipulate gene expression during development. This protocol can also be adapted for microinjection in other fish and amphibian species.
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29295818/comparison-of-various-nuclear-localization-signal-fused-cas9-proteins-and-cas9-mrna-for-genome-editing-in-zebrafish
#12
Peinan Hu, Xueying Zhao, Qinghua Zhang, Weiming Li, Yao Zu
CRISPR/Cas9 system has been proven to be an efficient and precise genome editing technology in various organisms. However, the gene editing efficiencies of Cas9 proteins with a nuclear localization signal (NLS) fused to different termini and Cas9 mRNA have not been systematically compared. Here, we compared the ability of Cas9 proteins with NLS fused to the N, C, or both N and C termini and N-NLS-Cas9-NLS-C mRNA to target two sites in the tyr gene and two sites in the gol gene related to pigmentation in zebrafish...
January 2, 2018: G3: Genes—Genomes—Genetics
https://www.readbyqxmd.com/read/29285825/targeted-copy-number-screening-highlights-an-intragenic-deletion-of-wdr63-as-the-likely-cause-of-human-occipital-encephalocele-and-abnormal-cns-development-in-zebrafish
#13
Wolfgang Hofmeister, Maria Pettersson, Deniz Kurtoglu, Miriam Armenio, Jesper Eisfeldt, Nikos Papadogiannakis, Peter Gustavsson, Anna Lindstrand
Congenital malformations affecting the neural tube can present as isolated malformations or occur in association with other developmental abnormalities and syndromes. Using high resolution copy number screening in 66 fetuses with neural tube defects we identified 6 fetuses with likely pathogenic mutations, three aneuploidies (one trisomy 13 and two trisomy 18) and three deletions previously reported in NTDs (one 22q11.2 deletion and two 1p36 deletions) corresponding to 9% of the cohort. In addition, we identified five rare deletions and two duplications of uncertain significance including a rare intragenic heterozygous in-frame WDR63 deletion in a fetus with occipital encephalocele...
December 28, 2017: Human Mutation
https://www.readbyqxmd.com/read/29243319/zebrafish-rfx4-controls-dorsal-and-ventral-midline-formation-in-the-neural-tube
#14
Irina Sedykh, Abigail N Keller, Baul Yoon, Laura Roberson, Oleg V Moskvin, Yevgenya Grinblat
BACKGROUND: Rfx winged-helix transcription factors, best known as key regulators of core ciliogenesis, also play ciliogenesis-independent roles during neural development. Mammalian Rfx4 controls neural tube morphogenesis via both mechanisms. RESULTS: We set out to identify conserved aspects of rfx4 gene function during vertebrate development and to establish a new genetic model in which to analyze these mechanisms further. To this end, we have generated frame-shift alleles in the zebrafish rfx4 locus using CRISPR/Cas9 mutagenesis...
December 15, 2017: Developmental Dynamics: An Official Publication of the American Association of Anatomists
https://www.readbyqxmd.com/read/29242584/mis-expression-of-grainyhead-like-transcription-factors-in-zebrafish-leads-to-defects-in-enveloping-layer-evl-integrity-cellular-morphogenesis-and-axial-extension
#15
Lee B Miles, Charbel Darido, Jan Kaslin, Joan K Heath, Stephen M Jane, Sebastian Dworkin
The grainyhead-like (grhl) transcription factors play crucial roles in craniofacial development, epithelial morphogenesis, neural tube closure, and dorso-ventral patterning. By utilising the zebrafish to differentially regulate expression of family members grhl2b and grhl3, we show that both genes regulate epithelial migration, particularly convergence-extension (CE) type movements, during embryogenesis. Genetic deletion of grhl3 via CRISPR/Cas9 results in failure to complete epiboly and pre-gastrulation embryonic rupture, whereas morpholino (MO)-mediated knockdown of grhl3 signalling leads to aberrant neural tube morphogenesis at the midbrain-hindbrain boundary (MHB), a phenotype likely due to a compromised overlying enveloping layer (EVL)...
December 14, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29232857/mir-34a-regulates-sperm-motility-in-zebrafish
#16
Wenjie Guo, Binyue Xie, Shuting Xiong, Xufang Liang, Jian-Fang Gui, Jie Mei
Increasing attention has been focused on the role of microRNAs in post-transcription regulation during spermatogenesis. Recently, the miR-34 family has been shown to be involved in the spermatogenesis, but the clear function of the miR-34 family in spermatogenesis is still obscure. Here we analyzed the function of miR-34a, a member of the miR-34 family, during spermatogenesis using miR-34a knockout zebrafish generated by the clustered regularly interspaced short palindromic repeats/associated protein 9 (CRISPR/Cas9) system...
December 10, 2017: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/29229728/cas9-enhances-bacterial-virulence-by-repressing-the-regr-transcriptional-regulator-in-streptococcus-agalactiae
#17
Ke Ma, Qing Cao, Su Luo, Zhaofei Wang, Guangjin Liu, Chengping Lu, Yongjie Liu
Clustered regularly interspaced palindromic repeats (CRISPR) and its associated cas genes have been demonstrated to regulate self-genes and virulence in many pathogens. In this study, we found that inactivation of cas9 caused reduced adhesion and intracellular survival of the piscine Streptococcus agalactiae strain GD201008-001 and significantly decreased the virulence of this strain in zebrafish and mice. Further investigation indicated that the regR transcriptional regulator was upregulated in the Δcas9 mutant...
December 11, 2017: Infection and Immunity
https://www.readbyqxmd.com/read/29222508/crispr-cpf1-mediates-efficient-homology-directed-repair-and-temperature-controlled-genome-editing
#18
Miguel A Moreno-Mateos, Juan P Fernandez, Romain Rouet, Charles E Vejnar, Maura A Lane, Emily Mis, Mustafa K Khokha, Jennifer A Doudna, Antonio J Giraldez
Cpf1 is a novel class of CRISPR-Cas DNA endonucleases, with a wide range of activity across different eukaryotic systems. Yet, the underlying determinants of this variability are poorly understood. Here, we demonstrate that LbCpf1, but not AsCpf1, ribonucleoprotein complexes allow efficient mutagenesis in zebrafish and Xenopus. We show that temperature modulates Cpf1 activity by controlling its ability to access genomic DNA. This effect is stronger on AsCpf1, explaining its lower efficiency in ectothermic organisms...
December 8, 2017: Nature Communications
https://www.readbyqxmd.com/read/29221193/steroidogenic-factor-1-hypermethylation-in-maternal-rat-blood-could-serve-as-a-biomarker-for-intrauterine-growth-retardation
#19
Dong-Mei Wu, Liang-Peng Ma, Gui-Li Song, Yong Long, Han-Xiao Liu, Yang Liu, Jie Ping
Intrauterine growth retardation (IUGR) is a common obstetric complication lacking an optimal method for prenatal screening. DNA methylation profile in maternal blood holds significant promise for prenatal screening. Here, we aimed to screen out potential IUGR biomarkers in maternal blood from the perspective of DNA methylation. The IUGR rat model was established by prenatal maternal undernutrition. High-throughput bisulfite sequencing of genomic DNA methylation followed by functional clustering analysis for differentially methylated region (DMR)-associated genes demonstrated that genes regulating transcription had the most significantly changed DNA methylation status in maternal blood with IUGR...
November 10, 2017: Oncotarget
https://www.readbyqxmd.com/read/29218308/opportunities-for-crispr-cas9-gene-editing-in-retinal-regeneration-research
#20
REVIEW
Leah J Campbell, David R Hyde
While retinal degeneration and disease results in permanent damage and vision loss in humans, the severely damaged zebrafish retina has a high capacity to regenerate lost neurons and restore visual behaviors. Advancements in understanding the molecular and cellular basis of this regeneration response give hope that strategies and therapeutics may be developed to restore sight to blind and visually-impaired individuals. Our current understanding has been facilitated by the amenability of zebrafish to molecular tools, imaging techniques, and forward and reverse genetic approaches...
2017: Frontiers in Cell and Developmental Biology
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