Efficient Gene Targeting in Zebrafish Mediated by a Zebrafish-Codon-Optimized Cas9 and Evaluation of Off-Targeting Effect

Liu, Da; Wang, Zhanxiang; Xiao, An; Zhang, Yutian; Li, Wenyuan; Zu, Yao; Yao, Su; Lin, Shuo; Zhang, Bo

doi:10.1016/j.jgg.2013.11.004

Cited by 76 publications

(60 citation statements)

References 14 publications

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“…We focused on ribosomal protein genes because they were the most significantly overrepresented KEGG pathway (Table 2), and they contribute to neural crest development [34]. We selected three ribosomal proteins having significantly reduced expression in alcohol-exposed cells and that are linked to facial deficits in Diamond-Blackfan anemia (RPL5; zrpl5a , RPL11/TCEB3; zrpl11 ) [34] or are known to affect facial development (RPS3A; zrps3a ) [27]; the zrpl3a and zrpl11 morphants recapitulate their transgenic insertional or CRISPR mutation [35–37]. To test for gene-alcohol interactions, we employed concentrations of morpholinos and alcohol that, when administered individually, had no or modest effects on craniofacial development.…”

Section: Resultsmentioning

confidence: 99%

“…Reductions in three ribosomal protein genes sensitized the embryos to alcohol-induced apoptosis, facial malformations, and cardiac irregularities. Although the use of morpholinos for gene knockdown is a limitation of this work due to a potential for off-target effects [29], we observed this gene-environment interaction at hypomorphic morpholino concentrations that in themselves caused only modest facial alterations [27,35–37]. Identification of the ribosome protein enrichment pathway is also consistent with a recent genetic screen that identified methionine tRNA synthase (MARS) loss-of-function as enhancing alcohol vulnerability [17].…”

Section: Discussionmentioning

confidence: 99%

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Transcriptome Profiling Identifies Ribosome Biogenesis as a Target of Alcohol Teratogenicity and Vulnerability during Early Embryogenesis

et al. 2017

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Fetal alcohol spectrum disorder (FASD) is a leading cause of neurodevelopmental disability. Individuals with FASD may exhibit a characteristic facial appearance that has diagnostic utility. The mechanism by which alcohol disrupts craniofacial development is incompletely understood, as are the genetic factors that can modify individual alcohol vulnerability. Using an established avian model, we characterized the cranial transcriptome in response to alcohol to inform the mechanism underlying these cells’ vulnerability. Gallus gallus embryos having 3–6 somites were exposed to 52 mM alcohol and the cranial transcriptomes were sequenced thereafter. A total of 3422 genes had significantly differential expression. The KEGG pathways with the greatest enrichment of differentially expressed gene clusters were Ribosome (P = 1.2 x 10−17, 67 genes), Oxidative Phosphorylation (P = 4.8 x 10−12, 60 genes), RNA Polymerase (P = 2.2 x 10−3, 15 genes) and Spliceosome (P = 2.6 x 10−2, 39 genes). The preponderance of transcripts in these pathways were repressed in response to alcohol. These same gene clusters also had the greatest altered representation in our previous comparison of neural crest populations having differential vulnerability to alcohol-induced apoptosis. Comparison of differentially expressed genes in alcohol-exposed (3422) and untreated, alcohol-vulnerable (1201) transcriptomes identified 525 overlapping genes of which 257 have the same direction of transcriptional change. These included 36 ribosomal, 25 oxidative phosphorylation and 7 spliceosome genes. Using a functional approach in zebrafish, partial knockdown of ribosomal proteins zrpl11, zrpl5a, and zrps3a individually heightened vulnerability to alcohol-induced craniofacial deficits and increased apoptosis. In humans, haploinsufficiency of several of the identified ribosomal proteins are causative in craniofacial dysmorphologies such as Treacher Collins Syndrome and Diamond-Blackfan Anemia. This work suggests ribosome biogenesis may be a novel target mediating alcohol’s damage to developing neural crest. Our findings are consistent with observations that gene-environment interactions contribute to vulnerability in FASD.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Transcriptome Profiling Identifies Ribosome Biogenesis as a Target of Alcohol Teratogenicity and Vulnerability during Early Embryogenesis

et al. 2017

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“…6A; Mali et al, 2013). This 19-bp region includes four single-nucleotide polymorphisms (SNPs) that distinguish mGFP5 from eGFP, with two of these SNPs included in the critical 39 region of the seed adjacent to the PAM (Cong et al, 2013;Jiang et al, 2013;Liu et al, 2013). This sgRNA was transformed into SlSCRpromGFP5 using A. rhizogenes.…”

Section: Optimization Of Intact and Trap For Tomatomentioning

confidence: 99%

Hairy Root Transformation Using Agrobacterium rhizogenes as a Tool for Exploring Cell Type-Specific Gene Expression and Function Using Tomato as a Model

et al. 2014

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ORCID ID: 0000-0002-8568-7125 (J.B-S.).Agrobacterium rhizogenes (or Rhizobium rhizogenes) is able to transform plant genomes and induce the production of hairy roots. We describe the use of A. rhizogenes in tomato (Solanum spp.) to rapidly assess gene expression and function. Gene expression of reporters is indistinguishable in plants transformed by Agrobacterium tumefaciens as compared with A. rhizogenes. A root cell type-and tissue-specific promoter resource has been generated for domesticated and wild tomato (Solanum lycopersicum and Solanum pennellii, respectively) using these approaches. Imaging of tomato roots using A. rhizogenes coupled with laser scanning confocal microscopy is facilitated by the use of a membrane-tagged protein fused to a red fluorescent protein marker present in binary vectors. Tomatooptimized isolation of nuclei tagged in specific cell types and translating ribosome affinity purification binary vectors were generated and used to monitor associated messenger RNA abundance or chromatin modification. Finally, transcriptional reporters, translational reporters, and clustered regularly interspaced short palindromic repeats-associated nuclease9 genome editing demonstrate that SHORT-ROOT and SCARECROW gene function is conserved between Arabidopsis (Arabidopsis thaliana) and tomato.

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“…The Cas9 and guide RNA (gRNA) plasmids were a generous gift from Bo Zhang of Perking University. These two plasmids were described previously (18,19). For gRNA vector construction, gRNA oligonucleotides targeting the protein-encoding exon of the zebrafish DHX33 gene (ENSDART00000156224) was selected using the ChopChop CRISPR/gRNA algorithm (http://chopchop.cbu.uib.no/).…”

Section: Methodsmentioning

confidence: 99%

DHX33 Transcriptionally Controls Genes Involved in the Cell Cycle

Yuan

Wang

Fan

et al. 2016

Molecular and Cellular Biology

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The RNA helicase DHX33 has been shown to be a critical regulator of cell proliferation and growth. However, the underlying mechanisms behind DHX33 function remain incompletely understood. We present original evidence in multiple cell lines that DHX33 transcriptionally controls the expression of genes involved in the cell cycle, notably cyclin, E2F1, cell division cycle (CDC), and minichromosome maintenance (MCM) genes. DHX33 physically associates with the promoters of these genes and controls the loading of active RNA polymerase II onto these promoters. DHX33 deficiency abrogates cell cycle progression and DNA replication and leads to cell apoptosis. In zebrafish, CRISPR-mediated knockout of DHX33 results in downregulation of cyclin A2, cyclin B2, cyclin D1, cyclin E2, cdc6, cdc20, E2F1, and MCM complexes in DHX33 knockout embryos. Additionally, we found the overexpression of DHX33 in a subset of non-small-cell lung cancers and in Ras-mutated human lung cancer cell lines. Forced reduction of DHX33 in these cancer cells abolished tumor formation in vivo. Our study demonstrates for the first time that DHX33 acts as a direct transcriptional regulator to promote cell cycle progression and plays an important role in driving cell proliferation during both embryo development and tumorigenesis.

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Efficient Gene Targeting in Zebrafish Mediated by a Zebrafish-Codon-Optimized Cas9 and Evaluation of Off-Targeting Effect

Cited by 76 publications

References 14 publications

Transcriptome Profiling Identifies Ribosome Biogenesis as a Target of Alcohol Teratogenicity and Vulnerability during Early Embryogenesis

Transcriptome Profiling Identifies Ribosome Biogenesis as a Target of Alcohol Teratogenicity and Vulnerability during Early Embryogenesis

Hairy Root Transformation Using Agrobacterium rhizogenes as a Tool for Exploring Cell Type-Specific Gene Expression and Function Using Tomato as a Model

DHX33 Transcriptionally Controls Genes Involved in the Cell Cycle

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