In this study, we used comparative genomics and developmental genetics to identify epigenetic regulators driving oncogenesis in a zebrafish retinoblastoma 1 (rb1) somatic-targeting model of RB1 mutant embryonal brain tumors. Zebrafish rb1 brain tumors caused by TALEN or CRISPR targeting are histologically similar to human central nervous system primitive neuroectodermal tumors (CNS-PNETs). Like the human oligoneural OLIG2+/SOX10+ CNS-PNET subtype, zebrafish rb1 tumors show elevated expression of neural progenitor transcription factors olig2, sox10, sox8b and the receptor tyrosine kinase erbb3a oncogene. Comparison of rb1 tumor and rb1/rb1 germline mutant larval transcriptomes shows that the altered oligoneural precursor signature is specific to tumor tissue. More than 170 chromatin regulators were differentially expressed in rb1 tumors, including overexpression of chromatin remodeler components histone deacetylase 1 (hdac1) and retinoblastoma binding protein 4 (rbbp4). Germline mutant analysis confirms that zebrafish rb1, rbbp4 and hdac1 are required during brain development. rb1 is necessary for neural precursor cell cycle exit and terminal differentiation, rbbp4 is required for survival of postmitotic precursors, and hdac1 maintains proliferation of the neural stem cell/progenitor pool. We present an in vivo assay using somatic CRISPR targeting plus live imaging of histone-H2A.F/Z-GFP fusion protein in developing larval brain to rapidly test the role of chromatin remodelers in neural stem and progenitor cells. Our somatic assay recapitulates germline mutant phenotypes and reveals a dynamic view of their roles in neural cell populations. Our study provides new insight into the epigenetic processes that might drive pathogenesis in RB1 brain tumors, and identifies Rbbp4 and its associated chromatin remodeling complexes as potential target pathways to induce apoptosis in RB1 mutant brain cancer cells.
Investigating the in vivo role of tumor suppressor genes in cancer is technically challenging due to their essential requirement during early animal development. To address this bottleneck, we generated genetic mosaic adult zebrafish using TALEN genome editing and demonstrate somatic inactivation of the tumor suppressor retinoblastoma1 (rb1) induces tumorigenesis at high frequency. 11–33% of 1-cell stage embryos injected with TALEN mRNAs targeting rb1 exon 2 or 3 develop tumors beginning as early as 3.5 months of age. Lesions predominantly arise in the brain and show features of neuroectodermal-like and glial-like tumors. Mutant allele analysis is consistent with tumor initiation due to somatic inactivation of rb1, revealing a conserved role for rb1 in tumor suppression across vertebrates. In contrast to genetic mosaics, heterozygous rb1−/+ adults show no evidence of neoplasia, while homozygous mutant rb1−/− are larval lethal. This is the first demonstration that somatic inactivation of a tumor suppressor causes cancer in zebrafish, and highlights the utility of site-specific nucleases to create genetic mosaic zebrafish for tumor suppressor gene discovery. Somatic inactivation with site-directed nucleases in zebrafish presents a rapid and scalable strategy to study tumor suppressor gene function in cancer.
In this study we describe the molecular and cellular characterization of a zebrafish mutant that develops tumors in the optic pathway. Heterozygous Tg(flk1:RFP)is18 transgenic adults develop tumors of the retina, optic nerve and optic tract. Molecular and genetic mapping demonstrate the tumor phenotype is linked to a high copy number transgene array integrated in the lincRNA gene lincRNAis18/Zv9_00007276 on chromosome 3. TALENs were used to isolate a 147kb deletion allele that removes exons 2–5 of the lincRNAis18 gene. Deletion allele homozygotes are viable and do not develop tumors, indicating loss of function of the lincRNAis18 locus is not the trigger for tumor onset. Optic pathway tumors in the Tg(flk1:RFP)is18 mutant occur with a penetrance of 80–100% by 1 year of age. The retinal tumors are highly vascularized and composed of rosettes of various sizes embedded in a fibrous matrix. Immunohistochemical analysis showed increased expression of the glial markers GFAP and BLBP throughout retinal tumors and in dysplastic optic nerve. We performed transcriptome analysis of pre-tumorous retina and retinal tumor tissue and found changes in gene expression signatures of radial glia and astrocytes (slc1a3), activated glia (atf3, blbp, apoeb), proliferating neural progenitors (foxd3, nestin, cdh2, her9/hes1), and glioma markers (S100β, vim). The transcriptome also revealed activation of cAMP, Stat3 and Wnt signal transduction pathways. qRT-PCR confirmed >10-fold overexpression of the Wnt pathway components hbegfa, ascl1a, and insm1a. Together the data indicate Müller glia and/or astrocyte-derived progenitors could contribute to the zebrafish Tg(flk1:RFP)is18 optic pathway tumors.
22 23Wierson, Welker, Almeida et al. GeneWeld: a method for efficient targeted integration directed by short homology 2 1 Wierson et al. describe a targeted integration strategy, called GeneWeld, and a vector 2 series for gene tagging, pGTag, which promote highly efficient and precise targeted integration 3 in zebrafish, pig fibroblasts, and human cells. This approach establishes an effective genome 4 engineering solution that is suitable for creating knock-in mutations for functional genomics and 5 gene therapy applications. The authors describe high rates of germline transmission (50%) for 6 targeted knock-ins at eight different zebrafish loci and efficient integration at safe harbor loci in 7 porcine and human cells. Abstract 11Choices for genome engineering and integration involve high efficiency with little or no 12 target specificity or high specificity with low activity. Here, we describe a targeted integration 13 strategy, called GeneWeld, and a vector series for gene tagging, pGTag (plasmids for Gene 14Tagging), which promote highly efficient and precise targeted integration in zebrafish embryos, 15 pig fibroblasts, and human cells utilizing the CRISPR/Cas9 system. Our work demonstrates that 16 in vivo targeting of a genomic locus of interest with CRISPR/Cas9 and a donor vector containing 17 as little as 24 to 48 base pairs of homology directs precise and efficient knock-in when the 18 homology arms are exposed with a double strand break in vivo. Our results suggest that the 19 length of homology is not important in the design of knock-in vectors but rather how the 20 homology is presented to a double strand break in the genome. Given our results targeting 21 multiple loci in different species, we expect the accompanying protocols, vectors, and web 22 interface for homology arm design to help streamline gene targeting and applications in 23 CRISPR and TALEN compatible systems. 25Keywords
Gene editing by microinjection is an efficient system to produce mutant livestock; however, microinjection is time-consuming and requires special skill, limiting its use for large-scale production of gene-edited animals. Therefore, the aim of this study was to develop a system to deliver guide (g)RNA/Cas9/ribonucleoprotein (RNP) by electroporation into parthenogenic porcine zygotes. For experiment 1, we delivered gRNA/Cas9 RNP (250ng μL−1 of each), targeting GATA4 using 2 electroporation conditions. Group 1 (n=130): 20V, 3ms, ×2 pulses, 1 repeat; group 2 (n=102): 20V, 1ms, ×2 pulses, 2 repeats; and Control (n=96): parthenogenic zygotes, no electroporation. For experiment 2, we delivered gRNA/Cas9 RNP (250ng μL−1 of each) targeting ROSA26 by electroporation with 4 conditions compared with delivery of RNP by microinjection: group 1 (n=17): 20V, 3ms, ×1 pulses, 1 repeat; group 2 (n=49): 20V, 3ms, ×3 pulses, 1 repeat; group 3 (n=64): 30V, 3ms, ×1 pulses, 1 repeat; group 4 (n=61): 30V, 3ms, ×3 pulses, 1 repeat; group 5 (n=120): zygotes microinjected with Cas9/ROSA26 sgRNA (25/25ng μL−1), and Control (n=76): parthenogenic zygotes, no electroporation. The electroporated zygotes were cultured in porcine zygote medium-3 (PZM-3) with controlled atmosphere, and development was evaluated on Day 2 (cleavage) and Day 7 (blastocyst rate). Gene editing was evaluated on embryos (blastocyst and morulas) by PCR and Sanger sequencing of amplicons including the RNP target site. Data were compared using chi-squared test, and differences were considered significant at P<0.05. Cleavage rates in experiment 1 were similar for the control (86/96; 89.5%), group 1 (94/102; 92.1%), and group 2 (119/130; 91.5%). Blastocyst rates were higher for the control (46/96; 47%) than for the other groups (P<0.01). However, for the treated groups, the blastocyst rates were similar, group 1 (19/102; 9.2%) and group 2 (12/130; 18.6%). Furthermore, the non-homologous end joining (NHEJ) efficiency was similar for groups 1 (14/18; 77.7%) and 2 (14/17; 82.3%). In experiment 2, the cleavage (53/76; 69%) and blastocyst rates (30/76; 39%) were significantly higher for the control than for the treated groups (P<0.01). Among the groups, the lower cleavage and blastocyst rates were for group 4 (20/61; 32.7% and 3/61; 4.9%, respectively) compared with the other electroporation and microinjection groups (P<0.03). However, NHEJ efficiency was higher for electroporation groups 2 (6/8; 75%), 3 (17/17; 100%), and 4 (2/2; 100%) compared with microinjection (2/15; 13%). In conclusion, electroporation of Cas9/RNP is an efficient alternative to microinjection for gene editing in porcine zygotes.
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