Efficient targeted gene disruption in the soma and germ line of the frog
            <i>Xenopus tropicalis</i>
            using engineered zinc-finger nucleases

Young, John J.; Cherone, Jennifer M.; Doyon, Yannick; Ankoudinova, Irina; Faraji, Farhoud; Lee, Andrew H.; Ngo, Catherine; Guschin, Dmitry; Paschon, David E.; Miller, Jeffrey C.; Zhang, Lei; Rebar, Edward J.; Gregory, Philip D.; Urnov, Fyodor D.; Harland, Richard M.; Zeitler, Bryan

doi:10.1073/pnas.1102030108

Cited by 127 publications

(89 citation statements)

References 32 publications

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“…The availability of targeted genome editing methods such as zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs) permits independent mutations to be made in genes of interest (22,23). Inner ear defects in sp8 TALEN-injected embryos can be observed from stage 28 and we classified TALEN-induced phenotypes as for ecl mutants ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…To characterize TALEN-induced mutations at targeted sp8 loci, we amplified genomic DNA fragments of 520 bp from sp8 TALENinjected individuals (Fig. 2H) and used CEL I endonuclease as previously to detect mutations induced by ZFNs (22). The CEL I assay produced two smaller fragments of ∼200 bp and 300 bp, respectively, only in sp8 TALEN-injected individuals, whereas only a larger fragment of 520 bp was found in controls (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Sp8 regulates inner ear development

Chung

Medina-Ruiz

Harland

2014

Proc. Natl. Acad. Sci. U.S.A.

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A forward genetic screen of N-ethyl-N-nitrosourea mutagenized Xenopus tropicalis has identified an inner ear mutant named eclipse (ecl). Mutants developed enlarged otic vesicles and various defects of otoconia development; they also showed abnormal circular and inverted swimming patterns. Positional cloning identified specificity protein 8 (sp8), which was previously found to regulate limb and brain development. Two different loss-of-function approaches using transcription activator-like effector nucleases and morpholino oligonucleotides confirmed that the ecl mutant phenotype is caused by down-regulation of sp8. Depletion of sp8 resulted in otic dysmorphogenesis, such as uncompartmentalized and enlarged otic vesicles, epithelial dilation with abnormal sensory end organs. When overexpressed, sp8 was sufficient to induce ectopic otic vesicles possessing sensory hair cells, neurofilament innervation in a thickened sensory epithelium, and otoconia, all of which are found in the endogenous otic vesicle. We propose that sp8 is an important factor for initiation and elaboration of inner ear development.T he vertebrate inner ear is a sensory organ responsible for balance and sound detection. Dysfunction of the inner ear is among the most common congenital disorders, affecting at least 1 in 500 births (1) and ∼40% of sensorineural deafness is associated with inner ear malformations (2). However, the study of hearing and balance impairment in humans is limited by the inability to follow inner ear development. Vertebrates share similarities in the sequence of developmental events that form the inner ear: the formation of an otic placode from an ectodermal thickening, morphogenesis to form the otocyst, and regional patterning of the otic vesicle (OV), resulting in the 3D membranous labyrinth (3, 4). Multipotent sensory progenitor cells are induced in the ectoderm surrounding the anterior neural plate, a domain termed the preplacodal region (PPR), and six1 has been characterized as marking this panplacodal domain. Signals from hindbrain and the regional expression of different transcription factors differentiate the PPR into the otic placode. The OV is partitioned by asymmetrical expression of various developmental regulators to pattern subdomains of the developing inner ear.The abilities of Xenopus to elucidate the cellular and molecular aspects of developmental processes position it as a valuable model organism (5-7). Earlier studies of lineage analysis and spatiotemporal expression of transcription factors during inner ear development led to construction of an inner ear fate map in Xenopus and this fate map allows us to interpret gene expression patterns within the context of the anatomy (8, 9). In recent years, genetic and genomic approaches have been developed in Xenopus tropicalis (10-13). To advance our understanding of inner ear development, we have screened N-ethyl-N-nitrosourea (ENU) mutagenized X. tropicalis colonies and recovered an inner ear mutant named eclipse (ecl). The ecl mutant perturbs specificity protei...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Sp8 regulates inner ear development

Chung

Medina-Ruiz

Harland

2014

Proc. Natl. Acad. Sci. U.S.A.

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“…S1). The plasmids containing ptf1a/p48-ZFN coding sequences were purchased from Sigma, and the DNA encoding noggin-ZFNs were synthesized according to Yong et al (14) and then subcloned into pCS2+KK and pCS2+EL.…”

Section: Methodsmentioning

confidence: 99%

“…1). noggin (14) and ptf1a/p48 were also targeted by the corresponding ZFNs for a side-by-side comparison with TALENs. The TALEN EBE sequences of noggin and ptf1a/p48 were located in the adjacent regions to the corresponding ZFN targeting sequences (Fig.…”

Section: Talens Effectively Induce Targeted Gene Disruption In Xenopusmentioning

confidence: 99%

Efficient targeted gene disruption in Xenopus embryos using engineered transcription activator-like effector nucleases (TALENs)

Lei

Guo

Liu

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

226

198

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Transcription activator-like effector nucleases (TALENs) are an approach for directed gene disruption and have been proved to be effective in various animal models. Here, we report that TALENs can induce somatic mutations in Xenopus embryos with reliably high efficiency and that such mutations are heritable through germ-line transmission. We modified the Golden Gate method for TALEN assembly to make the product suitable for RNA transcription and microinjection into Xenopus embryos. Eight pairs of TALENs were constructed to target eight Xenopus genes, and all resulted in indel mutations with high efficiencies of up to 95.7% at the targeted loci. Furthermore, mutations induced by TALENs were highly efficiently passed through the germ line to F 1 frogs. Together with simple and reliable PCR-based approaches for detecting TALEN-induced mutations, our results indicate that TALENs are an effective tool for targeted gene editing/knockout in Xenopus.genome editing | heritable mutagenesis | mutagenesis detection | reverse genetics | genome engineering A mong current animal models, Xenopus laevis and Xenopus tropicalis are classical animal models widely used in the study of embryonic development. However, because of the lack of methodologies for homologous recombination and embryonic stem cell derivation, it is difficult to perform specific gene targeting in these two models, which has impeded their use in genetic studies. Recently, site-specific gene targeting with transcription activator-like effector nucleases (TALENs) has been successfully applied in several animal models including rat, zebrafish, and Caenorhabditis elegans (1-4). Similar to zinc finger nucleases (ZFNs) (5), TALENs are engineered DNA nucleases that consist of a custom-designed DNA-binding domain and a nonspecific nuclease domain derived from Fok I endonuclease. Binding of adjacent TALENs allows dimerization of the endonuclease domains, leading to double-strand breaks at the predetermined site (6). These double-strand DNA breaks are frequently repaired through nonhomologous end joining (NHEJ) (7, 8), resulting in deletion or insertion (indel) mutations. The DNA binding specificity of TALENs, as distinct from ZFNs, is based on the transcription activator-like effectors (TALEs) from Xanthomonas plant pathogens (9, 10). The TALE proteins consist of an N-terminal translocation domain, a nuclear localization signal, and various numbers of tandem 34-aa repeats that determine the DNA binding specificity. Each repeat in the tandem array is identical except for two variable amino acid residues at positions 12 and 13 called repeat variable di-residues (RVDs), through which each repeat independently determines the targeted base (11,12). It is known that the RVDs NI, NG, HD, and NN preferentially recognize adenine (A), thymine (T), cytosine (C), and guanine (G)/adenine (A), respectively (13). With a given repeat combination, the TALE recognizes a specific target sequence predicted by this code. A pair of TALENs can then cleave doublestrand DNA between the two targ...

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“…1C). ZFNs have been shown to introduce DSBs specifically and efficiently in a variety of cell types and organisms, including nematodes , fruit flies (Beumer et al, 2008), zebrafish (Doyon et al, 2008;Meng et al, 2008;Foley et al, 2009), plants (Townsend et al, 2009), rats (Geurts et al, 2009;Mashimo et al, 2010), Xenopus (Young et al, 2011), pigs (Yang et al, 2011), and primary human cells, such as embryonic stem cells (ESCs) (Lombardo et al, 2007), T cells (Perez et al, 2008), induced pluripotent stem cells (iPSCs) (Hockemeyer et al, 2009;Zou et al, 2009Zou et al, , 2011, mesenchymal stromal cells (Benabdallah et al, 2010), and HSCs (Holt et al, 2010). The broad range of organisms and cell types in which ZFN-based genome editing has been applied suggests that it utilizes mechanisms of cellular DNA recognition and DNA repair that developed early in evolution.…”

Section: Zfns: a Rapid Coming Of Agementioning

confidence: 99%

Zinc-Finger Nucleases for Somatic Gene Therapy: The Next Frontier

et al. 2011

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Zinc-finger nucleases (ZFNs) are a powerful tool that can be used to edit the human genome ad libitum. The technology has experienced remarkable development in the last few years with regard to both the target site specificity and the engineering platforms used to generate zinc-finger proteins. As a result, two phase I clinical trials aimed at knocking out the CCR5 receptor in T cells isolated from HIV patients to protect these lymphocytes from infection with the virus have been initiated. Moreover, ZFNs have been successfully employed to knockout or correct disease-related genes in human stem cells, including hematopoietic precursor cells and induced pluripotent stem cells. Targeted genome engineering approaches in multipotent and pluripotent stem cells hold great promise for future strategies geared toward correcting inborn mutations for personalized cell replacement therapies. This review describes how ZFNs have been applied to models of gene therapy, discusses the opportunities and the risks associated with this novel technology, and suggests future directions for their safe application in therapeutic genome engineering.

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Efficient targeted gene disruption in the soma and germ line of the frog Xenopus tropicalis using engineered zinc-finger nucleases

Cited by 127 publications

References 32 publications

Sp8 regulates inner ear development

Sp8 regulates inner ear development

Efficient targeted gene disruption in Xenopus embryos using engineered transcription activator-like effector nucleases (TALENs)

Zinc-Finger Nucleases for Somatic Gene Therapy: The Next Frontier

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