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

Abstract

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...

Generation of RAG 1- and 2-deficient rabbits by embryo microinjection of TALENs

“…Unlike ZFNs, TALENs have fewer off-target effects and lower toxicity. Given the success of the TALEN technology in reported species [4][5][6], we attempted to explore the feasibility of producing KO rabbits using this technology. In the current study, we chose recombination activation genes (RAGs), including RAG1 and RAG2, as the first genes of interest.…”

Efficient Knockout of Transplanted Green Fluorescent Protein Gene in Medaka Using TALENs

“…This modified TALEN platform was used to efficiently induce mutations in Xenopus and zebrafish. Although previous studies have shown that TALEN has high specificity (Bogdanove and Voytas 2011;Huang et al 2011;Hwang et al 2013;Lei et al 2012;Liu et al 2013Liu et al , 2014Sander et al 2011), offtarget problems remained underestimated. On the one hand, while HD, NI, and NG RVDs of TALENs could specifically correspond with C, A, and T, only NN can identify both G and A, indicating that the more G in the target sequence, the more NN is required in the corresponding synthetic TALEN artificial nucleases, but this increases the opportunity for nonspecific knockouts.…”

“…Furthermore, non-specific cleavage of the genome can cause cytotoxic or embryotoxic effects. Compared with ZFN, the TALEN technique displays a broader recognition property for DNA sequences and, in fact, it is hardly ever affected by DNA sequence even though it has high sequence-specific recognition (Lei et al 2012;Liu et al 2013). Hence, we aimed to explore the application of TALENs in medaka and other fish to promote the study of genetic engineering and gene function in fish.…”

“…Previously, we simplified the assembly steps of the existing TALEN system (Cermak et al 2011;Li et al 2011). Indeed, we modified the Fok I catalytic domain to improve the efficiency of DSB caused by TALENs and built a fast and efficient TALEN technology platform (Lei et al 2012;Liu et al 2013). ZFNs and TALENs have been used successfully in gene knockout and genome-editing studies of zebrafish, and these experiments are aided by the availability of a genome sequence and gene structure information.…”

Enhanced gene disruption by programmable nucleases delivered by a minicircle vector

“…[16][17][18][19] TALEN-mediated gene targeting has also been successfully demonstrated in several systems including plants, 20,21 zebrafish, 22 yellow catfish, 23 Caenorhabditis elegans, 24 rats, 25 mice 26 and human cells. 3 To date, conventional plasmids, 3,20,[27][28][29][30][31][32] integrase-defective lentiviral vectors, 18,33 adenoviral vectors, 34,35 adeno-associated viral vectors, 36 direct microinjection into embryos, 12,[37][38][39] and recombinant proteins 40,41 have been used to deliver engineered nucleases. Among these approaches, plasmid-mediated delivery has been predominant because it is easy to generate the necessary components and vector integration into the host genome is relatively rare.…”