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...
For the emerging amphibian genetic model Xenopus tropicalis targeted gene disruption is dependent on zinc-finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs), which require either complex design and selection or laborious construction. Thus, easy and efficient genome editing tools are still highly desirable for this species. Here, we report that RNA-guided Cas9 nuclease resulted in precise targeted gene disruption in all ten X. tropicalis genes that we analyzed, with efficiencies above 45% and readily up to 100%. Systematic point mutation analyses in two loci revealed that perfect matches between the spacer and the protospacer sequences proximal to the protospacer adjacent motif (PAM) were essential for Cas9 to cleave the target sites in the X. tropicalis genome. Further study showed that the Cas9 system could serve as an efficient tool for multiplexed genome engineering in Xenopus embryos. Analysis of the disruption of two genes, ptf1a/p48 and tyrosinase, indicated that Cas9-mediated gene targeting can facilitate direct phenotypic assessment in X. tropicalis embryos. Finally, five founder frogs from targeting of either elastase-T1, elastase-T2 or tyrosinase showed highly efficient transmission of targeted mutations into F1 embryos. Together, our data demonstrate that the Cas9 system is an easy, efficient and reliable tool for multiplex genome editing in X. tropicalis.
Pigs share many physiological, biochemical, and anatomical similarities with humans and have emerged as valuable large animal models for biomedical research. Considering the advantages in immune system resemblance, suitable size, and longevity for clinical practical and monitoring purpose, SCID pigs bearing dysfunctional RAG could serve as important experimental tools for regenerative medicine, allograft and xenograft transplantation, and reconstitution experiments related to the immune system. In this study, we report the generation and phenotypic characterization of RAG1 and RAG2 knockout pigs using transcription activator-like effector nucleases. Porcine fetal fibroblasts were genetically engineered using transcription activator-like effector nucleases and then used to provide donor nuclei for somatic cell nuclear transfer. We obtained 27 live cloned piglets; among these piglets, 9 were targeted with biallelic mutations in RAG1, 3 were targeted with biallelic mutations in RAG2, and 10 were targeted with a monoallelic mutation in RAG2. Piglets with biallelic mutations in either RAG1 or RAG2 exhibited hypoplasia of immune organs, failed to perform V(D)J rearrangement, and lost mature B and T cells. These immunodeficient RAG1/2 knockout pigs are promising tools for biomedical and translational research.
Mutations in SCN1A, the gene encoding the α subunit of Nav1.1 channel, can cause epilepsies with wide ranges of clinical phenotypes, which are associated with the contrasting effects of channel loss-of-function or gain-of-function. In this project, CRISPR/Cas9- and TALEN-mediated genome-editing techniques were applied to induced pluripotent stem cell (iPSC)-based-disease model to explore the mechanism of epilepsy caused by SCN1A loss-of-function mutation. By fluorescently labeling GABAergic subtype in iPSC-derived neurons using CRISPR/Cas9, we for the first time performed electrophysiological studies on SCN1A-expressing neural subtype and monitored the postsynaptic activity of both inhibitory and excitatory types. We found that the mutation c.A5768G, which led to no current of Nav1.1 in exogenously transfected system, influenced the properties of not only Nav current amount, but also Nav activation in Nav1.1-expressing GABAergic neurons. The two alterations in Nav further reduced the amplitudes and enhanced the thresholds of action potential in patient-derived GABAergic neurons, and led to weakened spontaneous inhibitory postsynaptic currents (sIPSCs) in the patient-derived neuronal network. Although the spontaneous excitatory postsynaptic currents (sEPSCs) did not change significantly, when the frequencies of both sIPSCs and sEPSCs were further analyzed, we found the whole postsynaptic activity transferred from the inhibition-dominated state to excitation in patient-derived neuronal networks, suggesting that changes in sIPSCs alone were sufficient to significantly reverse the excitatory level of spontaneous postsynaptic activity. In summary, our findings fill the gap of our knowledge regarding the relationship between SCN1A mutation effect recorded on exogenously transfected cells and on Nav1.1-expressing neurons, and reveal the physiological basis underlying epileptogenesis caused by SCN1A loss-of-function mutation.
Xenopus tropicalis is an emerging vertebrate genetic model. A gene knock-in method has not yet been reported in this species. Here, we report that heritable targeted integration can be achieved in this diploid frog using a concurrent cleavage strategy mediated by the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR/ Cas9) system. The key point of the strategy is the addition of a Cas9/guide RNA cleavage site in the donor vector, allowing simultaneous cutting of the chromosomal target site and circular donor DNA in vivo. For the 3 distinct loci tested, all showed efficient targeted integration that was verified by both germ-line transmission and Southern blot analyses. By designing the target sites in introns, we were able to get precise editing of the tyrosinase coding sequence and green fluorescent protein expression from endogenous n-tubulin promoter and enhancers. We were unable to detect off-target effects with the T7 endonuclease I assay. Precise editing of protein coding sequences in X. tropicalis expands the utility of this diploid frog, such as for establishing models to study human inherited diseases.-Shi, Z., Wang, F., Cui, Y., Liu, Z., Guo, X., Zhang, Y., Deng, Y., Zhao, H., Chen, Y. Heritable CRISPR/Cas9-mediated targeted integration in Xenopus tropicalis. FASEB J. 29, 4914-4923 (2015). www.fasebj.orgRecent establishment of efficient targeted gene disruption methods in Xenopus tropicalis (1-6) has confirmed this diploid frog as an excellent vertebrate genetic model; however, recombination-mediated genome editing has not been reported in this species.Direct injection of mRNAs of engineered nucleases, such as zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), into fertilized eggs facilitates homologybased integration in zebrafish, mouse, and rat embryos (7-11). Unfortunately, taking similar strategies with either circular donor DNA, linear donor DNA, or synthetic oligodeoxynucleotides, we were unable to get heritable homologous recombination-mediated gene modification in X. tropicalis in the past several years, likely as a result of too low recombination efficiency.Here, we show that heritable targeted integration in X. tropicalis was successfully obtained when we took a homology-independent strategy recently developed for cell lines and zebrafish (12-15). The most prominent feature of the strategy is the introduction of a nuclease cleavage site into the donor DNA, thus allowing the concurrent cleavage of the chromosomal target site and circular donor DNA in vivo by a given nuclease. In addition, we designed a reporter system that helps identify early integration events. We tested the editing of 3 genes in X. tropicalis with this strategy. All showed efficient targeted integration that was passed to the next generation through germ-line transmission and was further confirmed by Southern blot analysis. Our data r...
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