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.

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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...

BackgroundPrecise genome editing is essential for both basic and translational research. The recently developed CRISPR/Cas9 system can specifically cleave a designated site of target gene to create a DNA double-strand break, which triggers cellular DNA repair mechanism of either inaccurate non-homologous end joining, or site-specific homologous recombination. Unfortunately, homology-directed repair (HDR) is challenging due to its very low efficiency. Herein, we focused on improving the efficiency of HDR using a combination of CRISPR/Cas9, eGFP, DNA ligase IV inhibitor SCR7, and single-stranded oligodeoxynucleotides (ssODN) in human cancer cells.ResultsWhen Cas9, gRNA and eGFP were assembled into a co-expression vector, the disruption rate more than doubled following GFP-positive cell sorting in transfected cells compared to those unsorted cells. Using ssODNs as templates, SCR7 treatment increased targeted insertion efficiency threefold in transfected cells compared to those without SCR7 treatment. Moreover, this combinatorial approach greatly improved the efficiency of HDR and targeted gene mutation correction at both the GFP-silent mutation and the β-catenin Ser45 deletion mutation cells.ConclusionThe data of this study suggests that a combination of co-expression vector, ssODN, and ligase IV inhibitor can markedly improve the CRISPR/Cas9-directed gene editing, which should have significant application in targeted gene editing and genetic disease therapy.Electronic supplementary materialThe online version of this article (10.1186/s13578-018-0200-z) contains supplementary material, which is available to authorized users.

Animal interphase chromosomes are organized into topologically associating domains (TADs). How TADs are formed is not fully understood. Here, we combined high-throughput chromosome conformation capture and gene silencing to obtain insights into TAD dynamics in Xenopus tropicalis embryos. First, TAD establishment in X. tropicalis is similar to that in mice and flies and does not depend on zygotic genome transcriptional activation. This process is followed by further refinements in active and repressive chromatin compartments and the appearance of loops and stripes. Second, within TADs, higher self-interaction frequencies at one end of the boundary are associated with higher DNA occupancy of the architectural proteins CTCF and Rad21. Third, the chromatin remodeling factor ISWI is required for de novo TAD formation. Finally, TAD structures are variable in different tissues. Our work shows that X. tropicalis is a powerful model for chromosome architecture analysis and suggests that chromatin remodeling plays an essential role in de novo TAD establishment.

Precise single‐base editing mXenopus tropicalis would greatly expand the utility of this true diploid frog for modeling human genetic diseases caused by point mutations. Here, we report the efficient conversion of C‐to‐T or G‐to‐A in X. tropicalis using the rat apolipoprotein B mRNA editing enzyme catalytic subunit 1–XTEN‐clustered regularly interspaced short palindromic repeat‐associated protein 9 (Cas9) nickase‐uracil DNA glycosylase inhibitor‐nuclear localization sequence base editor [base editor 3 (BE3)]. Coinjection of guide RNA and the Cas9 mutant complex mRNA into 1‐cell stage X. tropicalis embryos caused precise C‐to‐T or G‐to‐A substitution in 14 out of 19 tested sites with efficiencies of 5–75%, which allowed for easy establishment of stable lines. Targeting the conserved T‐box 5 R237 and Tyr C28 residues in X. tropicalis with the BE3 system mimicked human Holt‐Oram syndrome and oculocutaneous albinism type 1A, respectively. Our data indicate that BE3 is an easy and efficient tool for precise base editing in X. tropicalis.—Shi, Z., Xin, H., Tian, D., Lian, J., Wang, J., Liu, G., Ran, R., Shi, S., Zhang, Z., Shi, Y., Deng, Y., Hou, C., Chen, Y. Modeling human point mutation diseases in Xenopus tropicalis with a modified CRISPR/Cas9 system. FASEB J. 33,6962–6968 (2019). http://www.fasebj.org

BackgroundThe RNA guided CRISPR/Cas9 nucleases have been proven to be effective for gene disruption in various animal models including Xenopus tropicalis. The neural crest (NC) is a transient cell population during embryonic development and contributes to a large variety of tissues. Currently, loss-of-function studies on NC development in X. tropicalis are largely based on morpholino antisense oligonucleotide. It is worthwhile establishing targeted gene knockout X. tropicails line using CRISPR/Cas9 system to study NC development.MethodsWe utilized CRISPR/Cas9 to disrupt genes that are involved in NC formation in X. tropicalis embryos. A single sgRNA and Cas9 mRNA synthesized in vitro, were co-injected into X. tropicalis embryos at one-cell stage to induce single gene disruption. We also induced duplex mutations, large segmental deletions and inversions in X. tropicalis by injecting Cas9 and a pair of sgRNAs. The specificity of CRISPR/Cas9 was assessed in X. tropicalis embryos and the Cas9 nickase was used to reduce the off-target cleavages. Finally, we crossed the G0 mosaic frogs with targeted mutations to wild type frogs and obtained the germline transmission.ResultsTotal 16 target sites in 15 genes were targeted by CRISPR/Cas9 and resulted in successful indel mutations at 14 loci with disruption efficiencies in a range from 9.3 to 57.8 %. Furthermore, we demonstrated the feasibility of generation of duplex mutations, large segmental deletions and inversions by using Cas9 and a pair of sgRNAs. We observed that CRISPR/Cas9 displays obvious off-target effects at some loci in X. tropicalis embryos. Such off-target cleavages was reduced by using the D10A Cas9 nickase. Finally, the Cas9 induced indel mutations were efficiently passed to G1 offspring.ConclusionOur study proved that CRISPR/Cas9 could mediate targeted gene mutation in X. tropicalis with high efficiency. This study expands the application of CRISPR/Cas9 platform in X. tropicalis and set a basis for studying NC development using genetic approach.Electronic supplementary materialThe online version of this article (doi:10.1186/s13578-016-0088-4) contains supplementary material, which is available to authorized users.

23Metazoan genomes are folded into 3D structures in interphase nuclei. 24 However, the molecular mechanism remains unknown. Here, we show that 25 topologically associating domains (TADs) form in two waves during Xenopus 26 tropicalis embryogenesis, first at zygotic genome activation and then as the 27 expression of CTCF and Rad21 is elevated. We also found TAD structures 28 continually change for at least three times during development. Surprisingly, 29 the directionality index is preferentially stronger on one side of TADs where 30 orientation-biased CTCF and Rad21 binding are observed, a conserved 31 pattern that is found in human cells as well. Depletion analysis revealed CTCF, 32 Rad21, and RPB1, a component of RNAPII, are required for the establishment 33 of TADs. Overall, our work shows that Xenopus is a powerful model for 34 chromosome architecture analysis. Furthermore, our findings indicate that 35 cohesin-mediated extrusion may anchor at orientation-biased CTCF binding 36 sites, supporting a CTCF-anchored extrusion model as the mechanism for 37 TAD establishment. 38 39 40 41 42 3 KEYWORDS 43 Hi-C, Topologically associating domain (TAD), CTCF, Orientation-biased 44 binding, Cohesin-mediated extrusion, Directionality, Zygotic genome activation 45 (ZGA), RNAPII, Xenopus tropicalis, Genome assembly 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 Interphase chromosomes are partitioned into topologically associating 65 domains (TADs) 1-4 which segregate into compartments of active or repressive 66 chromatins 5-7 . TAD structures are relatively stable and resilient to 67 environmental perturbations 8,9 . Chromosome architecture at the TAD level is 68 also evolutionarily conserved in eukaryotic species 4,10,11 . Disruption of TAD 69 borders leads to developmental disorders and even tumorigenesis, thus 70 underlining the importance of 3D genome organization in gene regulation 12-15 . 71 The establishment of chromatin architecture during embryogenesis provides 72 an initial spatial frame which may guide proper genome organization, 73 chromatin interaction and gene regulation in following development and 74 differentiation processes 16 . The timing of de novo TADs formation during 75 development has been examined in Drosophila, mouse, zebrafish, and 76 human 17-21 . TAD structures form at zygotic genome activation (ZGA) and 77 continually consolidate during early embryo development in fruit fly, mouse and 78 human 17,19-21 . However, in zebrafish, TADs already exist before ZGA and are 79 lost after ZGA before being reestablished in later developmental stages 18 . This 80 difference raises the question if the process of TAD formation is evolutionarily 81 conserved. 82 Cohesin complex-mediated DNA loop extrusion was recently reported in 83 several in vitro studies 22,23 and proposed as a functional mechanism 84 underlying TAD establishment 24-26 . In cultured cells, the deletion of cohesin 85 5 Rad21 alone is enough to abolish the establishment of TADs 27 . In addition, 86 CTCF, WAPL, and PDS5 proteins p...