Human trophoblast stem cells (hTSCs) provide a valuable model to study placental development and function. While primary hTSCs have been derived from embryos/early placenta, and transdifferentiated hTSCs from naïve human pluripotent stem cells (hPSCs), the generation of hTSCs from primed PSCs is problematic. We report the successful generation of TSCs from primed hPSCs and show that BMP4 substantially enhances this process. TSCs derived from primed hPSCs are similar to blastocyst-derived hTSCs in terms of morphology, proliferation, differentiation potential, and gene expression. We define the chromatin accessibility dynamics and histone modifications (H3K4me3/H3K27me3) that specify hPSC-derived TSCs. Consistent with low density of H3K27me3 in primed hPSC-derived hTSCs, we show that knockout of H3K27 methyltransferases (EZH1/2) increases the efficiency of hTSC derivation from primed hPSCs. Efficient derivation of hTSCs from primed hPSCs provides a simple and powerful model to understand human trophoblast development, including the pathogenesis of trophoblast-related disorders, by generating disease-specific hTSCs.
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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