The X‐linked genetic bleeding disorder caused by deficiency of coagulator factor IX, hemophilia B, is a disease ideally suited for gene therapy with genome editing technology. Here, we identify a family with hemophilia B carrying a novel mutation, Y371D, in the human F9 gene. The CRISPR/Cas9 system was used to generate distinct genetically modified mouse models and confirmed that the novel Y371D mutation resulted in a more severe hemophilia B phenotype than the previously identified Y371S mutation. To develop therapeutic strategies targeting this mutation, we subsequently compared naked DNA constructs versus adenoviral vectors to deliver Cas9 components targeting the F9 Y371D mutation in adult mice. After treatment, hemophilia B mice receiving naked DNA constructs exhibited correction of over 0.56% of F9 alleles in hepatocytes, which was sufficient to restore hemostasis. In contrast, the adenoviral delivery system resulted in a higher corrective efficiency but no therapeutic effects due to severe hepatic toxicity. Our studies suggest that CRISPR/Cas‐mediated in situ genome editing could be a feasible therapeutic strategy for human hereditary diseases, although an efficient and clinically relevant delivery system is required for further clinical studies.

The coronaviruses are a large family of plus-strand RNA viruses that cause a wide variety of diseases both in humans and in other organisms. The coronaviruses are composed of three main lineages and have a complex organization of nonstructural proteins (nsp's). In the coronavirus, nsp3 resides a domain with the macroH2A-like fold and ADP-ribose-1؆-monophosphatase (ADRP) activity, which is proposed to play a regulatory role in the replication process. However, the significance of this domain for the coronaviruses is still poorly understood due to the lack of structural information from different lineages. We have determined the crystal structures of two viral ADRP domains, from the group I human coronavirus 229E and the group III avian infectious bronchitis virus, as well as their respective complexes with ADP-ribose. The structures were individually solved to elucidate the structural similarities and differences of the ADRP domains among various coronavirus species. The active-site residues responsible for mediating ADRP activity were found to be highly conserved in terms of both sequence alignment and structural superposition, whereas the substrate binding pocket exhibited variations in structure but not in sequence. Together with data from a previous analysis of the ADRP domain from the group II severe acute respiratory syndrome coronavirus and from other related functional studies of ADRP domains, a systematic structural analysis of the coronavirus ADRP domains was realized for the first time to provide a structural basis for the function of this domain in the coronavirus replication process.

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.

Smyd1/Bop is an evolutionary conserved histone methyltransferase previously shown by conventional knockout to be critical for embryonic heart development. To further explore the mechanism(s) in a cell autonomous context, we conditionally ablated Smyd1 in the first and second heart fields of mice using a knock-in (KI) Nkx2.5-cre driver. Robust deletion of floxed-Smyd1 in cardiomyocytes and the outflow tract (OFT) resulted in embryonic lethality at E9.5, truncation of the OFT and right ventricle, and additional defects consistent with impaired expansion and proliferation of the second heart field (SHF). Using a transgenic (Tg) Nkx2.5-cre driver previously shown to not delete in the SHF and OFT, early embryonic lethality was bypassed and both ventricular chambers were formed; however, reduced cardiomyocyte proliferation and other heart defects resulted in later embryonic death at E11.5-12.5. Proliferative impairment prior to both early and mid-gestational lethality was accompanied by dysregulation of transcripts critical for endoplasmic reticulum (ER) stress. Mid-gestational death was also associated with impairment of oxidative stress defense—a phenotype highly similar to the previously characterized knockout of the Smyd1-interacting transcription factor, skNAC. We describe a potential feedback mechanism in which the stress response factor Tribbles3/TRB3, when directly methylated by Smyd1, acts as a co-repressor of Smyd1-mediated transcription. Our findings suggest that Smyd1 is required for maintaining cardiomyocyte proliferation at minimally two different embryonic heart developmental stages, and its loss leads to linked stress responses that signal ensuing lethality.

The CRISPR-Cas RNA-guided system has versatile uses in many organisms and allows modification of multiple target sites simultaneously. Generating novel genetically modified mouse and rat models is one valuable application of this system. Through the injection of Cas9 protein instead of mRNA into embryos, we observed fewer off-target effects of Cas9 and increased point mutation knock-in efficiency. Large genomic DNA fragment (up to 95 kb) deletion mice were generated for in vivo study of lncRNAs and gene clusters. Site-specific insertion of a 2.7 kb CreERT2 cassette into the mouse Nfatc1 locus allowed labeling and tracing of hair follicle stem cells. In addition, we combined the Cre-Loxp system with a gene-trap strategy to insert a GFP reporter in the reverse orientation into the rat Lgr5 locus, which was later inverted by Cre-mediated recombination, yielding a conditional knockout/reporter strategy suitable for mosaic mutation analysis.

Background: GPR126 plays critical roles in development, but its function in vessels is not well characterized. Results: GPR126 regulates angiogenesis by modulating endothelial cell proliferation and migration via regulation of Vegfr2 expression. Conclusion: GPR126 is important for physiological and pathological angiogenesis. Significance: This finding provides a new functional mechanism for the regulation of angiogenesis.

Human pluripotent stem cells (hPSCs) exhibit very limited contribution to interspecies chimeras. One explanation is that the conventional hPSCs are in a primed state and so unable  to form chimeras in pre-implantation embryos. Here, we show that the conventional hPSCs undergo rapid apoptosis when injected into mouse pre-implantation embryos. While, forced-expression of BMI1, a polycomb factor in hPSCs overcomes the apoptosis and enables hPSCs to integrate into mouse pre-implantation embryos and subsequently contribute to chimeras with both embryonic and extra-embryonic tissues. In addition, BMI1 also enables hPSCs to integrate into pre-implantation embryos of other species, such as rabbit and pig. Notably, BMI1 high expression and anti-apoptosis are also indicators for naïve hPSCs to form chimera in mouse embryos. Together, our findings reveal that the apoptosis is an initial barrier in interspecies chimerism using hPSCs and provide a rational to improve it.

z Dear Editor, Mutations in the β-globin gene, the essential component of adult hemoglobin (HbA; α2β2), results in either a production of aberrant sickle hemoglobin (HbS) leading to sickle cell disease (SCD) or an insufficient β-globin synthesis leading to β-thalassemia. These two major forms of β-hemoglobinopathies cause impaired erythropoiesis and life-threatening anemia. Clinical evidence has suggested that reactivation of fetal γ-globin (HBG) gene expression which is normally silenced after birth by certain genetic mutations can ameliorate the clinical course of β-hemoglobinopathies. 1,2 In β-thalassemia, elevated levels of fetal γ-globin interact with α-globin to form fetal hemoglobin (HbF; α2γ2) restoring the α/β-like globin ratio and in SCD the γ-globin reduces HbS polymerization. There are two major strategies for re-activation of HBG expression: reducing the expression of critical trans-acting repression factors (such as BCL11A) 3,4 or deletion of inhibitory cis-regulatory elements in the HBG1/2 promoter region. To develop a practical strategy for clinical implementation, we leveraged electroporation of a Cas9:sgRNA RNP to edit the HBG1/2 promoter in hematopoietic stem/progenitor cells (HSPCs). We successfully achieved an average editing efficiency of 85% in β-thalassemia patient-derived HSPCs, leading to increased γ-globin mRNA expression (up to 126% relative to α-globin) and an improved terminal erythroid differentiation rate. Importantly, we discovered that the BCL11A binding site (TGACCA: −114 to −119), which is critical for the repression of γ-globin, 5 is an ideal target for base editor hA3A-BE3 induced mutation and elevation of HBG expression.To mimic the effect of the naturally occurring Δ13 bp allele in the HBG1 promoter (−102 to −114) which was identified in patients with hereditary persistence of fetal hemoglobin (HPFH), we synthesized two sgRNAs (with or without 2′-O-methyl 3′ phosphorothioate modifications at three terminal nucleotides at both the 5′ and 3′ ends) (sgRNA1, sgRNA2) targeting the HBG promoter region (Fig. 1a). After electroporation of either RNP complex into immortalized erythroid precursor HUDEP-2 cells, 4 we found that chemical modification 6 of sgRNA enhanced editing efficiency and that sgRNA1 was more efficient than sgRNA2 (Supplementary information, Fig. S1a). The enhancement effect of chemical modification on sgRNAs was also confirmed in T cell receptor (TCR) and beta-2-microglobulin (B2M) loci in human primary T cells (Supplementary information, Fig. S1b). Hence, unless specified, we used the chemically modified sgRNA1 in the following experiments. After titration of the optimal RNP concentration in the HUDEP-2 cell line and in human CD34 + HSPCs, we successfully achieved editing efficiency over 80% in both cells (Supplementary information, Fig. S1c, d). After erythroid differentiation, the γ-globin mRNA level in the edited HUDEP-2 cells reached a mean of 22.7% relative to the total β-like globin mRNA, which showed a 4-fold increase compared with controls (mean 5.6%) (Su...