Genome editing tools such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) have been widely used to modify genes in model systems including animal zygotes and human cells, and hold tremendous promise for both basic research and clinical applications. To date, a serious knowledge gap remains in our understanding of DNA repair mechanisms in human early embryos, and in the efficiency and potential off-target effects of using technologies such as CRISPR/Cas9 in human pre-implantation embryos. In this report, we used tripronuclear (3PN) zygotes to further investigate CRISPR/Cas9-mediated gene editing in human cells. We found that CRISPR/Cas9 could effectively cleave the endogenous β-globin gene (HBB). However, the efficiency of homologous recombination directed repair (HDR) of HBB was low and the edited embryos were mosaic. Off-target cleavage was also apparent in these 3PN zygotes as revealed by the T7E1 assay and whole-exome sequencing. Furthermore, the endogenous delta-globin gene (HBD), which is homologous to HBB, competed with exogenous donor oligos to act as the repair template, leading to untoward mutations. Our data also indicated that repair of the HBB locus in these embryos occurred preferentially through the non-crossover HDR pathway. Taken together, our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any clinical applications of CRSIPR/Cas9-mediated editing.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-015-0153-5) contains supplementary material, which is available to authorized users.

SummaryCre/LoxP-mediated DNA recombination allows for gene function and cell lineage analyses during embryonic development and tissue regeneration. Here, we describe the derivation of a K19 CreERT mouse line in which the tamoxifen-activable CreER T was knocked into the endogenous cytokeratin 19 locus. In the absence of tamoxifen, leaky Cre activity could be detected only in less than 1% of stomach and intestinal epithelial cells, but not in pancreatic or hepatic epithelial tissues. Tamoxifen administration in postnatal animals induced widespread DNA recombination in epithelial cells of pancreatic ducts, hepatic ducts, stomach, and intestine in a dose-dependent manner. Significantly, we found that Cre activity could be induced in the putative gut stem/ progenitor cells that sustained long-term gut epithelial expression of a Cre reporter. This mouse line should therefore provide a valuable reagent for manipulating gene activity and for cell lineage marking in multiorgans during normal tissue homeostasis and regeneration.Keywords lineage tracing; pancreas; small intestine; colon; liver; kidney; stomach; Cre The Cre/LoxP-based technology allows for functional analyses of essential genes in specific organs by gene inactivation or controlled ectopic gene expression (Branda and Dymecki, 2004;Lewandoski, 2001;Sauer and Henderson, 1988). When combined with detectable marker protein expression, Cre-LoxP allows for cell lineage analyses in living animals (Branda and Dymecki, 2004;Gu et al., 2003). Upon modifying Cre to produce a tamoxifen (TM)-dependent molecule, CreER T , it is now possible to control Cre activity both spatially and temporally (Metzger and Chambon, 2001). This feature allows for dissecting the genetic requirements for cell/tissue homeostasis and for following cell lineages during tissue regeneration.We have derived a K19 CreERT knockin allele to recombine DNA in epithelial cells of several adult organs. K19 encodes an intermediate filament protein (Moll et al., 1982) that is expressed in multiple cell types from the epiblast stage and is maintained in multiple epithelial cell types of later embryonic and postnatal stages (Bosch et al., 1988;Lane et al., 1983;Moll et al., 1982;Quinlan et al., 1985). For example, K19 is highly expressed in the pancreatic ducts of the adult pancreas (Deramaudt et al., 2006), but is absent or weak in acini and islets (Brembeck et al., 2001 We derived a K19 CreERT allele by replacing K19 ATG with a CreER T -cDNA followed by a SV40 polyadenylation signal (see Fig. 1). This design minimally altered K19 transcription regulatory elements while producing a CreER T message with a short 3′-UTR. Inclusion of a polyadenylation signal 3′ to the CreER T sequence prevented the transcription of the five noncoding exons of the endogenous K19 gene. Otherwise, the presence of these noncoding exons in CreER T mRNA could trigger nonsense-mediated mRNA degradation (Conti and Izaurralde, 2005;Doma and Parker, 2007). Thus, adding an extra polyadenylation signal immediately down-stream of CreER T c...

The basic helix-loop-helix transcription factor Neurog3 (Neurogenin3 or Ngn3) actively drives endodermal progenitor cells towards endocrine islet cell differentiation during embryogenesis. Here, we manipulate Neurog3 expression levels in endocrine progenitor cells without altering its expression pattern using heterozygosity and a hypomorph. Lowered Neurog3 gene dosage in the developing pancreatic epithelium reduces the overall production of endocrine islet cells without significantly affecting the proportions of various islet cell types that do form. A reduced Neurog3 production level in the endocrine-directed pancreatic progenitor population activates the expression of Neurog3 in an increased number of epithelial progenitors. Yet a significant number of these Neurog3+ cells detected in heterozygous and hypomorphic pancreata, possibly those that express low levels of Neurog3, move on to adopt pancreatic ductal or acinar fates. These data directly demonstrate that achieving high levels of Neurog3 expression is a critical step for endocrine commitment from multipotent pancreatic progenitors. These findings also suggest that a high level of Neurog3 expression could mediate lateral inhibition or other unknown feedback mechanisms to regulate the number of cells that initiate Neurog3 transcription and protein production. The control of Neurog3+ cell number and the Neurog3 threshold-dependent endocrine differentiation mechanism combine to select a specific proportion of pancreatic progenitor cells to adopt the islet cell fate.

Colorectal cancers (CRCs) arise from precursor polyps whose cellular origins, molecular heterogeneity, and immunogenic potential may reveal diagnostic and therapeutic insights when analyzed at high resolution. We present a single-cell transcriptomic and imaging atlas of the two most common human colorectal polyps, conventional adenomas and serrated polyps, and their resulting CRC counterparts. Integrative analysis of 128 datasets from 62 participants reveals adenomas arise from WNT-driven expansion of stem cells, while serrated polyps derive from differentiated cells through gastric metaplasia. Metaplasia-associated damage is coupled to a cytotoxic immune microenvironment preceding hypermutation, driven partly by ll

β-Thalassemia is a global health issue, caused by mutations in the HBB gene. Among these mutations, HBB −28 (A>G) mutations is one of the three most common mutations in China and Southeast Asia patients with β-thalassemia. Correcting this mutation in human embryos may prevent the disease being passed onto future generations and cure anemia. Here we report the first study using base editor (BE) system to correct disease mutant in human embryos. Firstly, we produced a 293T cell line with an exogenous HBB −28 (A>G) mutant fragment for gRNAs and targeting efficiency evaluation. Then we collected primary skin fibroblast cells from a β-thalassemia patient with HBB −28 (A>G) homozygous mutation. Data showed that base editor could precisely correct HBB −28 (A>G) mutation in the patient’s primary cells. To model homozygous mutation disease embryos, we constructed nuclear transfer embryos by fusing the lymphocyte or skin fibroblast cells with enucleated in vitro matured (IVM) oocytes. Notably, the gene correction efficiency was over 23.0% in these embryos by base editor. Although these embryos were still mosaic, the percentage of repaired blastomeres was over 20.0%. In addition, we found that base editor variants, with narrowed deamination window, could promote G-to-A conversion at HBB −28 site precisely in human embryos. Collectively, this study demonstrated the feasibility of curing genetic disease in human somatic cells and embryos by base editor system.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-017-0475-6) contains supplementary material, which is available to authorized users.

Islet β cells from newborn mammals exhibit high basal insulin secretion and poor glucose-stimulated insulin secretion (GSIS). Here we show that β cells of newborns secrete more insulin than adults in response to similar intracellular Ca concentrations, suggesting differences in the Ca sensitivity of insulin secretion. Synaptotagmin 4 (Syt4), a non-Ca binding paralog of the β cell Ca sensor Syt7, increased by ∼8-fold during β cell maturation. Syt4 ablation increased basal insulin secretion and compromised GSIS. Precocious Syt4 expression repressed basal insulin secretion but also impaired islet morphogenesis and GSIS. Syt4 was localized on insulin granules and Syt4 levels inversely related to the number of readily releasable vesicles. Thus, transcriptional regulation of Syt4 affects insulin secretion; Syt4 expression is regulated in part by Myt transcription factors, which repress Syt4 transcription. Finally, human SYT4 regulated GSIS in EndoC-βH1 cells, a human β cell line. These findings reveal the role that altered Ca sensing plays in regulating β cell maturation.

This retrospective study assessed the predictive value of endometrial thickness (EMT) on HCG administration day for the clinical outcome of fresh IVF and intracytoplasmic sperm injection (ICSI) cycles. A total of 8690 consecutive women undergoing 10,787 cycles over a 5-year period were included. The 5th, 50th and 95th centiles for EMT were determined as 8, 11 and 15 mm, respectively. Group analysis according to these centiles (Group 1: < 8 mm; Group 2: ≥ 8 and ≤11 mm; Group 3: > 11 and ≤15 mm; Group 4: > 15 mm) demonstrated significant differences (P < 0.001) in clinical pregnancy rates (23.0%, 37.2%, 46.2% and 53.3%, respectively), live birth rates per clinical pregnancy (63.3%, 72.0%, 78.1% and 80.3%, respectively), spontaneous abortion rates (26.7%, 23.8%, 19.9% and 17.5%, respectively), and ectopic pregnancy rates (10.0%, 4.3%, 2.1% and 2.2%, respectively). Logistic regression analyses showed EMT as one of the independent variables predictive of clinical pregnancy (OR = 1.097; P < 0.001), live birth (OR = 1.078; P < 0.001), spontaneous abortion (OR = 0.948; P < 0.001), and ectopic pregnancy (OR = 0.851; P < 0.001). Future research should aim to understand the underlying mechanisms relating EMT to conception, ectopic implantation and spontaneous abortion.

Objective: To investigate the impact of antithyroid antibody on pregnancy outcome following the in vitro fertilization and embryo transfer (IVF-ET).Methods: A total of 90 patients (156 cycles) positive for antithyroid antibody (ATA+ group) and 676 infertile women (1062 cycles) negative for antithyroid antibody (ATA- group) undergoing IVF/ICSI from August 2009 to August 2010 were retrospectively analyzed.Results: There was no significant difference in the days of ovarian stimulation, total gonadotropin dose, serum E2 level of HCG day and number of oocytes retrieved between the two groups. The fertilization rate, implantation rate and pregnancy rate following IVF-ET were significantly lower in women with antithyroid antibody than in control group (64.3% vs 74.6%, 17.8% vs 27.1% and 33.3% vs 46.7%, respectively), but the abortion rate was significantly higher in patients with antithyroid antibody (26.9% vs 11.8%).Conclusion: Patients with antithyroid antibody showed significantly lower fertilization rate, implantation rate and pregnancy rate and higher risk for abortion following IVF-ET when compared with those without antithyroid antibody. Thus, the presence of antithyroid antibody is detrimental for the pregnancy outcome following IVF-ET.