Canquan Zhou

CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes

ABSTRACTGenome 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.

Derivation of human embryonic stem cell lines from parthenogenetic blastocysts

Parthenogenesis is one of the main, and most useful, methods to derive embryonic stem cells (ESCs), which may be an important source of histocompatible cells and tissues for cell therapy. Here we describe the derivation and characterization of two ESC lines (hPES-1 and hPES-2) from in vitro developed blastocysts following parthenogenetic activation of human oocytes. Typical ESC morphology was seen, and the expression of ESC markers was as expected for alkaline phosphatase, octamer-binding transcription factor 4, stage-specific embryonic antigen 3, stage-specific embryonic antigen 4, TRA-1-60, and TRA-1-81, and there was absence of expression of negative markers such as stage-specific embryonic antigen 1. Expression of genes specific for different embryonic germ layers was detected from the embryoid bodies (EBs) of both hESC lines, suggesting their differentiation potential in vitro. However, in vivo, only hPES-1 formed teratoma consisting of all three embryonic germ layers (hPES-2 did not). Interestingly, after continuous proliferation for more than 100 passages, hPES-1 cells still maintained a normal 46 XX karyotype; hPES-2 displayed abnormalities such as chromosome translocation after long term passages. Short Tandem Repeat (STR) results demonstrated that the hPES lines were genetic matches with the egg donors, and gene imprinting data confirmed the parthenogenetic origin of these ES cells. Genome-wide SNP analysis showed a pattern typical of parthenogenesis. All of these results demonstrated the feasibility to isolate and establish human parthenogenetic ESC lines, which provides an important tool for studying epigenetic effects in ESCs as well as for future therapeutic interventions in a clinical setting.

Visualization of meiotic spindle and subsequent embryonic development in in vitro and in vivo matured human oocytes.

PurposeThe aim of this study is to investigate the relationship between spindle location and embryonic development of in vivo and in vitro matured human oocytes.MethodsThe spindles of 134 in vivo matured, 105 in vitro matured oocytes were examined by Polscope at the time of ICSI.ResultsThe spindles were visualized in 83.6 and 77.1% of in vivo and in vitro matured oocytes respectively. The rate of fertilization of in vivo matured oocytes with spindles beneath or adjacent to the first polar body (angle of 0–5°) was significantly higher (93.3%) than all other groups. The proportions of various spindle positions did not differ statistically in in vivo and in vitro matured oocytes.ConclusionsMeiotic spindle location with regard to the first polar body appears to influence fertilization rate.

Relationship between antithyroid antibody and pregnancy outcome following in vitro fertilization and embryo transfer.

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.

High follicle-stimulating hormone increases aneuploidy in human oocytes matured in vitro

OBJECTIVE To study the effect of FSH on the aneuploidy risk of human oocytes matured in vitro. DESIGN Prospective study. SETTING Hospital-based IVF center. PATIENT(S) Patients with male factor infertility undergoing intracytoplasmic sperm injection (ICSI) cycles. INTERVENTION(S) Immature oocytes were put into five groups according to the FSH concentration (0, 5.5, 22, 100, and 2,000 ng/mL) in in vitro maturation (IVM) medium. Spindles were observed under a polarized microscope before polar body biopsy. Fixed polar bodies and corresponding oocytes were examined on chromosomes 13, 16, 18, 21, and 22 by fluorescence in situ hybridization. Oocytes matured in 5.5 and 2,000 ng/mL FSH were immunostained for tubulin and chromatin. MAIN OUTCOME MEASURE(S) Aneuploidy rate, spindle visualization rate, and spindle morphology. RESULT(S) The frequency rates of aneuploidy were 26.7%, 23.3%, 36.75%, 46.67%, and 63.3% in the five FSH groups, respectively. There was a significantly higher aneuploidy rate in oocytes matured in the 2,000 ng/mL FSH group. The spindle visualization rates assessed under PolScope were not significantly different between aneuploid and normal oocytes. There was no difference in spindle morphology between the 2,000 and 5.5 ng/mL FSH groups. CONCLUSION(S) High-concentration FSH in IVM medium significantly increased the first meiotic division error, resulting in more aneuploid oocytes during IVM.

Assessment of the developmental competence of human somatic cell nuclear transfer embryos by oocyte morphology classification

BACKGROUND The oocyte plays a key role in reprogramming the epigenetic status of donor cell nuclei, and the absence of reprogramming elements in the cytoplasm or aberrant accumulation of proteins can trigger the abnormal development of nuclear transfer (NT) embryos. Previous studies have demonstrated the relationship between oocyte morphology and both embryo development and pregnancy outcome. In the present study, we compared the morphology of oocytes with subsequent development of human somatic cell NT (SCNT) embryos. METHODS Piezo-assisted SCNT technology was used to produce reconstructed embryos, with almost 92% of oocytes reconstructed successfully. Depending on their morphologies, we separated metaphase II oocytes into four grades according to criteria which assess oocyte morphology, first polar body and perivitelline space, and especially, cytoplasm granula distribution. RESULTS Embryos from oocytes of Grades A and B could develop to the blastocyst stage with similar development efficiency for every developmental stage. However, embryos from Grade C oocytes arrested at or before the 8-cell stage then degraded, and the donor cell genome could not be activated and reprogrammed in such oocytes. For Grade D oocytes, cleavage was not observed in the reconstructed embryos, suggesting that the oocytes themselves have no developmental potential. CONCLUSIONS Our study revealed that different levels of developmental competence of SCNT embryos resulting from different oocyte reprogramming potentials associated with different morphologies. The results suggest that effective methods for improving oocyte quality should be studied, and that human SCNT efficiency would be increased following simple assessment of established oocyte morphology criterion.

Correction of β-thalassemia mutant by base editor in human embryos

Abstractβ-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.

Cryopreservation of Human Embryonic Stem Cells with a New Bulk Vitrification Method

To increase the manipulation efficiency and storage capability of vitrified human embryonic stem cells, a new bulk vitrification method was established using transformed cryovials. This method vitrified a large number of cell clumps, as opposed to those cryopreserved by a slow-freezing method with conventional cryovials at one time (round). After warming, vitrified human embryonic stem cells exhibited a much higher survival rate than the slow-freezing cells. The vitrified stem cells continued to express markers of pluripotency and formed teratomas in mice with severe combined immunodeficiency, confirming the pluripotency of vitrified-warmed human embryonic stem cell clumps. The new bulk vitrification method is superior to and more practical than the open pulled straw vitrification method and the slow-freezing method for the cryopreservation of human embryonic stem cells.

Medium-based Noninvasive Preimplantation Genetic Diagnosis for Human α-thalassemias-sea

AbstractTo develop a noninvasive medium-based preimplantation genetic diagnosis (PGD) test for &agr;-thalassemias-SEA.The embryos of &agr;-thalassemia-SEA carriers undergoing in vitro fertilization (IVF) were cultured. Single cells were biopsied from blastomeres and subjected to fluorescent gap polymerase chain reaction (PCR) analysis; the spent culture media that contained embryo genomic DNA and corresponding blastocysts as verification were subjected to quantitative-PCR (Q-PCR) detection of &agr;-thalassemia-SEA. The diagnosis efficiency and allele dropout (ADO) ratio were calculated, and the cell-free DNA concentration was quantitatively assessed in the culture medium.The diagnosis efficiency of medium-based &agr;-thalassemias–SEA detection significantly increased compared with that of biopsy-based fluorescent gap PCR analysis (88.6% vs 82.1%, P < 0.05). There is no significant difference regarding ADO ratio between them. The optimal time for medium-based &agr;-thalassemias–SEA detection is Day 5 (D5) following IVF.Medium-based &agr;-thalassemias–SEA detection could represent a novel, quick, and noninvasive approach for carriers to undergo IVF and PGD.

Preimplantation genetic diagnosis for Duchenne muscular dystrophy by multiple displacement amplification

OBJECTIVE To evaluate the use of multiple displacement amplification (MDA) in preimplantation genetic diagnosis (PGD) for female carriers with Duchenne muscular dystrophy (DMD). DESIGN MDA was used to amplify a whole genome of single cells. Following the setup on single cells, the test was applied in two clinical cases of PGD. One mutant exon, six short tandem repeats (STR) markers within the dystrophin gene, and amelogenin were incorporated into singleplex polymerase chain reaction (PCR) assays on MDA products of single blastomeres. SETTING Center for reproductive medicine in First Affiliated Hospital, Sun Yat-sen University, China. PATIENT(S) Two female carriers with a duplication of exons 3-11 and a deletion of exons 47-50, respectively. INTERVENTION(S) The MDA of single cells and fluorescent PCR assays for PGD. MAIN OUTCOME MEASURE(S) The ability to analyze single blastomeres for DMD using MDA. RESULT(S) The protocol setup previously allowed for the accurate diagnosis of each embryo. Two clinical cases resulted in a healthy girl, which was the first successful clinical application of MDA in PGD for DMD. CONCLUSION(S) We suggest that this protocol is reliable to increase the accuracy of the PGD for DMD.