Recent advances in genome editing, especially CRISPR-Cas nucleases, have revolutionized both laboratory research and clinical therapeutics. CRISPR-Cas nucleases, together with the DNA damage repair pathway in cells, enable both genetic diversification by classical non-homologous end joining (c-NHEJ) and precise genome modification by homology-based repair (HBR). Genome editing in zygotes is a convenient way to edit the germline, paving the way for animal disease model generation, as well as human embryo genome editing therapy for some life-threatening and incurable diseases. HBR efficiency is highly dependent on the DNA donor that is utilized as a repair template. Here, we review recent progress in improving CRISPR-Cas nuclease-induced HBR in mammalian embryos by designing a suitable DNA donor. Moreover, we want to provide a guide for producing animal disease models and correcting genetic mutations through CRISPR-Cas nuclease-induced HBR in mammalian embryos. Finally, we discuss recent developments in precise genome-modification technology based on the CRISPR-Cas system.
Animals ; CRISPR-Cas Systems/genetics* ; DNA/genetics* ; Embryo, Mammalian/metabolism* ; Endonucleases/metabolism* ; Gene Editing ; Mammals/metabolism*

Aging ; genetics ; Embryo Research ; ethics ; Embryo, Mammalian ; metabolism ; Fertilization in Vitro ; Humans ; Morals ; Preimplantation Diagnosis ; RNA Editing ; genetics ; Zygote ; metabolism

CRISPR-Cas Systems ; Embryo, Mammalian ; cytology ; metabolism ; Germ Cells ; cytology ; metabolism ; Humans ; RNA Editing ; genetics

Human pluripotent stem cells provide an inexhaustible model to study human embryogenesis in vitro. Recent studies have provided diverse models to generate human blastoids by self-organization of different pluripotent stem cells or somatic reprogramming intermediates. However, whether blastoids can be generated from other cell types or whether they can recapitulate postimplantation development in vitro is unknown. Here, we develop a strategy to generate human blastoids from heterogeneous intermediates with epiblast, trophectoderm, and primitive endoderm signatures of the primed-to-naïve conversion process, which resemble natural blastocysts in morphological architecture, composition of cell lineages, transcriptome, and lineage differentiation potential. In addition, these blastoids reflect many features of human peri-implantation and pregastrulation development when further cultured in an in vitro 3D culture system. In summary, our study provides an alternative strategy to generate human blastoids and offers insights into human early embryogenesis by modeling peri- and postimplantation development in vitro.
Humans ; Pluripotent Stem Cells/metabolism* ; Embryo, Mammalian/metabolism* ; Cell Differentiation ; Blastocyst ; Cell Lineage ; Embryonic Development

To investigate the method of RNA isolation from human embryonic tissues and the factors influencing the quality of RNA, the RNA from human embryonic tissues obtained with drug-induced labor or non-drug induced labor were isolated by using grind with liquid nitrogen or homogenizer without liquid nitrogen. The results showed that the positive rates of RNA integrity in grind with liquid nitrogen group and in homogenizer without liquid nitrogen group were 68.42% and 29.79% respectively, and there was significant difference between these two groups; however, there was no statistic difference in positive rate of RNA integrity, OD(260)/OD(280) ratio and beta-actin gene expression level between the drug-induced labor group and non-drug induced labor group. It is concluded that pulverize of tissue in liquid nitrogen remains the integrity of RNA isolated and may be applied for RNA isolation from human embryonic tissues. The quality of RNA is not affected by different methods of induction of maternal labor.
Embryo, Mammalian ; metabolism ; Freezing ; Humans ; Nitrogen ; pharmacology ; RNA ; isolation & purification ; RNA Stability ; drug effects

Assisted reproduction technologies (ART) include controlled ovarian hyperstimulation, in vitro fertilization-embryo transfer, intracytoplasmic sperm injection, in vitro maturation of oocytes, pre-implantation genetic diagnosis, etc. They have been used for the treatment of impaired fertility but may damage the health of offspring. The ART procedures may alter the epigenetic status of these offspring and DNA methylation may be a crucial mechanism. This paper summarizes epigenetic alterations in ART embryos and offspring, and discusses the risks.
Animals ; DNA Methylation ; Embryo, Mammalian ; metabolism ; Female ; Fertilization in Vitro ; Genomic Imprinting ; Humans ; Pregnancy ; Preimplantation Diagnosis

OBJECTIVETo explore the effect of basic fibroblast growth factor 1 (bFGF1) during embryonic development on hematopoiesis, to study the expression of FGF1, vascular endothelial growth factor receptor (KDR), CD133, CD34 and transcription factors Ihh, SCL, GATA-1, GATA-2 and PU. 1 in the yolk sac, and to learn about the role and relationships of FGF1, hematopoietic cells and transcription factors during embryonic hematopoiesis.

METHODS10 microm sections and total RNA were prepared from 107 human embryos aged 3-12 weeks. Immunohischemical SP staining and RT-PCR were performed.

RESULTSThe yolk sac blood islands of human 3 approximately 12 weeks embryos consisted of peripheral vascular endothelial cells and central hematopoietic cells. The expression of FGF1 was firstly found in visceral mesoderm around periphery of yolk sac blood island at day 16, while was little inside it. KDR was not or lowly expressed and CD34 and CD133 were not expressed then. The expression increased, gray value decreased and staining enhanced at day 21. Strong staining of CD34+, CD133+ and KDR+ cells were found in blood island and mesoderm at day 30, their gray values changed from 156 +/- 16, 173 +/- 18 and 160 +/- 14 to 53 +/- 7, 52 +/- 6 and 69 +/- 8 respectively. FGF1 expression was strong positive, the gray value declined dramatically from 161 +/- 13 to 40 +/- 5. Some positive cells formed vessel-like structure along the periphery of blood island. Moderate expression of CD34+, CD133+, KDR+ cells increased at day 45, the cells aggregated into mass in blood island and FGF1+ cells did the same in blood island, while little in mesoderm. Its gray valve was increased. After 7 weeks, CD133+, KDR+, CD34+ cells significantly decreased their gray values increased, the staining became week. FGF1 was weakly expressed in yolk sac and its gray value increased to 179 +/- 22. RT-PCR showed Ihh, SCL, GATA-1 and GATA-2 were expressed at different time in yolk sac. PU. 1 were not expressed at day 16, and then expressed.

CONCLUSIONSThe hematopoietic properties of yolk sac may be dependent on signaling through FGF receptors and FGF1 plays an important role in hematopoietic stem cell homeostasis. The FGF pathway regulates primitive hematopoiesis by modulating transcription factors such as Gata1 expression level and activity.

Embryo, Mammalian ; metabolism ; physiology ; Fibroblast Growth Factor 1 ; metabolism ; Hematopoiesis ; Humans ; In Vitro Techniques ; Yolk Sac ; metabolism ; physiology

Animals ; Embryo, Mammalian ; Humans ; Photoreceptor Cells, Vertebrate ; metabolism ; physiology ; Retinal Ganglion Cells ; metabolism ; physiology ; Retinal Pigments ; metabolism

Animals ; Cdc20 Proteins/metabolism* ; Cell Line ; Embryo, Mammalian/embryology* ; Embryonic Development ; Female ; Infertility, Female/metabolism* ; Mice ; Mutation ; Oocytes/metabolism*

OBJECTIVETo investigate the role of calcium dependent adhesion molecules (cadherin) during the implantation and development of human embryo by studying the expression of E-cadherin in human embryo and blastocysts.

METHODSExpression of E-cadherin in preimplantation embryos and blastocysts was detected by indirect immunofluorescence assay and quantitated by laser scanning confocal microscopy.

RESULTSExpression of E-cadherin was found in all the preimplantation embryos and blastocysts the present authors studied. The expression was higher in blastocysts than that in preimplantation embryos.

CONCLUSIONThe above results suggested that human embryos and blastocysts could express E-cadherin and the expression increased during their development. These may be of significance to the adhesion and implantation of the human embryo and blastocysts.

Blastocyst ; metabolism ; Cadherins ; genetics ; metabolism ; Embryo Implantation ; Embryo, Mammalian ; metabolism ; Female ; Fluorescent Antibody Technique, Indirect ; Gene Expression Regulation, Developmental ; Humans ; Microscopy, Confocal ; Pregnancy