Whole chromosome loss and genomic instability in mouse embryos after CRISPR-Cas9 genome editing

AbstractKaryotype alterations have emerged as on-target complications from CRISPR-Cas9 genome editing. However, the events that lead to these karyotypic changes in embryos after Cas9-treatment remain unknown. Here, using imaging and single-cell genome sequencing of 8-cell stage embryos, we track both spontaneous and Cas9-induced karyotype aberrations through the first three divisions of embryonic development. We observe the generation of abnormal structures of the nucleus that arise as a consequence of errors in mitosis, including micronuclei and chromosome bridges, and determine their contribution to common karyotype aberrations including whole chromosome loss that has been recently reported after editing in embryos. Together, these data demonstrate that Cas9-mediated germline genome editing can lead to unwanted on-target side effects, including major chromosome structural alterations that can be propagated over several divisions of embryonic development.

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The Relationship between Embryonic Development and the Efficiency of Target Mutations in Porcine Endogenous Retroviruses (PERVs) Pol Genes in Porcine Embryos

Animals ◽

10.3390/ani9090593 ◽

2019 ◽

Vol 9 (9) ◽

pp. 593 ◽

Cited By ~ 2

Author(s):

Maki Hirata ◽

Manita Wittayarat ◽

Takayuki Hirano ◽

Nhien Thi Nguyen ◽

Quynh Anh Le ◽

...

Keyword(s):

Embryonic Development ◽

Genome Editing ◽

Endogenous Retrovirus ◽

Endogenous Retroviruses ◽

Cell Stage ◽

Gene Target ◽

Porcine Endogenous Retrovirus ◽

Cas9 Protein ◽

Pig Genome ◽

The Relationship

Porcine endogenous retrovirus (PERV) is a provirus found in the pig genome that may act as an infectious pathogen in humans who receive pig organ xenotransplantation. Inactivation of the PERV pol gene in porcine cells reportedly affects cell growth. Therefore, the mutation of PERV pol gene in porcine embryos using genome editing may affect the embryonic development. The present study was carried out to investigate the relationship between the mutation of the PERV pol gene in porcine embryos and their development. We introduced, either alone or in combination, three different gRNAs (gRNA1, 2, and 3) into porcine zygotes by genome editing using electroporation of the Cas9 protein (GEEP) system. All three gRNAs targeted the PERV pol gene, and we assessed their effects on porcine embryonic development. Our results showed that the blastocyst formation rates of zygotes electroporated with gRNA3—alone and in combination—were significantly lower (p < 0.05) than those of zygotes electroporated with gRNA1. The mutation rates assessed by the PERV pol gene target site sequencing in individual blastocysts and pooled embryos at the 2-to-8-cell stage did not differ among the three gRNAs. However, the frequency of indel mutations in mutant embryos at the 2-to-8-cell stage trended higher in the embryos electroporated with gRNA3 alone and in combination. Embryonic development may be affected by gRNAs that induce high-frequency indel mutations.

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A unified model of human hemoglobin switching through single-cell genome editing

Nature Communications ◽

10.1038/s41467-021-25298-9 ◽

2021 ◽

Vol 12 (1) ◽

Cited By ~ 1

Author(s):

Yong Shen ◽

Jeffrey M. Verboon ◽

Yuannyu Zhang ◽

Nan Liu ◽

Yoon Jung Kim ◽

...

Keyword(s):

Single Cell ◽

Genome Editing ◽

Fetal Hemoglobin ◽

Functional Assay ◽

Human Genetic Variation ◽

Genetic Changes ◽

Hemoglobin Switching ◽

Multiple Mutation ◽

Cell Genome ◽

Single Cell Genome

AbstractKey mechanisms of fetal hemoglobin (HbF) regulation and switching have been elucidated through studies of human genetic variation, including mutations in the HBG1/2 promoters, deletions in the β-globin locus, and variation impacting BCL11A. While this has led to substantial insights, there has not been a unified understanding of how these distinct genetically-nominated elements, as well as other key transcription factors such as ZBTB7A, collectively interact to regulate HbF. A key limitation has been the inability to model specific genetic changes in primary isogenic human hematopoietic cells to uncover how each of these act individually and in aggregate. Here, we describe a single-cell genome editing functional assay that enables specific mutations to be recapitulated individually and in combination, providing insights into how multiple mutation-harboring functional elements collectively contribute to HbF expression. In conjunction with quantitative modeling and chromatin capture analyses, we illustrate how these genetic findings enable a comprehensive understanding of how distinct regulatory mechanisms can synergistically modulate HbF expression.

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166MOUSE EMBRYONIC DEVELOPMENT FROM ONE-CELL STAGE TO BLASTOCYST, UTILIZING KSOM SUPPLEMENTED WITH AMINO ACIDS IN A MICROCHANNEL DEVICE

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab166 ◽

2004 ◽

Vol 16 (2) ◽

pp. 205

Author(s):

P.H.C. Lopes ◽

S.A. Malusky ◽

A.S. Lima ◽

D.J. Beebe ◽

M.B. Wheeler

Keyword(s):

Amino Acids ◽

Embryonic Development ◽

Mouse Strain ◽

Mouse Embryos ◽

Type I ◽

Alternative Technique ◽

Cell Stage ◽

Mammalian Embryos ◽

Microchannel Device

Great efforts have been made to improve in vitro culture for enhancement of embryonic development. However, in vitro development of mammalian embryos still remains a challenge for the scientific community. Recently, the use of microfluidic culture devices, as an alternative technique compared to the standard drop, has allowed mammalian embryos to develop to the hatched blastocyst stage. With the use of a different medium, mouse strain, and microchannel device than previously reported (Raty S et al., 2001 Theriogenology 55, 241 abst), this study was undertaken to determine if a microchannel device fabricated from borosilicate and poly-dimethylsiloxane would support development of mouse embryos from one-cell to blastocyst, as an alternative to standard microdrop culture. Mice (F1 inbred C57BL/6CRL X SJL) from 3 to 8 weeks old were superovulated with 5IU of PMSG and 5IU of hCG. The female SJL strain of the mice has demonstrated low reproductive performance. One-cell embryos were collected in M2 medium (Sigma, St. Louis, MO, USA.). For each treatment, 240 embryos in 24 replicates were cultured. Groups of 10 embryos were cultured in the microchannel device using 500μL of KSOM with amino acids (MR-106-D, Speciality Media, Phillipsburg, NJ, USA.); no additional supplements were added. Groups of 10 embryos were cultured in standard microdrops (control) using 30μL of the same medium covered with mineral oil. Embryos were cultured in a 100% humidified, 5% CO2 in air atmosphere at 37°C for 96h. Embryos were allocated to the control treatment or the microchannel device treatment using a randomized block design. The percentage of embryos at each stage of development was evaluated at 24-h intervals. The stage of embryo development at each observation was analyzed by ANOVA using the general linear model in SAS (PROC GLM, type I sum of squares). Blastocyst development in the microchannel device was not different when compared to results obtained in the standard drop. The percentage of blastocysts developing, when analyzed from one-cell stage, was 29±5% for the control and 26±6% for the microchannel. The percentage of blastocysts, when analyzed from cleavage, was 35±5% for the standard drop and 31±7% for the microchannel device. The results obtained are encouraging, when considering the non-optimized medium and mouse strain utilized in this experiment. In conclusion, the results show the microchannel device may be considered an alternative technique for use in embryo culture as it supports development of mouse embryos from one-cell stage to blastocyst.

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Human Adenovirus Uptake by Preirnplantation Mouse Embryos

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100094607 ◽

1972 ◽

Vol 30 ◽

pp. 268-269

Author(s):

D. G. Chase ◽

W. Winters ◽

L. Piko

Keyword(s):

Hela Cells ◽

Human Adenovirus ◽

Mouse Embryos ◽

Equilibrium Density ◽

Cell Stage ◽

Virus Attachment ◽

Preimplantation Mouse Embryos ◽

Embryo Culture Medium ◽

Preimplantation Mouse ◽

Mouse Cells

Although the outlines of human adenovirus entry and uncoating in HeLa cells has been clarified in recent electron microscope studies, several details remain unclear or controversial. Furthermore, morphological features of early interactions of human adenovirus with non-permissive mouse cells have not been extensively documented. In the course of studies on the effects of human adenoviruses type 5 (AD-5) and type 12 on cultured preimplantation mouse embryos we have examined virus attachment, entry and uncoating. Here we present the ultrastructural findings for AD-5.AD-5 was grown in HeLa cells and purified by successive velocity gradient and equilibrium density gradient centrifugations in CsCl. After dialysis against PBS, virus was sedimented and resuspended in embryo culture medium. Embryos were placed in culture at the 2-cell stage in Brinster's medium.

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Modification of the method for analysis of genome editing results using CRISPR/Cas9 system on preimplantation mouse embryos

Bulletin of Russian State Medical University ◽

10.24075/brsmu.2016-03-02 ◽

2016 ◽

pp. 15-20 ◽

Cited By ~ 1

Author(s):

T. V. Dimitireva ◽

◽

D. A. Reshetov ◽

V. E. Zhernovkov ◽

D. E. Vlodavets ◽

...

Keyword(s):

Genome Editing ◽

Mouse Embryos ◽

Preimplantation Mouse Embryos ◽

Preimplantation Mouse

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Totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage

Scientific Reports ◽

10.1038/s41598-021-90653-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Marino Maemura ◽

Hiroaki Taketsuru ◽

Yuki Nakajima ◽

Ruiqi Shao ◽

Ayaka Kakihara ◽

...

Keyword(s):

Inner Cell Mass ◽

Cell Mass ◽

Mouse Embryos ◽

Primitive Endoderm ◽

Cell Stage ◽

Supportive Environment ◽

Mouse Zygotes ◽

Single Blastomeres ◽

Multicellular Organisms

AbstractIn multicellular organisms, oocytes and sperm undergo fusion during fertilization and the resulting zygote gives rise to a new individual. The ability of zygotes to produce a fully formed individual from a single cell when placed in a supportive environment is known as totipotency. Given that totipotent cells are the source of all multicellular organisms, a better understanding of totipotency may have a wide-ranging impact on biology. The precise delineation of totipotent cells in mammals has remained elusive, however, although zygotes and single blastomeres of embryos at the two-cell stage have been thought to be the only totipotent cells in mice. We now show that a single blastomere of two- or four-cell mouse embryos can give rise to a fertile adult when placed in a uterus, even though blastomere isolation disturbs the transcriptome of derived embryos. Single blastomeres isolated from embryos at the eight-cell or morula stages and cultured in vitro manifested pronounced defects in the formation of epiblast and primitive endoderm by the inner cell mass and in the development of blastocysts, respectively. Our results thus indicate that totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage.

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STAT3 Is an Upstream Regulator of Granzyme G in the Maternal-To-Zygotic Transition of Mouse Embryos

International Journal of Molecular Sciences ◽

10.3390/ijms22010460 ◽

2021 ◽

Vol 22 (1) ◽

pp. 460

Author(s):

Huan Ou-Yang ◽

Shinn-Chih Wu ◽

Li-Ying Sung ◽

Shiao-Hsuan Yang ◽

Shang-Hsun Yang ◽

...

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Fluorescent Protein ◽

Mouse Embryos ◽

Cell Nuclei ◽

Cell Stage ◽

Promoter Assay ◽

Maternal To Zygotic Transition ◽

Mouse Zygotes ◽

Time Specific

The maternal-to-zygotic transition (MZT), which controls maternal signaling to synthesize zygotic gene products, promotes the preimplantation development of mouse zygotes to the two-cell stage. Our previous study reported that mouse granzyme g (Gzmg), a serine-type protease, is required for the MZT. In this study, we further identified the maternal factors that regulate the Gzmg promoter activity in the zygote to the two-cell stage of mouse embryos. A full-length Gzmg promoter from mouse genomic DNA, FL-pGzmg (−1696~+28 nt), was cloned, and four deletion constructs of this Gzmg promoter, Δ1-pGzmg (−1369~+28 nt), Δ2-pGzmg (−939~+28 nt), Δ3-pGzmg (−711~+28 nt) and Δ4-pGzmg (−417~+28 nt), were subsequently generated. Different-sized Gzmg promoters were used to perform promoter assays of mouse zygotes and two-cell stage embryos. The results showed that Δ4-pGzmg promoted the highest expression level of the enhanced green fluorescent protein (EGFP) reporter in the zygotes and two-cell embryos. The data suggested that time-specific transcription factors upregulated Gzmg by binding cis-elements in the −417~+28-nt Gzmg promoter region. According to the results of the promoter assay, the transcription factor binding sites were predicted and analyzed with the JASPAR database, and two transcription factors, signal transducer and activator of transcription 3 (STAT3) and GA-binding protein alpha (GABPα), were identified. Furthermore, STAT3 and GABPα are expressed and located in zygote pronuclei and two-cell nuclei were confirmed by immunofluorescence staining; however, only STAT3 was recruited to the mouse zygote pronuclei and two-cell nuclei injected with the Δ4-pGzmg reporter construct. These data indicated that STAT3 is a maternal transcription factor and may upregulate Gzmg to promote the MZT. Furthermore, treatment with a STAT3 inhibitor, S3I-201, caused mouse embryonic arrest at the zygote and two-cell stages. These results suggest that STAT3, a maternal protein, is a critical transcription factor and regulates Gzmg transcription activity in preimplantation mouse embryos. It plays an important role in the maternal-to-zygotic transition during early embryonic development.

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Nobiletin enhances the development and quality of bovine embryos in vitro during two key periods of embryonic genome activation

Scientific Reports ◽

10.1038/s41598-021-91158-7 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Karina Cañón-Beltrán ◽

Yulia N. Cajas ◽

Serafín Peréz-Cerezales ◽

Claudia L. V. Leal ◽

Ekaitz Agirregoitia ◽

...

Keyword(s):

Embryonic Development ◽

Signaling Pathway ◽

Akt Signaling ◽

Mitochondrial Activity ◽

Bovine Embryos ◽

Akt Signaling Pathway ◽

Cell Stage ◽

Development In Vitro

AbstractIn vitro culture can alter the development and quality of bovine embryos. Therefore, we aimed to evaluate whether nobiletin supplementation during EGA improves embryonic development and blastocyst quality and if it affects PI3K/AKT signaling pathway. In vitro zygotes were cultured in SOF + 5% FCS (Control) or supplemented with 5, 10 or 25 µM nobiletin (Nob5, Nob10, Nob25) or with 0.03% dimethyl-sulfoxide (CDMSO) during minor (2 to 8-cell stage; MNEGA) or major (8 to 16-cell stage; MJEGA) EGA phase. Blastocyst yield on Day 8 was higher in Nob5 (42.7 ± 1.0%) and Nob10 (44.4 ± 1.3%) for MNEGA phase and in Nob10 (61.0 ± 0.8%) for MJEGA phase compared to other groups. Mitochondrial activity was higher and lipid content was reduced in blastocysts produced with nobiletin, irrespective of EGA phase. The mRNA abundance of CDK2, H3-3B, H3-3A, GPX1, NFE2L2 and PPARα transcripts was increased in 8-cells, 16-cells and blastocysts from nobiletin groups. Immunofluorescence analysis revealed immunoreactive proteins for p-AKT forms (Thr308 and Ser473) in bovine blastocysts produced with nobiletin. In conclusion, nobiletin supplementation during EGA has a positive effect on preimplantation bovine embryonic development in vitro and corroborates on the quality improvement of the produced blastocysts which could be modulated by the activation of AKT signaling pathway.

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