Project description:The first mitotic division causes both parental genomes present in the zygote to segregate into two biparental diploid daughter cells. This fundamental tenet was challenged by the observation that blastomeres with different genome ploidy and distinct parental genotypes can coexist within individual embryos. We hypothesized that whole parental genomes can segregate into distinct blastomere lines during the multipolar division of the zygote, a phenomenon referred to as “heterogoneic” cell division. Here, we provide evidence of genome-wide segregation errors in two human blastocysts and further pinpoint its origin in a bovine model by mapping the genomic landscape of 82 blastomeres from 25 embryos that underwent multipolar division at the zygote stage using genome-wide SNP arrays and sequencing. In most embryos, the coexistence of androgenetic and diploid or polyploid blastomeres with or without anuclear blastomeres, androgenetic and anuclear blastomeres, and androgenetic and gynogenetic blastomeres within the same embryo provided proof that multipolar zygotic division coincides with heterogoneic segregation of the parental genome. By mapping the segregational origin of the genomic content, we deduced distinct segregation mechanisms underlying heterogoneic cell division including segregation by a tripolar spindle, the pronuclear extrusion of a paternal genome and, the operation of an ectopic paternal or private parental spindles. Polyspermic embryos expel excessive paternal genomes resulting in an androgenetic or polyploid blastomere. Confirming the results in human blastocysts we found genome-wide segregation errors to persist in bovine blastocysts.
Project description:To assess genome-wide off-target activity of base editors and Cas9 nucleases identified by CHANGE-seq-BE, Digenome-seq, and CHANGE-seq, we performed hybrid capture sequencing for five therupatic loci (B2M, CBLB, CD7, CIITA, PDCD1) in human primary T-cells and PCSK9 in human hepatocytes. We observed high on-target editing for ABE, CBE, Cas9 mRNA edited cells and potential off-targets confirmed by hybrid capture sequencing. Our results demonstrate that CHANGE-seq-BE is highly sensitive than Digenome-seq to detect more bona fide off-targets with cellular activity ranging from 0.5% to 92.2%. Moreover, ABE exhibit more off-targets than Cas9 under same delivery conditions.
Project description:To assess genome-wide off-target activity of base editors and Cas9 nucleases identified by CHANGE-seq-BE, Digenome-seq, and CHANGE-seq, we performed hybrid capture sequencing for five therupatic loci (B2M, CBLB, CD7, CIITA, PDCD1) in human primary T-cells and PCSK9 in human hepatocytes. We observed high on-target editing for ABE, CBE, Cas9 mRNA edited cells and potential off-targets confirmed by hybrid capture sequencing. Our results demonstrate that CHANGE-seq-BE is highly sensitive than Digenome-seq to detect more bona fide off-targets with cellular activity ranging from 0.5% to 92.2%. Moreover, ABE exhibit more off-targets than Cas9 under same delivery conditions.
Project description:Little is understood of the molecular mechanisms involved in the earliest cell fate decision in human development, leading to the establishment of the preimplantation embryo’s outer trophectoderm (TE) layer, involved in implantation, while maintaining the inner cell mass (ICM) stem cell population. Using multiple systems biology approaches to compare developmental stages in the early human embryo with single cell transcript data from blastomeres, we have shown that blastomeres considered to be totipotent are not transcriptionally equivalent. Furthermore we have linked the developmental interactome to individual blastomeres and to later cell lineage. This new understanding has clinical implications for understanding the impact of fertility treatments and developmental programming of long term health.
Project description:Supplemented with splicing inhibitor PlaB, we realize in vitro culturing of human totipotent stem cells comparable to 2-4-cell blastomeres Assay for transposase accessible chromatin with high-throughput sequencing in hESC sand hTBLCs
Project description:Supplemented with splicing inhibitor PlaB, we realize in vitro culturing of human totipotent stem cells comparable to 2-4-cell blastomeres Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for h3k4me3 and h3k27me3 in hESC sand hTBLCs
Project description:Upon 2-cell embryo splitting, individual blastomeres were compared and contrasted with each other respecting pair associations (e.g. blastomere '1a' and '1b' of embryo 1, '2a' and '2b' of embryo 2, and so forth) Transcriptome analysis followed by cluster analysis (Ward) was able to match a minority of the blastomeres with the correct sister blastomere
Project description:This study describes the combined sequencing of the genomes and transcriptomes of single blastomeres from mouse 8-cell stage embryos.
