Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here, we establish a versatile blastocyst complementation platform ...based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution, embryogenesis, and human disease, interspecies blastocyst complementation might allow human organ generation in animals whose organ size, anatomy, and physiology are closer to humans. To date, however, whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals, the ungulates. We find that naïve hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead, an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos.
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•Naive rat PSCs robustly contribute to live rat-mouse chimeras•A versatile CRISPR-Cas9 mediated interspecies blastocyst complementation system•Naive rodent PSCs show no chimeric contribution to post-implantation pig embryos•Chimerism is observed with some human iPSCs in post-implantation pig embryos
Human pluripotent stem cells robustly engraft into both cattle and pig pre-implantation blastocysts, but show limited chimeric contribution to post-implantation pig embryos.
Embryonic stem cells (ESCs) are derived from the inner cell mass of preimplantation blastocysts. From agricultural and biomedical perspectives, the derivation of stable ESCs from domestic ungulates ...is important for genomic testing and selection, genome engineering, and modeling human diseases. Cattle are one of the most important domestic ungulates that are commonly used for food and bioreactors. To date, however, it remains a challenge to produce stable pluripotent bovine ESC lines. Employing a culture system containing fibroblast growth factor 2 and an inhibitor of the canonical Wnt-signaling pathway, we derived pluripotent bovine ESCs (bESCs) with stable morphology, transcriptome, karyotype, population-doubling time, pluripotency marker gene expression, and epigenetic features. Under this condition bESC lines were efficiently derived (100% in optimal conditions), were established quickly (3–4 wk), and were simple to propagate (by trypsin treatment). When used as donors for nuclear transfer, bESCs produced normal blastocyst rates, thereby opening the possibility for genomic selection, genome editing, and production of cattle with high genetic value.
One of the ultimate goals of regenerative medicine is the generation of patient-specific organs from pluripotent stem cells (PSCs). Sheep are potential hosts for growing human organs through the ...technique of blastocyst complementation. We report here the creation of pancreatogenesis-disabled sheep by oocyte microinjection of CRISPR/Cas9 targeting PDX1, a critical gene for pancreas development. We compared the efficiency of target mutations after microinjecting the CRISPR/Cas9 system in metaphase II (MII) oocytes and zygote stage embryos. MII oocyte microinjection reduced lysis, improved blastocyst rate, increased the number of targeted bi-allelic mutations, and resulted in similar degree of mosaicism when compared to zygote microinjection. While the use of a single sgRNA was efficient at inducing mutated fetuses, the lack of complete gene inactivation resulted in animals with an intact pancreas. When using a dual sgRNA system, we achieved complete PDX1 disruption. This PDX1
fetus lacked a pancreas and provides the basis for the production of gene-edited sheep as a host for interspecies organ generation. In the future, combining gene editing with CRISPR/Cas9 and PSCs complementation could result in a powerful approach for human organ generation.
Genome editing using programmable nucleases has revolutionized biomedical research. CRISPR-Cas9 mediated zygote genome editing enables high efficient production of knockout animals suitable for ...studying development and relevant human diseases. Here we report efficient disabling pancreatogenesis in pig embryos via zygotic co-delivery of Cas9 mRNA and dual sgRNAs targeting the PDX1 gene, which when combined with chimeric-competent human pluriopotent stem cells may serve as a suitable platform for the xeno-generation of human tissues and organs in pigs.
The production of knock-out (KO) livestock models is both expensive and time consuming due to their long gestational interval and low number of offspring. One alternative to increase efficiency is ...performing a genetic screening to select pre-implantation embryos that have incorporated the desired mutation. Here we report the use of sheep embryo biopsies for detecting CRISPR/Cas9-induced mutations targeting the gene
PDX1
prior to embryo transfer.
PDX1
is a critical gene for pancreas development and the target gene required for the creation of pancreatogenesis-disabled sheep. We evaluated the viability of biopsied embryos in vitro and in vivo, and we determined the mutation efficiency using PCR combined with gel electrophoresis and digital droplet PCR (ddPCR). Next, we determined the presence of mosaicism in ~ 50% of the recovered fetuses employing a clonal sequencing methodology. While the use of biopsies did not compromise embryo viability, the presence of mosaicism diminished the diagnostic value of the technique. If mosaicism could be overcome, pre-implantation embryo biopsies for mutation screening represents a powerful approach that will streamline the creation of KO animals.
The substantial epigenetic remodeling that occurs during early stages of mammalian embryonic development likely contributes to reprogramming the parental genomes from a differentiated to a totipotent ...state and activation of the embryonic genome. Trimethylation of lysine 27 of histone 3 (H3K27me3) is a repressive mark that undergoes global dynamic changes during preimplantation development of several species. To ascertain the role of H3K27me3 in bovine preimplantation development we perturbed the activity of KDM6B, which demethylates H3K27me3. Knockdown of maternal KDM6B mRNA inhibited the reduction in global levels of H3K27me3 from 2-cell to 8-cell embryo stages and compromised development to the blastocyst stage; embryos that developed to the blastocyst stage had fewer inner cell mass (ICM) and trophectoderm (TE) cells. In addition, the transcriptome of KDM6B knockdown embryos was altered at the 8-cell stage and characterized by downregulation of transcripts related to transcriptional regulation, chromatin remodeling, and protein catabolism. Inhibiting the catalytic activity of KDM6B with a specific small molecule inhibitor also prevented the global decrease in H3K27me3 and compromised development to the blastocyst stage. These results indicate that histone demethylation activity, mediated by KDM6B, is required for the global decrease in H3K27me3, correct activation of the embryonic genome, and development to the blastocyst stage in bovine embryos.
CRISPR technologies used for mammalian embryology have wide implications from basic research to applications in agriculture and biomedicine. Confirmation of successful gene editing following ...CRISPR/Cas9 delivery is often limited to either protein expression or sequencing analyses of embryos but not both, due to technical challenges. Herein we report an integrative approach for evaluating both protein expression and genotype of single embryos from fixed bovine embryos previously subjected to CRISPR/Cas9 microinjection. The techniques described facilitate investigation of functional genomics in bovine embryos compatible with gene editing in livestock after zygotic CRISPR microinjection. These methods avoid traditional avenues that necessitate the use of gene-edited cell lines followed by nuclear transfer that hinder efficiency, limit physiological relevance and contribute to technical challenges.
Two experiments were performed to determine if there is a relation between the goat's hierarchical position, and (1) the conception rate and litter size obtained with hormonal oestrous ...synchronisation treatments and fixed timed artificial insemination, and (2) the number of corpus luteum (CL) and the embryo quality obtained in superovulatory treatments during the breeding and the non-breeding seasons. In the first experiment, the success index of goats from four herds was determined, and was related to the reproductive results after oestrous hormonal synchronisation and fixed time artificial insemination. There were no significant differences in fertility (
n
=
253; 49.5, 50.7 and 43.2%) or litter size (
n
=
185), which was 1.6
±
0.1, 1.8
±
0.1, and 1.8
±
0.1 (mean
±
SEM) for Low, Medium and High ranked goats, respectively (
P
>
0.1). In the second experiment, 37 and 34 superovulatory treatments were performed during the breeding and the non-breeding season, respectively. There was a link between hierarchical position and the number of corpus luteum in the non-breeding season and between dominance hierarchy and transferable embryos/fertilised embryos in the breeding season. We conclude that probably the social rank is not related to the conception rate or the litter size obtained after oestrous synchronisation treatments and fixed time artificial insemination in dairy goats under intensive reproductive management. Social hierarchy does not affect the response to superovulatory treatments in terms of the number of corpus luteum or the embryo quality during the breeding season. However, during the non-breeding season, hierarchical position may influence on the number of corpus luteum obtained.
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