Abstract
POU5F1 is a transcription factor and master regulator of cell pluripotency with indispensable roles in early embryo development and cell lineage specification. The role of embryonic POU5F1 in blastocyst formation and cell lineage specification differs between mammalian species but remains completely unknown in cattle. The CRISPR/Cas9 system was utilized for targeted disruption of the POU5F1 gene by direct injection into zygotes. Disruption of the bovine POU5F1 locus prevented blastocyst formation and was associated with embryonic arrest at the morula stage. POU5F1 knockout morulas developed at a similar rate as control embryos and presented a similar number of blastomeres by day 5 of development. Initiation of SOX2 expression by day 5 of development was not affected by lack of POU5F1. On the other hand, CDX2 expression was aberrant in embryos lacking POU5F1. Notably, the phenotype observed in bovine POU5F1 knockout embryos reveals conserved functions associated with loss of human embryonic POU5F1 that differ from Pou5f1- null mice. The similarity observed in transcriptional regulation of early embryo development between cattle and humans combined with highly efficient gene editing techniques make the bovine a valuable model for human embryo biology with expanded applications in agriculture and assisted reproductive technologies.
Highlights
POU5F1 is a transcription factor and developmental control gene with regulatory roles in mammalian embryo development, cell lineage specification and maintenance of germ cell pluripotency[1]
Synthetic guide RNA targeting exon 2 (E2) of bovine POU5F1 was microinjected into the cytoplasm of bovine zygotes in complex with Cas[9] protein (Fig. 1A)
Adaptation of the CRISPR/Cas[9] system for directed intracytoplasmic injection of ribo-nucleoprotein constructs using a single sgRNA in a system devoid of cell lines and reconstructed embryos has allowed us to evaluate the function of bovine POU5F1 by targeted inactivation of the POU5F1 gene with high efficiency and repeatability
Summary
POU5F1 is a transcription factor and developmental control gene with regulatory roles in mammalian embryo development, cell lineage specification and maintenance of germ cell pluripotency[1]. POU5F1 is consistently expressed at EGA, suggesting some conserved roles in early development, including cell lineage specification and blastocyst formation[2]. The requirement of embryonic POU5F1 in bovine blastocyst development and cell lineage specification was investigated using a loss-of-function approach based on direct zygotic injection of CRISPR/Cas[9] components.
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