Base editing reveals an essential role for NANOG in human embryogenesis

Area of Science:

• Developmental Biology
• Genetics
• Regenerative Medicine

Background:

• Understanding early human cell lineage specification is crucial for regenerative medicine, infertility, and pregnancy loss research.
• Mouse models offer insights but translating findings to human embryos is challenging due to ethical, technical, and biological constraints.
• Previous genome-editing approaches in human embryos caused genotoxicity, limiting functional studies of developmental regulators.

Purpose of the Study:

• To investigate the role of the transcription factor NANOG in early human development.
• To establish a genotoxicity-free method for functional studies of developmental regulators in human embryos.
• To explore the utility of adenine base editing (ABE8e) for precise gene knockout in human embryos.

Main Methods:

• Application of adenine base editing (ABE8e) to target an exon splice donor site of NANOG.
• Induction of a splicing defect for functional knockout of NANOG without causing genotoxicity.
• Assessment of off-target editing and analysis of cellular differentiation post-NANOG knockout.

Main Results:

• Adenine base editing successfully created a functional knockout of NANOG in human embryos with limited off-target effects and no detectable genotoxicity.
• Loss of NANOG disrupted pluripotent epiblast specification, leading to differentiation towards primitive endoderm or trophectoderm lineages.
• Human embryos exhibited retention of primitive endoderm differentiation, a distinct functional compensation compared to mouse models.

Conclusions:

• NANOG plays an essential role in maintaining human pluripotency and epiblast specification.
• Adenine base editing is a valuable and safe tool for functional interrogation of developmental genes in human embryos.
• Direct investigation of human development reveals species-specific mechanisms, distinct from those observed in mouse models.