Graduate Student The Francis Crick Institute London, England, United Kingdom
Abstract: The molecular mechanisms regulating the establishment of the human epiblast are poorly understood in human embryogenesis. To date, a small number of studies have used CRISPR/Cas9 mutagenesis to investigate gene function during pre-implantation development across species, including human embryos. These studies have recurrently identified loss of heterozygosity (LOH), genomic instability and complex chromosomal rearrangements following CRISPR/Cas9-mediated double stranded breaks (DSBs). To identify alternative methods to study gene function in human embryogenesis we adapt the adenine base editing (ABE) system and targeted the transcription factor NANOG as a proof of principle. The ABE system allows single base A>G edits that can be used to ablate the eukaryotic exon/intron splice motif, resulting in nonsense transcript production and loss of gene expression. We find efficient loss of NANOG protein following base editing in human embryonic stem cells. We demonstrate highly efficacious editing rates (>70%) in mouse and human pre-implantation embryos. We further observe the absence of any LOH events at the on-target locus in both species of embryos. Using single cell RNA sequencing, we identified that NANOG null mutations led to the misexpression of molecular markers of the epiblast and yolk sac and suggest that the inner cell mass is stuck in an unspecified state in the mouse. We are currently analysing single cell RNA sequencing of NANOG targeted human pre-implantation embryos and will be interested to compare this phenotype to the mouse to determine conserved and divergent mechanisms of NANOG function. Overall, we demonstrate that adenine base editing is a powerful tool to perform precise single base pair changes to perturb and study gene function in human embryogenesis.
