De novo Assembly and Delivery of Synthetic Megabase-Scale Human DNA into Mouse Early Embryos

Epigenetic modifications on natural chromosomes are inherited and maintained in a default state, making it challenging to remove intrinsic marks to study the fundamental principles of their establishment and further influence on transcriptional regulation. Here, we developed SynNICE, a method for precisely assembling and transferring intact, naïve, synthetic Mb-scale human DNA into early mouse embryos, to study de novo epigenetic regulation. By synthesizing and transferring the 1.14 Mb human AZFa (hAZFa) locus, we observed the spontaneous incorporation of murine histones and the establishment of DNA methylation at the 1-cell stage. Notably, DNA methylation from scratch was strongly enriched for repeat sequences without H3K9me3 reinforcement. Furthermore, the transcription of hAZFa initiated at the 4-cell stage was regulated by newly established DNA methylation. This method provides a unique platform for exploring de novo epigenomic regulation mechanisms in higher animals.