Yeast nuclei-mediated precise delivery of synthetic megabase-scale human DNA into mammalian embryos

The cross-species delivery of megabase-scale synthetic DNA molecules, from microorganisms into mammalian cells, remains a major challenge for synthetic genomics. Recently, we developed nucleus isolation for chromosome extraction (NICE), a method that enables the isolation of yeast nuclei containing intact synthetic megabase-scale DNA with preserved chromatin structure. By leveraging the unique epigenomic features of Saccharomyces cerevisiae, which lacks cytosine methylation and repressive histone marks, synthetic DNA encapsulated within isolated yeast nuclei was successfully delivered into mouse early embryos, maintaining a naive state. This work established a unique platform for studying the establishment of de novo epigenetic modifications and their influence on transcriptional regulation over time. Here, we provide a detailed protocol for NICE, including the isolation of yeast nuclei and their subsequent delivery into mammalian embryos. The high-concentration and high-purity isolated nuclei can be stored at -80 °C for >6 months. Using microinjection, we achieved 100% delivery efficiency, reliably transferring isolated yeast nuclei into mouse embryos. The entire procedure, including pulsed-field gel electrophoresis verification, can be completed within ~5 d. When the isolated yeast nuclei are intended for cross-species delivery into embryos, prior familiarity with mammalian embryo microinjection techniques may be required. This protocol offers an efficient and reliable method for the delivery of large-scale genetic information, advancing the study of complex biological functions.