YLC-assembly: large DNA assembly via yeast life cycle

Abstract

As an enabling technique of synthetic biology, the scale of DNA assembly largely determines the scale of genetic manipulation. However, large DNA assembly technologies are generally cumbersome and inefficient. Here, we developed a YLC (yeast life cycle)-assembly method that enables in vivo iterative assembly of large DNA by nesting cell-cell transfer of assembled DNA in the cycle of yeast mating and sporulation. Using this method, we successfully assembled a hundred-kilobase (kb)-sized endogenous yeast DNA and a megabase (Mb)-sized exogenous DNA. For each round, over 104 positive colonies per 107 cells could be obtained, with an accuracy ranging from 67% to 100%. Compared with other Mb-sized DNA assembly methods, this method exhibits a higher success rate with an easy-to-operate workflow that avoid in vitro operations of large DNA. YLC-assembly lowers the technical difficulty of Mb-sized DNA assembly and could be a valuable tool for large-scale genome engineering and synthetic genomics.

Crafting Genetic Diversity: Unlocking the Potential of Protein Evolution

“…The simultaneous expression of phage-derived T5 DNA exonuclease and T4 DNA ligase facilitates in vivo DNA assembly in a diverse range of microorganisms, such as Cupriavidus necator, Pseudomonas putida, Lactobacillus plantarum, and Yarrowia lipolytica. Another cutting-edge technique, the Yeast Life Cycle (YLC) assembly method [28], capitalizes on CRISPR-Cas9 and yeast meiosis to iteratively assemble large DNA fragments. The advantage of the YLC method lies in bypassing challenging in vitro steps associated with handling and importing large DNA fragments into a host system.…”

Crafting Genetic Diversity: Unlocking the Potential of Protein Evolution

“…Simultaneous expression of phage-derived T5 DNA exonuclease and T4 DNA ligase facilitates in vivo DNA assembly in a diverse range of microorganisms such as Cupriavidus necator, Pseudomonas putida, Lactobacillus plantarum, and Yarrowia lipolytica. Another cutting-edge technique, the Yeast Life Cycle (YLC) assembly method [27], capitalizes on CRISPR-Cas9 and yeast meiosis to iteratively assemble large DNA fragments. The advantage of the YLC method lies in bypassing challenging in vitro steps associated with handling and importing large DNA fragments into a host system.…”

Application and Technical Challenges in Design, Cloning, and Transfer of Large DNA

“…Alternatively, large DNA already present in the cells can be directly used for in vivo assembly through transfer methods such as cell fusion. In vivo assembly of large DNA has demonstrated success across various species, including Mycoplasm, Saccharomyces cerevisiae [2][3][4][5][6][7][8], Escherichia coli [9,10], and mammalian cells [11,12]. Currently, host cells commonly used for intracellular assembly include E. coli, Bacillus subtilis, and S. cerevisiae.…”