Optimizing Yeast Homologous Recombination for Splicing Large Coronavirus Genome Fragments

Abstract

Reverse genetics is a useful tool for studying viruses and developing vaccines for coronaviruses. However, constructing and manipulating the coronavirus genome in Escherichia coli can be time-consuming and challenging due to its large size and instability. Homologous recombination, a genetic manipulation mechanism found in organisms, is essential for DNA repair, gene recombination, and genetic engineering. In yeast, particularly Saccharomyces cerevisiae, homologous recombination technology is commonly used for constructing gene expression plasmids and genome editing. In this study, we successfully split and spliced a 30 kb viral genome fragment using yeast homologous recombination. By optimizing the program parameters, such as homologous arm lengths and fragment-to-vector ratios, we achieved a splicing efficiency of up to 97.9%. The optimal parameters selected were a 60 bp homologous sequence size and a vector fragment ratio of 1:2:2:2:2:2 for yeast homologous recombination of large DNA fragments.

Keywords: DNA assembly; automated splicing; reverse genetics; synthetic biology; yeast homologous recombination.

Figure 1

The experimental operation process of this study.

Figure 2

Recombination efficiency of different homologous arm lengths and vector fragment ratios.

Figure 3

Identification of YAC plasmid. (A) PCR identification of YAC plasmid; M: 2K marker (BM101, TransGen Biotech, Beijing, China); 1–7: identification results of overlapping areas 1–7. (B) Identification results of double enzyme digested YAC plasmids; M: DNA marker (NEB, N3239S); 1: YAC plasmid with BamHI-HF (NEB, R3136M); 2: YAC plasmid with SbfI-HF (NEB, R3642S); 3: double enzyme digestion of YAC plasmid by BamHI-HF and SbfI-HF.