Xiong Guoqing, Huang Xuan, Hu Ao, Meng Zhixin, Cui Jiazhen, Feng Yuzhong, Chen Zhili, Lu Yuanyuan, Yang Qi, Liu Gang
Institutes of Physical Science and Information Technology, Anhui University, Hefei 230000, China.
Academy of Military Medical Sciences, Beijing 100850, China.
Int J Mol Sci. 2024 Dec 23;25(24):13742. doi: 10.3390/ijms252413742.
Reverse genetics is a useful tool for studying viruses and developing vaccines for coronaviruses. However, constructing and manipulating the coronavirus genome in 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.
反向遗传学是研究病毒和开发冠状病毒疫苗的有用工具。然而，由于冠状病毒基因组规模大且不稳定，在体内构建和操作其基因组可能既耗时又具有挑战性。同源重组是生物体中发现的一种基因操作机制，对DNA修复、基因重组和基因工程至关重要。在酵母中，特别是酿酒酵母，同源重组技术常用于构建基因表达质粒和基因组编辑。在本研究中，我们利用酵母同源重组成功地拆分和拼接了一个30 kb的病毒基因组片段。通过优化程序参数，如同源臂长度和片段与载体比例，我们实现了高达97.9%的拼接效率。为大DNA片段的酵母同源重组选择的最佳参数是60 bp的同源序列大小和1:2:2:2:2:2的载体片段比例。
