Frontier Science Center for Synthetic Biology (Ministry of Education), Tianjin University, Tianjin, 300072, China.
Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, China.
ACS Synth Biol. 2021 Sep 17;10(9):2222-2230. doi: 10.1021/acssynbio.1c00132. Epub 2021 Aug 22.
DNA inversion is a type of site-specific recombination system that plays an important role in the generation of genetic diversity and phenotypic adaptation by programmed rearrangements in bacteria. However, no such inversion system exhibiting a strong directionality bias has been identified or developed in eukaryotes yet. Here, using directed evolution of Rci recombinase, a tyrosine recombinase from a bacterial DNA inversion system, we identified a mutant Rci8 with a ratio of inversion/deletion up to ∼4320 in yeast. Based on Rci8 recombinase and sites, we have established a DNA inversion system in yeast and mammalian cells, enabling specificity for DNA inversions between inverted sites over deletions between directly repeated sites. Our results validated that the reversible DNA inversion system can act as an on/off transcriptional switch. Moreover, we demonstrate that the inversion system can also work on linear chromosomes. The eukaryotic DNA inversion system would provide a new tool for fields of genetic circuits, cellular barcoding, and synthetic genomes.
DNA 倒位是一种特定于位点的重组系统，通过细菌中的程序性重排，在遗传多样性和表型适应的产生中发挥重要作用。然而，在真核生物中尚未鉴定或开发出具有强方向性偏差的此类倒位系统。在这里，我们使用来自细菌 DNA 倒位系统的酪氨酸重组酶 Rci 进行定向进化，在酵母中鉴定出一个突变体 Rci8，其倒位/缺失比高达约 4320。基于 Rci8 重组酶和 位点，我们在酵母和哺乳动物细胞中建立了一个 DNA 倒位系统，能够特异性地在倒位位点之间进行 DNA 倒位，而不是在直接重复位点之间进行缺失。我们的结果验证了可逆 DNA 倒位系统可以作为转录的开/关开关。此外，我们还证明该倒位系统也可用于线性染色体。该真核 DNA 倒位系统将为遗传电路、细胞条形码和合成基因组等领域提供新的工具。
