A DNA Inversion System in Eukaryotes Established via Laboratory Evolution

• Published in: ACS synthetic biology 2021-09, Vol.10 (9), p.2222-2230
• Main Authors: Han, Peiyan, Ma, Yuan, Fu, Zongheng, Guo, Zhou, Xie, Jiangnan, Wu, Yi, Yuan, Ying-jin
• Format: Article
• Language: English
• Subjects: Directed Molecular Evolution; DNA Nucleotidyltransferases - genetics; DNA Nucleotidyltransferases - metabolism; DNA, Bacterial - genetics; DNA, Bacterial - metabolism; HEK293 Cells; Humans; Plasmids - genetics; Plasmids - metabolism; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Salmonella - genetics
• ISSN: 2161-5063; 2161-5063
• DOI: 10.1021/acssynbio.1c00132

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 sfxa101 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.