Abstract
Rearrangement of genomic DNA elements in a dynamic controlled fashion is a fundamental challenge. Site-specific DNA recombinases have been tamed as a powerful tool in genome editing. Here, we reported a DNA element rearrangement on the basis of a pairwise orthogonal recombination system which is comprised of two site-specific recombinases of Vika/vox and Cre/loxp in yeast Saccharomyces Creevisiae. Taking the advantage of the robust pairwise orthogonality, we showed that multi gene elements could be organized in a programmed way, in which rationally designed pattern of loxP and vox determined the final genotype after expressing corresponding recombinases. Finally, it was demonstrated that the pairwise orthogonal recombination system could be utilized to refine synthetic chromosome rearrangement and modification by loxP-mediated evolution, SCRaMbLE, in yeast cell carrying a completely synthesized chromosome III.
Highlights
Yeast is one of the well-studied cell models, which has significantly contributed to modern eukaryotic biology[18] and the pioneer organism, which has been utilized in food manufacturing through fermentation since the very early of human history[19]
Yeast is attracting more attentions in metabolic engineering applications[20,21]. We reported that another site-specific DNA recombinase Vika, originally identified in a gram-negative bacterium Vibro coralliilyticus, could functionally and deleted genomic DNA fragment via recognizing specific DNA site vox in yeast Saccharomyces cerevisiae[22,23] and other spices[24], including mammal cell and bacteria
Since the two systems belong to the same tyrosine recombinase family, they share high similarity in both sequence and secondary structure in their DNA recognition sites, in which one 8 base pairs spacer sequence are flanked by 13 base pairs inverted repeated sequence (Fig. 1A)
Summary
Yeast is one of the well-studied cell models, which has significantly contributed to modern eukaryotic biology[18] and the pioneer organism, which has been utilized in food manufacturing through fermentation since the very early of human history[19]. The Vika-vox system functioned properly even in yeast cell carrying the synthesized chromosome III on which 98 loxPsym sites are encoded Based on this robust pairwise orthogonal of Cre and Vika recombinases, we designed versatile genetic editing cassette by which DNA elements could be rearranged following a designed process and the final genotype output could be controlled through designing the pattern of loxP and vox location. We introduced the Vika-vox system into the SCRaMbLE of synthetic chromosome III and demonstrated that Vika-vox was able to efficiently terminate the genome scrambling through completely deletion of gene Cre. We believed that the robust orthogonal recombinase system could be powerful in developing highly controlled DNA element editing tool and especially for developing enhanced recombinase-drive genome directed evolution system
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