Lifeasible provides markerless gene editing services in polyploidy organisms and plasmids, including cyanobacteria, yeast and chloroplasts of plants. Our service is based on site-specific recombinases system, exploiting the polyploidy of their chromosome as a natural counterselection system.
RecoNase™ platform is an expert of gene editing, using site-specific recombinase system and counterselection of polyploidy as tools to complete markerless gene editing quickly and efficiently.
Site-specific recombinase technology is a gene editing tool that replacing target sequence of DNA with homologous recombination. They catalyze directionally DNA exchange reactions between a pair of short target site sequences. Each recombinase system can provide three types of DNA rearrangements, including deletion, inversion and translocation.
Deletion
If the short target sites face in the same direction, DNA sequence between them will be deleted.
Inversion
If the sites are in opposite direction, DNA sequence between them will be reversed.
Translocation
If the sites are in different DNA molecules, translocation will happen between genes the sites.
Site-specific recombinases generally belong to two subfamilies: tyrosine site-specific recombinases (T-SSR) and serine site-specific recombinases (S-SSR), depending on the mechanism. The individual of each these two families performs reaction with similar outcomes, but they don’t interfere each other. Each of the enzymes recombine one pair of sites, which are either identical or distinct.
Here is the list of available SSRs, by combining them in gene editing, multiple gene editing is feasible.
| T-SSR (Recombinases-sites) | S-SSR (Recombinases-sites) | ||
|---|---|---|---|
| • Cre-loxP • Dre-rox • Vika-vox • Flp-FRT • R-RRT |
• Kw-KwRT • Kd-KdRT • B2-B2RT • B3-B3RT • λ-Int/attP |
• γδ-attP/attB • ParA-attP/attB • Tn3-attP/attB • Gin-attP/attB |
• ΦC31-attP/attB • Bxb1-attP/attB • R4-attP/attB |
Polyploidy is the heritable genetic character of possessing more than two complete chromosome sets, which is very common in microorganisms and plants, as well as among some groups of fish and amphibians. When genes encoding DNA modifying enzymes are integrated in essential gene of polyploidy (cells become merodiploid), as facilitated by antibiotic selection. As long as the antibiotic selection maintains, strain remains merodiploid, and once the required DNA modification is complete, removal of antibiotic selection lead promptly elimination of DNA modification enzymes gene from genome.
Counterselection of Polyploidy Genome
Lifeasible provides gene editing service by exploits this feature of polyploidy organisms to achieve markerless gene editing.
By introducing site-specific recombinases system into genome via plasmid constructed with overlapping primers and Gibson Assembly, gene editing can be completed markerlessly in cyanobacteria, chloroplast of plant and yeast with the RecoNase™ platform.
Under condition of gene knocking in, plasmid containing exogenous gene and antibiotic resistance gene (Abx resistance cassette) flanked with site-specific recombinant sites will be introduced into genome. Then the plasmid containing recombinases gene and another antibiotic resistance gene will be introduced into genome. Under the condition that antibiotic exists, the recombinases will be expressed to knock out the antibiotic resistance gene flanked by site-specific recombinant sites. Once the desire gene modification is completed, removal of antibiotic selection will result in the elimination of recombinases gene by counterselection. In this way, gene can be knocked in markerlessly.
Under condition of gene knocking out, plasmid containing only antibiotic resistance gene flanked with site-specific recombinant sites will be introduced into genome first. The following steps will be the same as those in gene knocking in.
RecoNase™ platform has extensive experience in gene editing. With our advanced and mature technology, we provide you multiple options of gene editing services using recombinases system. For assisting our global customers making progress in their field better and faster, RecoNase™ platform provides pre-made mutant for research use as well. With first-class instrument and equipment, as well as professional experts in gene editing territory, the platform can promote the project satisfying your requirement quickly and efficiently. Please feel free to contact us, get assisted with our strong and advanced platform.
