Yeast Genome Editing
As biotechnological applications of synthetic biology tools including multiplex genome engineering are expanding rapidly, the construction of strategically designed yeast cell factories becomes increasingly possible. Coupling the CRISPR/Cas system with traditional yeast multiplex genome integration or donor DNA delivery methods expedites strain development through increased efficiency and accuracy. Novel approaches such as pre-placing synthetic sequences in the genome along with improved bioinformatics tools and automation technologies have the potential to further streamline the strain development process.
Yeast Genome Editing Methods
- Double strand break (DSB) mechanism mediated either by non‐homologous end joining (NHEJ) or homologous recombination (HR).
- Cre‐loxP mediated recombination. Depending on the orientation and location of the loxP sequences, activated Cre recombinase can catalyse deletions, inversions or translocation of a chromosomal fragment.
- In delitto perfetto a CORE cassette is used to replace a gene or sequence of interest by HR. Subsequently, the CORE cassette is removed by HR using oligonucleotides complementary to the flanking regions of the cassette.
- The meganuclease mediated DSB method utilizes a CORE cassette. The addition of an I‐SceI site and induction of DSB significantly increase the recombination rate.
- CRISPR/Cas9 system. A single or double plasmid system are used to express the Cas9 endonuclease and guide RNA (s) (gRNA). Alternatively, the Cas9 gene is integrated into the yeast genome and the gRNA is delivered on a plasmid. After expression, the gRNA locates the target sequence and the endonuclease cleaves the foreign DNA that subsequently leads to either NHEJ or HR events.
Service Specifications
| Service Details |
Provided by Clients |
Deliverables |
| Construction of knock-out strains |
- Strain that needs to be modified along with strain information.
-
Information of the knock-out gene along with knock-out sequences. Or knock-in sites and replacement sequences.
|
- Report (Reagents, instruments, experimental protocol, sequencing results, etc.)
-
Sequencing File
-
Edited Strains
|
| Construction of knock-in strains |
| Construction of site directed mutagenesis stains |
* Note: plasmids for editing are not delivered.
Figure 1 Overview of the yeast genome editing methods
The budding yeast Saccharomyces cerevisiae is one of the most extensively used model organism for studying eukaryotic functional genomics, metabolic pathways, aging, exploration of protein interactions and as a bio‐producer of a wide range of chemicals and by‐products. The process robustness and the ease of maintenance and manipulation of this yeast make it a perfect candidate for the development of new genetically engineered strains for research and industrial applications.
The methylotrophic yeast Pichia pastoris is one of the most commonly used expression systems for heterologous protein production. However the recombination machinery in P. pastoris is less effective in contrast to Saccharomyces cerevisiae, where efficient homologous recombination naturally facilitates genetic modifications. The lack of simple and efficient methods for gene disruption and specifically integrating cassettes has remained a bottleneck for strain engineering in P. pastoris. The establishment of CRISPR/Cas9 technologies for P. pastoris solve this problem and demonstrate targeting efficiencies approaching 100%. Our experts use optimized CRISPR/Cas9 system for gene editing. This system allows rapid, marker-less genome engineering in P. pastoris enabling unprecedented strain and metabolic engineering applications.
Creative Biogene has excellent microbiology experts, well-established gene editing platforms, and professional bioinformatics analysis to provide you with a one-stop service in yeast genome editing. If you are interested in our services, please contact us for more details.
For Research Use Only.
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