Pichia pastoris Genome Editing Service

Creative Biogene is a leading biotechnology company offering the best Pichia pastoris genome editing services. With years of experience and expertise in microbial genome editing, our talented scientists will work closely with you to provide any help in Pichia pastoris genome editing services.

Pichia pastoris (Komagataella phaffii), Due to its potent secretion of pure protein, less extensive glycosylation and high cell density growth, is one of the most commonly used expression systems for heterologous protein production. It has been widely used for protein production in basic and applied research.

Our P. pastoris genome editing services based on CRISPR/Cas9 technology and homologous recombination technique. The state-of-art P. pastoris editing system helps you successfully achieve gene knockout, gene insertion and point mutation for either research or industrial purposes.

Pichia pastoris Genome Editing Based on Homologous Recombination

Red/ET Recombination permits the engineering of DNA in P. pastoris using homologous recombination mediated by phage protein pairs, either RecE/RecT or Reda/Redb. The central step in Red/ET recombination is the crossover step between a targeting construct containing homology arms and the target which can be a gene locus on the P. pastoris chromosome by designing a homologous fusion fragment of the target gene, it is cloned into a suicide vector, and the suicide vector is transformed into the target P. pastoris. An insertion mutant is selected by antibiotic screening. Under the second round of reverse selection pressure, only the mutation that contain second homologous recombination and the loss of the suicide plasmid can survive. By PCR screening and sequencing, we can obtain the mutant of the strain.

Workflow of homologous recombination genome editing

Fig.1. Workflow of homologous recombination genome editing

Work flow:

• Homology arms design and suicide plasmid construction
• Recombineering
• Selection/Screening
• Validation

CRISPR/Cas9-mediated P. pastoris Genome Editing

CRISPR technology, which derived from the immune system present in bacteria and archaea, is an efficient genome-scale editing tool that has revolutionized conventional genetic engineering methods and unprecedentedly facilitated strain engineering. It enables fast and reliable genetic manipulation in P. pastoris. Two components are requested to work: a guide RNA (gRNA), e.g. under an RNA polymerase III promoter, and the nuclear localization tag fused DNA endonuclease, with Cas9 being the most commonly used.

When Cas9 protein and gRNA are expressed in yeast cells, Cas9 introduces DSBs that must be repaired by the cells via non-homologous end joining (NHEJ) or homologous recombination (HR). By supplying a DNA repair template for use in HR, various DNA modifications can be obtained.

Workflow of homologous recombination genome editing

Fig.2. Workflow of homologous recombination genome editing

Project steps:

• sgRNA design and construction
• Transformation
• Selection/Screening
• Validation

The advantages of CRISPR based P. pastoris genome editing in Creative Biogene:

• Fast turnaround time
• Scarless genome editing
• Multigene editing: can knock-out up to 5 genes simultaneously
• Easy selection: no selectable marker is required

What we could help?

• Gene disruption, deletion or insertion
• Reporter gene and tag integration
• Promoter fine tuning
• Introduction of point mutations

With years of experience in genome editing field, Creative Biogene could provide the most excellent service for P. pastoris’ genome editing. Our talent experts are dedicated to edit your P. pastoris with the greatest chance to succeed. Meanwhile, based on the commitment of prompt communication and on-time reporting, our staffs will ensure a high-efficiency service to meet the strict project timelines.

If you have any special requirements in our P. pastoris genome editing service, please feel free to contact us at . We are looking forward to working together with your attractive projects.

References
1. Yu D, et al. An efficient recombination system for chromosome engineering in Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 2000;97:5978–5983
2. Stovicek V, Holkenbrink C. Borodina I. CRISPR/Cas system for yeast genome engineering: advances and applications. FEMS Yeast Res. 2017;17:fox030.
3. Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production. Appl. Microbiol. Biotechnol., 98 (2014), pp. 5301-5317,
4. Astrid Weninger, Anna-Maria Hatzl, Christian Schmid, Thomas Vogl, Anton Glieder, Combinatorial optimization of CRISPR/Cas9 expression enables precision genome engineering in the methylotrophic yeast Pichia pastoris, Journal of Biotechnology, Volume 235, 2016, Pages 139-149,

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