Authors
Creative Biogene
Summary
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.
Introduction
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.
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.
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