Authors
Creative Biogene
Summary
Creative Biogene is a leading biotechnology company offering the best Saccharomyces cerevisiae 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 Saccharomyces cerevisiae genome editing services.
Saccharomyces cerevisiae is an important eukaryotic model organism, which is widely used as industrial host for production of fuels, chemicals and recombinant proteins. Genome editing of S. cerevisiae has profound meaning in the research and industry. Thanks to its excellent HR capability, S. cerevisiae is relatively easy to engineer genetically.
Materials
Red/ET Recombination permits the engineering of DNA in S. cerevisiae 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 S. cerevisiae 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 S. cerevisiae. 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 S. cerevisiae. 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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