Applying CRISPR in Yeasts | Gene-knockout, knock-in, etc.

Introduction:

 

Yeasts are eukaryotic, single-celled microorganisms classified as members of the fungus kingdom. The first yeast originated hundreds of millions of years ago, and at least 1,500 species are currently recognized. The yeast species Saccharomyces cerevisiae converts carbohydrates to carbon dioxide and alcohols in a process known as fermentation. The products of this reaction have been used in baking and the production of alcoholic beverages for thousands of years. S. cerevisiae is also an important model organism in modern cell biology research, and is one of the most thoroughly studied eukaryotic microorganisms. Researchers have cultured it in order to understand the biology of the eukaryotic cell and ultimately human biology in great detail. Other species of yeasts, such as Candida albicans, are opportunistic pathogens and can cause infections in humans. Yeasts have recently been used to generate electricity in microbial fuel cells and to produce ethanol for the biofuel industry.

 

The useful physiological properties of yeast have led to their use in the field of biotechnology. Fermentation of sugars by yeast is the oldest and largest application of this technology. Many types of yeasts are used for making many foods: baker's yeast in bread production, brewer's yeast in beer fermentation, and yeast in wine fermentation and for xylitol production. So-called red rice yeast is actually a mold, Monascus purpureus. Yeasts include some of the most widely used model organisms for genetics and cell biology.

The methods based on the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system have quickly gained popularity for genome editing and transcriptional regulation in many organisms, including yeast.

 

Red/ET recombination system is a classical method of microbial gene editing, which can achieve the knockin, knockout, point mutation and other modifications of the target gene. This technology has been widely used in genetic modification of genomic DNA, such as bacterial artificial chromosome(BAC), Escherichia coli chromosome. However, the efficiency of this system still needs to be improved. How to improve the efficiency of gene recombination and editing has always been a hotspot of microbial gene editing. Therefore, CRISPR/Cas9 technology is adopted to improve the efficiency of microbial gene editing. The price for gene editing yeast services will be similar to those in cell lines.

 

CRISPR/Cas9 is an acquired immune system in bacteria and archaea and can be used to fight against invading viruses and exogenous DNA. In recent years, the CRISPR/Cas9 gene-editing technology has been widely used because it is simple and efficient. It has been the most advanced method for gene editing. Ubigene developed CRISPR-B™ which optimizes the microbial gene-editing vectors and process. The efficiency and accuracy are much higher than traditional methods. CRISPR-B™ can be used in gene editing of bacteria and fungi.

 

Genome-editing in Yeast using precise targeting CRISPR

 

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. In the case of efficient cutting, the generated DSBs serve as a negative selection. Thus, there is no need for using a selective marker as in non-CRISPR genome editing methods. Relatively precise and flexible targeting and elimination of the need for positive selection are the two key advantages of the CRISPR/Cas9 technology for yeast genome engineering.

 

The rrk1 CRISPR-Cas9 method enables rapid and efficient genome manipulation and unlocks the CRISPR toolset for use in fission yeast

Application of the CRISPR-Cas9 genome editing system in the model organism  has been hampered by the lack of constructs to express RNA of arbitrary sequence. Therefore, researchers present expression constructs that use the promoter/leader RNA of K RNA (rrk1) and a ribozyme to produce the targeting guide RNA. Together with constitutive expression of Cas9, this system achieves selection-free specific mutagenesis with efficiencies approaching 100%. The fission yeast Schizosaccharomyces pombe has proven to be a useful model organism because of its higher degree of similarity to genomes of higher eukaryotes than the classic yeast model S. cerevisiae. S. pombe remains less well studied than S. cerevisiae, and lags behind in availability of molecular tools. In particular, lack of a portable RNA Pol III promoter to express sgRNA has prevented the implementation of the CRISPR-Cas9 system. A CRISPR-Cas9 system that enables specific, precise and efficient genome editing in S. pombe is developed. Its flexibility enables the use in fission yeast of the full range of CRISPR-derived tools for genome manipulation.

 

Combinatorial optimization of CRISPR/Cas9 expression enables precision genome engineering in  yeast

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. Therefore tools and methods for targeted genome modifications are of great interest. Researchers developed a CRISPR/Cas9 system that enables specific and precise genome engineering in P. pastoris as a potent alternative to the currently applied genome engineering strategies.

The establishment of CRISPR/Cas9 technologies for P. pastoris and demonstrate targeting efficiencies approaching 100%. However there appeared to be a narrow window of optimal conditions required for efficient CRISPR/Cas9 function for this host. Scientists systematically tested combinations of various codon optimized DNA sequences of CAS9, different gRNA sequences, RNA Polymerase III and RNA Polymerase II promoters in combination with ribozymes for the expression of the gRNAs and RNA Polymerase II promoters for the expression of CAS9. Only 6 out of 95 constructs were functional for efficient genome editing.

This optimized CRISPR/Cas9 system for gene disruption studies, was used to introduce multiplexed gene deletions and to test the targeted integration of homologous DNA cassettes. This system allows rapid, marker-less genome engineering in P. pastoris enabling unprecedented strain and metabolic engineering applications.

 

Ubigene developed CRISPR-B™ which optimizes the microbial gene-editing vectors and process. The efficiency and accuracy are much higher than traditional methods. CRISPR-B™ can be used in gene editing of bacteria and fungi. Easily achieve microbial gene knockout (KO), point mutation (PM) and knockin (KI).

 

References:

Vratislav Stovicek, Carina Holkenbrink, Irina Borodina, CRISPR/Cas system for yeast genome engineering: advances and applications, FEMS Yeast Research, Volume 17, Issue 5, August 2017, fox030, https://doi.org/10.1093/femsyr/fox030

Jacobs, J., Ciccaglione, K., Tournier, V. et al. Implementation of the CRISPR-Cas9 system in fission yeast. Nat Commun 5, 5344 (2014). https://doi.org/10.1038/ncomms6344

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, ISSN 0168-1656.