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Method 'crisper'; for genetic editing in fungi



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CRISPR / Cas9 is now a home name associated with genetic engineering studies. Through innovative research described in their published paper Scientific Reportsa team of researchers from the University of Tokyo Science, the University of Meiji, and Tokyo University of Agriculture and Technology, led by Dr Takayuki Arazoe and Professor Shigeru Kuwata, have recently established a series of new strategies to increase the efficiency of gene disruption. to target new and "gene" genes using the CRISPR / Cas9 system in the rice blast fungus Pyricularia (Magnaporthe) oryzae. These strategies include a quicker introduction (one step) for genes, the use of small homologous sequences, and the avoidance of "patterns" of hosting a certain essential host and modifying a host component.

The team led by Dr Arazoe and Professor Kuwata have devised simple and quick techniques for gene editing (target gene targeting, sequence replacement, and reintroduction of desired genes) using CRISPR / Cas9 in the blast fungus. rice Pyricularia (Magnaporthe) oryzae, a type of filamentous fungus. After triggering results, the researchers are overcoming, "Plant and their pathogens are still compatible with their nature. It could take advantage of model pathogenic fungal mutations like Genome editing technique leads to the development of new techniques in genetic engineering. "

The active component of the CRISPR / Cas9 system binds to the target gene (DNA) region and causes a site-specific double doubled (DSB) in the DNA. The binding of this component effectively requires a specific "motif" or "pattern" of the name of the protospacer-nearby motif (PAM), which follows downstream of; r region target genes.

Most genome editing techniques require DSBs to be incentivized on the target site, which triggers DNA "repair" routes in the host. Homologous recombination is a mechanism for repairing DSBs, and is useful because it adds complementary sequences. However, the underlying methodology is time consuming, and its conventional efficiency depends on external factors such as the host properties as well as PAM. HR can be divided into two routes: "non-transsover" (gene conversion) and type "crossing". Cross-type repairs are known to occur in cells that are meiosis. However, the understanding of their role in cells with mitosis is limited, and information on such filamentous fungi is almost available. This gap in information that the researchers intended to address.

In their study, the researchers created a vector (gene supply system) first based on CRISPR / Cas9 to confirm cross-type AD in the receptor genes region in the rice blast fungus.

Then, to check gene targeting or "sequence replacement", they created a "mutant" vector, which was optimized for single cross-type AD, for disruption to target on the host gene & # 39 ency scytalone dehydration (SDH), a protein associated with the formation of melanin. This vector was introduced to the vector containing the gene for hygromycin B phosphotransferase (hph), which gives resistance to the antibiotic hygromycin B. The researchers were guessing t the same type of cross would insert the entire vector along with hph into the target site. The mutants with a SDH gene and disturbed them would be known as white colonies (due to loss of melanin) on a medium containing hygromycin B. The researchers found that the number of white colonies is 39 biobycin resistant has increased dramatically using the CRISPR / Cas9 vector, which means that the CRISPR / Cas9 system is effective in stimulating single AD type crosses. The greatest benefit of this technique is the need for very short homologous sequences (100 basic pairs; very small in molecular biology).

The researchers also used a similar strategy to check whether the introduction of genes (or “hit-in”) is possible through Human Resources one type of cross-over using the CRISPR / Cas9 vector. They used the green fluorescent protein gene (GFP), which is widely used as a "reporter" gene to make guest cells fainting fluorescent green when they are put into their genome. They guessed that a single AD crossing would lead to the introduction of GFP to the receiver sequence. Indeed, the use of a CRISPR / Cas9 vector has been found to have resulted in green fluorescent colonies on hygromycin medium. These findings show that the CRISPR / Cas9 system can be used for efficient "one step" gene generation.

This research suggests that PAMs are not all necessary for editing CRISPR / Cas9 genes in fungi. According to the success of the research, the team says, "We have found that filamentous fungi have unique genomic features, where diagonals are often stimulated, even in somatic cells," t By clearing the target DNA, these features were used to disrupt the DNA target or introduce "reporter" genes. We have also managed to increase the efficiency and speed of the goal-in, using a one-step process; This technology overcomes the constraint posed by PAMs – one of the biggest disadvantages of the CRISPR / Cas9 System – and enables more flexible genomes to be edited, which have been difficult in previous studies on filamentous fungi. "

Finally, when asked about the wider applications of this research, Dr Arazoe and Professor Kuwata state eloquently, "The fungus of rice blast is an important pathogen causing a devastating rice disease, which is the main food of the rice." CRISPR / Cas9 a genome editing technique developed in our study can accelerate molecular biological research on this pathogen, ultimately contributing to a stable food supply and plant-based food safety. apply to other filamentous fungi that are widely used in industry – especially in the processing of food industries, and fermentation. "

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Authors

  • Takayuki Arazoe, Department of Applied Biological Sciences, University of Tokyo Science, Tokyo
  • Misa Kuroki, Department of Applied Biological Sciences, University of Tokyo Science, Tokyo
  • Akihito Nozaka, Department of Applied Biological Sciences, University of Tokyo Science, Tokyo
  • Takashi Kamakura, Faculty of Agriculture, University of Tokyo Science, Tokyo
  • Ai Handa, Graduate School of Agriculture, University of Meiji, Kanagawa
  • Tohru Yamato, Graduate School of Agriculture, University of Meiji, Kanagawa
  • Shuichi Ohsato, Graduate School of Agriculture, University of Meiji, Kanagawa
  • Shigeru Kuwata, Graduate School of Agriculture, University of Meiji, Kanagawa
  • Tsutomu Arie, Faculty of Agriculture, Tokyo Agriculture and Technology University, Tokyo

About Tokyo Science University

Tokyo University of Science (TUS) is a well-known and respected university, the most specialized private research university in Japan, with four campuses in central Tokyo and suburbs and Hokkaido. Founded in 1881, the university has continually contributed to the development of Japan in science by motivating the love of science in researchers, technicians and educators.

With a mission of "Creating science and technology for the development of harmonious nature, humans, and society", TUS has undertaken a wide range of research of basic science to applied science. TUS has embraced a multidisciplinary approach to research and has undertaken an intensive study in some of today's most critical areas. TUS is one merit where the best in science is recognized and nurtured. This is the only private university in Japan that has produced a Nobel Prize winner and the only private university in Asia to produce Nobel Prize winners in the field of natural sciences.

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