AsianScientist (Sep. 15, 2017) – In a study published in the journal Scientific Reports, scientists in Japan have used the CRISPR-Cas9 genome-editing tool to change flower color in an ornamental plant.
The CRISPR-Cas9 system is based on a bacterial defense mechanism. It is composed of two molecules that alter the DNA sequence. Cas9, an enzyme, cuts the two strands of DNA in a precise location so that DNA can be added or removed. Cas9 is guided to the correct location by guide RNA (gRNA), a small piece of RNA that has been designed to be complementary to the target DNA sequence. Cas9 cuts the two strands of DNA at the target location, allowing DNA to be removed and/or added.
In this study, researchers from the University of Tsukuba, the National Agriculture and Food Research Organization (NARO) and Yokohama City University, Japan, altered the flower color of the traditional Japanese garden plant, Japanese morning glory (Ipomoea nil or Pharbitis nil), from violet to white by disrupting a single gene.
The research team targeted dihydroflavonol-4-reductase-B (DFR-B), a gene encoding an anthocyanin biosynthesis enzyme, that is responsible for the color of the plant’s stems, leaves and flowers. Two other very closely related genes (DFR-A and DRF-C) are adjacent to the DFR-B gene. Therefore, the challenge was to specifically and accurately target the DFR-B gene without altering the other genes.
The CRISPR-Cas9 system was inserted into tissue-cultured embryos of Japanese morning glory plants using the DNA-transferring capabilities of the plant bacterium Rhizobium. As expected, the DFR-B enzyme was successfully inactivated, resulting in approximately 75 percent of the transgenic plants producing green stems and white flowers. Non-transformed plants with an active enzyme had violet stems and flowers. These changes in stem color were observed very early in the tissue culture process.
A series of genetic analyses confirmed that the DNA target sequence had been altered in the transgenic plants, with either DNA insertions or deletions in both copies of the DFR-B gene. The other related genes, DFR-A and DFR-C, were examined and no mutations were found, confirming the high specificity of the CRISPR-Cas9 system.
Next, the researchers examined the inheritance of the CRISPR-Cas9-induced mutations by analyzing plants from the next generation. These plants looked exactly like their parents, but some did not contain any of the introduced DNA.
This raises interesting questions in terms of the regulation of genetically modified organisms as these next-generation plants are considered transgenic based on process-based definitions (how they were made), whereas product-based definitions (the presence of foreign DNA in the final product) consider them to be non-transgenic.
The researchers said that the CRISPR-Cas9 system is extremely useful in confirming the function of genes, enabling a ‘reverse’ genetic approach to find out what an organism looks like after a known gene is disrupted. They were thus able to conclusively determine that the DFR-B gene is the main gene responsible for color in Japanese morning glory plants.
The article can be found at: Watanabe et al. (2017) CRISPR/Cas9-Mediated Mutagenesis of the Dihydroflavonol-4-reductase-B (DFR-B) Locus in the Japanese Morning Glory Ipomoea (Pharbitis) nil.
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Source: University of Tsukuba.
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