| Budget Amount *help |
¥5,200,000 (Direct Cost: ¥4,000,000、Indirect Cost: ¥1,200,000)
Fiscal Year 2016: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000)
Fiscal Year 2015: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
Fiscal Year 2014: ¥2,600,000 (Direct Cost: ¥2,000,000、Indirect Cost: ¥600,000)
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| Outline of Final Research Achievements |
Genome editing is now widely utilized to modify various plant genomes. Using a truncated gRNA (tru-gRNA)/Cas9, we generated new alleles for OST2, a proton pump in Arabidopsis, with no off-target effects. Newly generated mutations in CRIPSR/Cas9 transgenic plants were detected with the average mutation rates of 32.8% using a constitutive promoter for Cas9 expression. The reducing nuclear localization signals (NLS) in Cas9 decreased the mutation rates. The tru-gRNA/Cas9 driven by the meristematic- and reproductive tissue-specific promoters increased the heritable mutation rate in Arabidopsis, showing that high expression in the germ line can produce bi-allelic mutations with high mutation efficiency. Finally, we generated the new mutant alleles for OST2, and the mutants exhibited altered stomatal closing in response to stress conditions. The results suggest further applications in molecular breeding to improve plant function using optimized CRISPR/Cas9.
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