Development of DNA-free genome editing in fruit-bearing plants

• Project/Area Number: 18K19200
• Research Category: Grant-in-Aid for Challenging Research (Exploratory)
• Allocation Type: Multi-year Fund
• Review Section: Medium-sized Section 39:Agricultural and environmental biology and related fields
• Research Institution: University of Tsukuba
• Principal Investigator: ARIIZUMI Tohru 筑波大学, 生命環境系, 准教授 (70575381)
• Co-Investigator(Kenkyū-buntansha): 三浦 謙治 筑波大学, 生命環境系, 教授 (00507949)
• Project Period (FY): 2018-06-29 – 2020-03-31
• Project Status: Completed (Fiscal Year 2019)
• Budget Amount: ¥6,240,000 (Direct Cost: ¥4,800,000、Indirect Cost: ¥1,440,000); Fiscal Year 2019: ¥3,380,000 (Direct Cost: ¥2,600,000、Indirect Cost: ¥780,000); Fiscal Year 2018: ¥2,860,000 (Direct Cost: ¥2,200,000、Indirect Cost: ¥660,000)
• Keywords: トマト / ゲノム編集技術 / 形質転換 / DNAフリー

Outline of Final Research Achievements

Genome editing technology is a technique to modify DNA within regions specified by sgRNA sequence. The CRISPR/Cas9 induces site-specific DNA modification by nuclease activity of Cas9 protein, although application of this system to plants is highly dependent on T-DNA integration. In this study, we aimed to develop a method to edit the target site without integrating T-DNA. We employed the transient expression of Cas9 nuclease/sgRNA in tomato via Agrobacterium infection, followed by regeneration of culture medium devoid of antibiotics. We succeeded to select the edited individuals in which DNA modifications were induced by CRISPR/Cas9 as well as by Target-AID, while T-DNA was not incorporated in the plants. Thus, it was demonstrated that transient expression system worked well for obtaining the DNA-edited plants without harboring T-DNA insertion.

Academic Significance and Societal Importance of the Research Achievements

本研究はT-DNAが組み込まれないゲノム編集技術の開発に取り組んだ。通常のCRISPR/Cas9法を植物に適応させる方法では、アグロバクテリウムを介してT-DNAを組み込ませてCas9/sgRNAを発現させる方法である。この方法では外来遺伝子の発現が保証される一方、ヌルセグリガント獲得までの時間が課題であった。本研究では、培養当代でのヌルセグリガント獲得を実証できた。植物育種の迅速化は地球温暖化など、気候変動が進む世界において喫緊の課題であるため、本研究で開発された技術は育種の高速化に大きく寄与すると考えられる。