development of genome-editing techniques using engineered nucleases

2016 Fiscal Year Final Research Report

• Project/Area Number: 26460088
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Biological pharmacy
• Research Institution: Institute of Physical and Chemical Research
• Principal Investigator: Hisano Yu 国立研究開発法人理化学研究所, 脳科学総合研究センター, 基礎科学特別研究員 (60467636)
• Co-Investigator(Renkei-kenkyūsha): KAWAHARA Atsuo 山梨大学, 総合研究部, 教授 (10362518) ; KOBAYASHI Takuma 国立研究開発法人理化学研究所, 脳科学総合研究センター, 研究員 (80582288)
• Project Period (FY): 2014-04-01 – 2017-03-31
• Keywords: 人工ヌクレアーゼ / ゲノム編集 / TALEN / CRISPR/Cas9 system / ゼブラフィッシュ / ノックイン / 逆遺伝学

Outline of Final Research Achievements

This study aimed to develop new genome-editing techniques using engineered nucleases such as CRISPR/Cas9 system in zebrafish. CRISPR/Cas9 system causes DNA double strand breaks, which can be repaired by non-homologous end joining or homology directed recombination. We achieved to knock-in exogenous genes into target genomic locus using donor vector harboring short homologous sequences. The donor vector was designed to have gRNA-recognition sites, homology arms, and the exogenous gene to be integrated. By introducing two gRNA-recognition sites in both side of the integrating gene, backbone sequences of donor vector can be eliminated. Furthermore, the precise genome modification was also occurred in germ cells, resulting in the establishment of knocked-in zebrafish line. This method enables efficient and precise genome modification in a homology-dependent manner, which should be able to be applied to other model organisms

Free Research Field

脂質生化学