Genome editing by microinjection in the model organism Marchantia polymorpha

2017 Fiscal Year Final Research Report

• Project/Area Number: 15K06902
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Genome biology
• Research Institution: Kindai University
• Principal Investigator: Yamato Katsuyuki 近畿大学, 生物理工学部, 教授 (50293915)
• Co-Investigator(Renkei-kenkyūsha): KOHCHI Takayuki 京都大学, 大学院生命科学研究科, 教授 (40202056) ; ISHIZAKI Kimitsune 神戸大学, 大学院理学研究科, 准教授 (00452293) ; TAKIKAWA Yoshihiro 近畿大学, 先端技術総合研究所, 准教授 (60446010)
• Project Period (FY): 2015-04-01 – 2018-03-31
• Keywords: RNA-protein complex / CRISPR/Cas9 / microinjection

Outline of Final Research Achievements

DNA-free genome editing allows target-specific manipulation of a given genome. CRISPR RNA and Cas9 protein can be directly introduced into target cells and lead to genome-editing. We have successfully performed DNA-free genome editing mediated by microinjection of CRISPR/Cas9 complex in the liverwort Marchantia polymorpha. NOPPERABO1 (NOP1), of which loss-of-function mutation causes impaired air-chamber formation and thus can be found readily, was selected as a target gene. Custom gRNA and commercially available Cas9 protein were first allowed to form RNA-protein complex and then injected into single-cell sporelings by a laser thermal microinjector. One of the thalli grown from microinjected sporelings formed a sector that showed the nop1 mutant phenotype, and DNA isolated from the sector showed 5-bp deletion in the target coding sequence. This is the first demonstration of DNA-free genome-editing by microinjection in plants.

Free Research Field

plant molecular genetics