Development of new comprehensive gene-silencing technology by using artificial chimeric repressor in plant

2014 Fiscal Year Final Research Report

• Project/Area Number: 25660011
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Science in genetics and breeding
• Research Institution: National Institute of Advanced Industrial Science and Technology
• Principal Investigator: MITSUDA Nobutaka 独立行政法人産業技術総合研究所, 生物プロセス研究部門, 主任研究員 (80450667)
• Co-Investigator(Kenkyū-buntansha): KATO Yoshio 国立研究開発法人産業技術総合研究所, 生命工学領域・バイオメディカル研究部門, 主任研究員 (20415657)
• Project Period (FY): 2013-04-01 – 2015-03-31
• Keywords: CRES-T / 転写抑制 / 転写因子 / CRISPR

Outline of Final Research Achievements

To determine which region is most effective to suppress expression of target gene by chimeric repressor, repeated GAL4 binding sequence was inserted into LUC gene. However, it was found that LUC activity becomes much reduced even without any effector in that case. We therefore decided to employ CRISPR-Cas9 system for our purpose. We prepared vector that expresses gRNA and mutated CAS9 (D10A H840A) fused with SRDX which doesn’t digest DNA. As a test experiment, we designed several gRNAs for the reporter construct 35S:GAL4UASx5:TATA:LUC, however, we didn’t see any repression of the reporter when we expressed these gRNAs and mutated CAS9 fused with SRDX together at least in the level of transient assay.

Free Research Field

植物分子生物学