Single-cell genomics and genome editing of unculturable protist

Research Project

Project/Area Number	26660075
Research Category	Grant-in-Aid for Challenging Exploratory Research
Allocation Type	Multi-year Fund
Research Field	Applied microbiology
Research Institution	Institute of Physical and Chemical Research
Principal Investigator	Yuki Masahiro 国立研究開発法人理化学研究所, 環境資源科学研究センター, 特別研究員 (50572773)
Project Period (FY)	2014-04-01 – 2016-03-31
Project Status	Completed (Fiscal Year 2015)
Budget Amount *help	¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000) Fiscal Year 2015: ¥2,730,000 (Direct Cost: ¥2,100,000、Indirect Cost: ¥630,000) Fiscal Year 2014: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
Keywords	CRISPR / シロアリ / 共生原生生物 / シロアリ腸内原生生物 / CRISPR-Cas9 / セロビオハイドロラーゼ
Outline of Final Research Achievements	The aim of this study was to perform genome editing of uncultivated symbiotic protists in the termite gut using the CRISPR/Cas system. Cellobiohydrolase genes were detected in the symbiotic protist genome, and their target Cas9 sites were determined. We also calculated the codon usage of the protist genome, and t optimized the cas9 and GFP genes accordingly. The mixture of cas9 mRNA, gRNA and DNA for homologue recombination was injected into the termite gut. Furthermore, in order to remove contaminating sequences of symbiotic bacteria from the protist genome, we analyzed the genome of ectosymbionts by single-cell genomics.