Establishing diatom genome editing technique by using CRISPR/Cas9 system

• Main Authors: Yen, Shao-Chieh, 顏紹傑
• Other Authors: Lin, Han-Jia
• Format: Others
• Language: zh-TW
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/30022599106880632583

碩士 === 國立臺灣海洋大學 === 生命科學暨生物科技學系 === 103 === Genome editing is considered as an important technology to study a gene’s function. Among genome editing tools, recently developed CRISPR/Cas9 system is considered the most powerful one, since a single-stranded guide RNA (sgRNA) and one nuclease (Cas9) are the only two components needed to make a double strand break (DSB) in a specific site on chromosomes. As a consequence, DSB may induce nonhomologous end-joining (NHEJ) or homology-directed repair (HDR) that can further use in gene knockout or knockin. Here, we try to apply this system to Phaeodactylum tricornutum, a model of marine diatom. Since diatoms are major primary producers in the ocean with unique evolutionary status, studying diatom‘s gene functions by using genome editing technology is important. Therefore, three impotant tools were prepared. First, we successful cloning diatom U6 gene promoter and confirmed this promoter can be applied to drive sgRNA expression in diatom. Then the Cas9 expression vector was constructed. Thirdly, a diatom homologous recombination vector, which contains two homologous arms and selection marker genes, was also prepared to used in genome editing. By using diatom transgenic technology, we send these plasmids into P. tricornutum to evaluate the efficiency and possibilities of future applications of CRISPR/Cas9 system in diatom.