Establishment of next generation chromosome engineering

2015 Fiscal Year Final Research Report

• Project/Area Number: 26670165
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Pathological medical chemistry
• Research Institution: Institute of Physical and Chemical Research
• Principal Investigator: Takumi Toru 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, シニアチームリーダー (00222092)
• Project Period (FY): 2014-04-01 – 2016-03-31
• Keywords: コピー数多型 / 染色体工学 / ゲノム編集技術 / 自閉症

Outline of Final Research Achievements

In the CRISPR-Cas9 system, a single guide RNA (sgRNA) directs the endonuclease Cas9 to a targeted DNA sequence for site-specific manipulation. The sgRNA has also been shown to play a role in activating the endonuclease activity of Cas9. This dual function of the sgRNA likely underlies observations that different sgRNAs have varying on-target activities. We evaluated the cleavage activities of 218 sgRNAs using in vitro Surveyor assays. We found that nucleotides at both PAM-distal and PAM-proximal region of the sgRNA are significantly correlated with on-target efficiency. Furthermore, we demonstrated that the genomic contexts of the targeted DNA, the GC percentage, and the secondary structure of sgRNA are critical factors contributing to cleavage efficiency.

Free Research Field

病態医化学