2020 Fiscal Year Final Research Report
Complete removal of HBV genome by nickase-Cas9
| Project/Area Number |
19K18123
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| Research Category |
Grant-in-Aid for Early-Career Scientists
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| Allocation Type | Multi-year Fund |
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| Review Section |
Basic Section 55020:Digestive surgery-related
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| Research Institution | Kyushu University |
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Principal Investigator |
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| Project Period (FY) |
2019-04-01 – 2021-03-31
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| Keywords | B型肝炎 / ゲノム治療 |
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| Outline of Final Research Achievements |
Recent reports have shown that a novel genome-editing tool using Cas9 with a single-guide RNA (sgRNA) system can cleave the HBV genome in vitro and in vivo. However, Cas9 risks undesirable off-target cleavage on the host genome. Nickase-Cas9 cleaves a single strand of DNA, and thereby two sgRNAs are required for inducing DSBs. To avoid Cas9-induced off-target mutagenesis, we examined the effects of the expressions of nickase-Cas9 and with sgRNAs on HBV replication. The expression of nickase-Cas9 with a pair of sgRNAs cleaved the target HBV genome and suppressed the viral-protein expression and HBV replication in vitro. Moreover, nickase-Cas9 with the sgRNA pair cleaved the targeted HBV genome in mouse liver.These results suggest the possible use of nickase-Cas9 with a pair of sgRNAs for eliminating HBV DNA from the livers of chronic hepatitis B patients with low risk of undesirable off-target mutation on the host genome.
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| Free Research Field |
消化器外科
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| Academic Significance and Societal Importance of the Research Achievements |
B型肝炎患者に対する現行の治療ではB型肝炎ウイルスDNAを完全に排除するのは困難である。ゲノム編集ツールであるCas9蛋白は特定のゲノム配列を認識するsingle-guide RNAとの組み合わせによってB型肝炎ウイルスゲノムのDNA2本鎖切断を誘導しうるが、Cas9蛋白は宿主細胞に想定外の非ターゲットDNA切断が起こるリスクも高い。今回の研究によりnickase-Cas9蛋白とペアとなるsingle-guide RNAの組み合わることによって、宿主DNAへの変異導入のリスクを低減した感染細胞核内のB型肝炎ウイルスゲノムの完全排除の可能性が見出された点において学術的意義があると考えられる。
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