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標題: | 利用 CRISPR/Cpf1 系統對 B 型肝炎病毒進行多位點基因編輯 Multiplex targeting HBV via CRISPR/Cpf1 |
作者: | Pei-Chun Hou 侯佩君 |
指導教授: | 楊宏志(Hung-Chih Yang) |
關鍵字: | B 型肝炎病毒,嵌入型 B 型肝炎基因體,基因編輯,CRISPR,Cpf1,基因體甲基化, Hepatitis B virus,integrated HBV DNA,gene editing,CRISPR,Cpf1,DNA methylation, |
出版年 : | 2018 |
學位: | 碩士 |
摘要: | B型肝炎病毒(Hepatitis B virus, HBV)感染肝細胞後,其基因體以共價閉合環狀去氧核糖核酸(covalently closed circular DNA, cccDNA)結構或嵌入宿主基因體中存在宿主肝細胞內。嵌入型的B型肝炎基因體(Integrated HBV DNA),並不會再進行複製,但仍可轉錄、轉譯出HBV的表面抗原(HBsAg)。目前治療HBV的藥物- Nucleos(t)ide analogues (NAs)可有效抑制病毒反轉錄複製的過程,但因為無法徹底清除病毒的複製模板-cccDNA,所以仍然無法治癒HBV感染。我們先前的研究以及其他多份文獻中,都證明了HBV的基因體可以被核酸內切酶-Cas9此基因編輯工具破壞;此外,還有另一種核酸內切酶稱作Cpf1,它被分類在第五型CRISPR系統,其特色是具有去氧核醣核酸酶(DNase)及核糖核酸酶(RNase)兩種活性,除了DNA之外,也可以切割RNA,因此只要利用一長條crRNA前驅物(pre-crRNA)就可以切割成數個成熟的crRNA,以引導Cpf1進行多位點專一性編輯,Cpf1會是個適合進行多位點基因編輯的工具。在本篇研究中,我們的目標為利用CRISPR/Cpf1系統對HBV基因體做多位點的基因編輯;我們首先挑選出八條具B型肝炎病毒專一性的crRNA,其引導Cpf1去切割病毒基因體並誘發基因的插入或刪除(Indels);接著我們將數個crRNA組成pre-crRNA,在體外測試其基因編輯能力,結果能同時切割三個HBV CpG island,使B型肝炎的核心抗原(HBc),e抗原(HBe)、表面抗原(HBs)同時下降。我們也更進一步發展出以CRISPR/Cpf1為基礎的工具,其由無DNase活性的LbCpf1(簡稱dCpf1)及DNA甲基轉移酶(DNA methyltransferase)所組成,目的是希望能將HBV的基因體進行位點專一性的甲基化,利用此非切斷基因的方法來抑制HBV的基因表現。我們認為,多位點的甲基化可以更有效且專一的去抑制HBV的基因表現。本篇研究成功利用CRISPR/Cpf1系統對HBV基因體進行多位點基因編輯,在體外能有效破壞B型肝炎病毒基因體,以降低其表現,將來有潛力做為根除B型肝炎病毒之抗病毒藥物。 Following the entry of hepatitis B virus (HBV) into hepatocytes, a minor portion of viral DNAs integrate into host chromosomes. Although the integrated HBV genome is not required for HBV replication, it can continuously produce HBV surface antigens, and is found in 85%-90% of HBV-related HCCs. Nucleos(t)ide analogues (NAs) inhibit the reverse transcription of HBV replication cycle very effectively, but they cannot cure HBV infection because they fail to eliminate the replicative template covalently closed circular DNA (cccDNA) and the integrated HBV genomes, which can still produce viral antigens. Previous studies, including ours, have shown that the HBV genome can be disrupted by the RNA-guided endonuclease Cas9. Cpf1 nuclease belongs to the type V CRISPR system and has dual DNase and RNase activities, so it can produce multiple CRISPR RNAs (crRNAs) from a single long RNA (pre-crRNA). Therefore, Cpf1 is an ideal tool for multiplex gene editing. In this studies, we aimed to utilize the CRISPR/Cpf1 system to target HBV DNA. We first screened an array of HBV-specific protospacer sequences and found eight of them were able to induce indel formation by the CRISPR/Cpf1 system. Furthermore, we used Cpf1 and multiple HBV-specific crRNAs to cut on three CpG islands and simultaneously reduced HBc, HBe, and HBs antigens. Furthermore, we developed the CRISPR/Cpf1-based tool, consisting of DNase inactive LbCpf1(dLbCpf1) and a catalytic domain of DNA methyltransferase DNMT3A, to methylate HBV genome in a site-specific manner for the reduction of gene expression without cleavage. We hypothesize that multiplex methylation can suppress HBV gene expression more specifically and efficiently. In conclusion, we demonstrated the ability and potential of the CRISPR/Cpf1 system for multiplex targeting HBV genome in the clearance of HBV in chronic hepatitis B patients. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/71112 |
DOI: | 10.6342/NTU201802152 |
全文授權: | 有償授權 |
顯示於系所單位: | 微生物學科所 |
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