CAJ | 학술논문

目的：利用CRISPR/Cas9基因编辑技术对乙型肝炎病毒( HBV)的特定基因进行编辑，评价其抑制HBV复制的效应。方法针对HBV的S 基因设计2套CRISPR/Cas9表达质粒并转染HepG2-N10细胞株。采用噻唑蓝( MTT)法测定细胞毒性、化学发光法检测HBsAg、荧光定量RT-PCR( qRT-PCR)法检测病毒mRNA表达水平，荧光定量PCR( qRT-PCR)法检测HBV DNA含量，高通量测序分析HBV基因组编辑情况。结果未见明显细胞毒性效应；2套针对HBV的CRISPR/Cas9表达质粒组培养基中HBsAg含量较对照组降低了24.2%(P=0.031)和16.9%(P>0.05)，细胞中HBsAg含量分别降低了16.4%(P>0.05)和32.1%(P>0.05)；S、C、X基因mRNA基因表达呈现不同程度降低；培养上清中HBV DNA含量较对照组降低了23%(P>0.05)和35%(P=0.047)，细胞中HBV DNA含量降低了7.2%(P>0.05)和18%(P>0.05)；高通量测序提示HBV基因组出现不同类型的插入/缺失突变。结论针对HBV的S 基因设计的2套CRISPR/Cas9表达质粒能通过对HBV基因组的编辑实现抑制HBV基因表达及病毒复制作用，可能为HBV治疗提供新的途径。
목적：이용CRISPR/Cas9기인편집기술대을형간염병독( HBV)적특정기인진행편집，평개기억제HBV복제적효응。방법침대HBV적S 기인설계2투CRISPR/Cas9표체질립병전염HepG2-N10세포주。채용새서람( MTT)법측정세포독성、화학발광법검측HBsAg、형광정량RT-PCR( qRT-PCR)법검측병독mRNA표체수평，형광정량PCR( qRT-PCR)법검측HBV DNA함량，고통량측서분석HBV기인조편집정황。결과미견명현세포독성효응；2투침대HBV적CRISPR/Cas9표체질립조배양기중HBsAg함량교대조조강저료24.2%(P=0.031)화16.9%(P>0.05)，세포중HBsAg함량분별강저료16.4%(P>0.05)화32.1%(P>0.05)；S、C、X기인mRNA기인표체정현불동정도강저；배양상청중HBV DNA함량교대조조강저료23%(P>0.05)화35%(P=0.047)，세포중HBV DNA함량강저료7.2%(P>0.05)화18%(P>0.05)；고통량측서제시HBV기인조출현불동류형적삽입/결실돌변。결론침대HBV적S 기인설계적2투CRISPR/Cas9표체질립능통과대HBV기인조적편집실현억제HBV기인표체급병독복제작용，가능위HBV치료제공신적도경。
Objective To evaluate the practicability of using CRISPR/Cas9 genome editing tech-nology for inhibition of hepatitis B virus ( HBV) replication. Methods Two sgRNA targeting sites were de-signed for the S region of HBV genome. The CRISPR/Cas9 expression plasmids specific for HBV were con-structed and then transfected into a cell line expressing HBV genome(HepG2-N10). The cytotoxicity of cells transfected with different expression plasmids were detected by MTT assay. The levels of hepatitis B surface antigen ( HBsAg ) were determined by using chemiluminescent immunoassay ( CLIA ) . The expression of HBV at mRNA level was analyzed by quantitative real-time PCR ( qRT-PCR) . The qPCR was performed for the detection of extracellular and intracellular HBV DNA. The next-generation sequencing ( NGS) Illumina MiSeq Platform was used to analyze HBV genome editing. Results No significant cytotoxic effects were de-tected in HepG2-N10 cells transfected with different expression plasmids. Compared with the cells carrying pCas-Guide-GFP-Scramble, the levels of HBsAg in the supernatants of transfected cell culture harboring pCas-Guide-GFP-G1 and pCas-Guide-GFP-G2 were decreased by 24. 2% (P<0. 05) and 16. 9% (P>0. 05), respectively. The levels of HBsAg in cells transfected with pCas-Guide-GFP-G1 and pCas-Guide-GFP-G2 were respectively decreased by 16. 4% (P>0. 05) and 32. 1% (P>0. 05) as compared with that of pCas-Guide-GFP-Scramble transfected group. The expression of HBV at mRNA level was inhibited as indica-ted by the results of qRT-PCR. Moreover, the levels of extracellular HBV DNA were respectively suppressed by 23% (P>0. 05) and 35% (P<0. 05), and the levels of intracellular HBV DNA were respectively sup-pressed by 7. 2% (P>0. 05) and 18% (P>0. 05). Different types of insertion/deletion mutation were de-tected in HBV genome by high-throughput sequencing. Conclusion HBV-specific CRISPR/Cas9 system could inhibit the expression of HBV gene and the replication of virus. Therefore, the CRISPR/Cas9 genome editing technology might be used as a potential tool for the treatment of persistent HBV infection.