INSERM U1052, CNRS UMR-5286, Cancer Research Center of Lyon, Lyon, France.
Evotec, Lyon, France.
mBio. 2022 Apr 26;13(2):e0288821. doi: 10.1128/mbio.02888-21. Epub 2022 Apr 7.
Chronic hepatitis B virus (HBV) infection persists due to the lack of therapies that effectively target the HBV covalently closed circular DNA (cccDNA). We used HBV-specific guide RNAs (gRNAs) and CRISPR-Cas9 and determined the fate of cccDNA after gene editing. We set up a ribonucleoprotein (RNP) delivery system in HBV-infected HepG2-NTCP cells. HBV parameters after Cas9 editing were analyzed. Southern blot (SB) analysis and DNA/RNA sequencing (DNA/RNA-seq) were performed to determine the consequences of cccDNA editing and transcriptional activity of mutated cccDNA. Treatment of infected cells with HBV-specific gRNAs showed that CRISPR-Cas9 can efficiently affect HBV replication. The appearance of episomal HBV DNA variants after dual gRNA treatment was observed by PCR, SB analysis, and DNA/RNA-seq. These transcriptionally active variants are the products of simultaneous Cas9-induced double-strand breaks in two target sites, followed by repair and religation of both short and long fragments. Following suppression of HBV DNA replicative intermediates by nucleoside analogs, mutations and formation of smaller transcriptionally active HBV variants were still observed, suggesting that established cccDNA is accessible to CRISPR-Cas9 editing. Targeting HBV DNA with CRISPR-Cas9 leads to cleavage followed by appearance of episomal HBV DNA variants. Effects induced by Cas9 were sustainable after RNP degradation/loss of detection, suggesting permanent changes in the HBV genome instead of transient effects due to transcriptional interference. Hepatitis B virus infection can develop into chronic infection, cirrhosis, and hepatocellular carcinoma. Treatment of chronic hepatitis B requires novel approaches to directly target the viral minichromosome, which is responsible for the persistence of the disease. Designer nuclease approaches represent a promising strategy to treat chronic infectious diseases; however, comprehensive knowledge about the fate of the HBV minichromosome is needed before this potent tool can be used as a potential therapeutic approach. This study provides an in-depth analysis of CRISPR-Cas9 targeting of HBV minichromosome.
慢性乙型肝炎病毒（HBV）感染持续存在，是因为缺乏能够有效靶向 HBV 共价闭合环状 DNA（cccDNA）的治疗方法。我们使用 HBV 特异性向导 RNA（gRNA）和 CRISPR-Cas9，并确定基因编辑后 cccDNA 的命运。我们在 HBV 感染的 HepG2-NTCP 细胞中建立了核糖核蛋白（RNP）递送系统。分析 Cas9 编辑后 HBV 参数。进行 Southern 印迹（SB）分析和 DNA/RNA 测序（DNA/RNA-seq），以确定 cccDNA 编辑和突变 cccDNA 转录活性的结果。用 HBV 特异性 gRNA 处理感染细胞表明，CRISPR-Cas9 可以有效地影响 HBV 复制。通过 PCR、SB 分析和 DNA/RNA-seq 观察到双 gRNA 处理后，cccDNA 编辑后出现了游离型 HBV DNA 变体。这些转录活性变体是 Cas9 诱导的两个靶位点同时产生的双链断裂的产物，随后是短片段和长片段的修复和再连接。在核苷类似物抑制 HBV DNA 复制中间体后，仍然观察到突变和较小的转录活性 HBV 变体的形成，这表明已建立的 cccDNA 可被 CRISPR-Cas9 编辑。用 CRISPR-Cas9 靶向 HBV DNA 会导致切割，随后出现游离型 HBV DNA 变体。在 RNP 降解/检测丢失后，Cas9 诱导的效应仍然可持续，这表明 HBV 基因组发生了永久性变化，而不是由于转录干扰导致的瞬时变化。乙型肝炎病毒感染可发展为慢性感染、肝硬化和肝细胞癌。慢性乙型肝炎的治疗需要新的方法直接靶向负责疾病持续存在的病毒微染色体。设计酶核酸酶方法代表了治疗慢性传染病的一种很有前途的策略；然而，在这种强大的工具被用作潜在的治疗方法之前，需要全面了解 HBV 微染色体的命运。本研究对 CRISPR-Cas9 靶向 HBV 微染色体进行了深入分析。
