CRISPR/Cas13-assisted hepatitis B virus covalently closed circular DNA detection

• Published in: Hepatology international 2022-04, Vol.16 (2), p.306-315
• Main Authors: Zhang, Xiangying, Tian, Yuan, Xu, Ling, Fan, Zihao, Cao, Yaling, Ma, Yingmin, Li, Hao, Ren, Feng
• Format: Article
• Language: English
• Subjects: Amplification; Antiviral agents; Antiviral drugs; Assaying; Blood; Circular DNA; Colorectal Surgery; CRISPR; CRISPR-Cas Systems; Deoxyribonuclease; Deoxyribonucleic acid; DNA; DNA viruses; DNA, Circular - genetics; DNA, Viral - analysis; DNA, Viral - genetics; Fluorescence; Hepatitis; Hepatitis B; Hepatitis B - diagnosis; Hepatitis B virus - genetics; Hepatitis B, Chronic; Hepatology; Humans; Infections; Leukocytes, Mononuclear; Liver; Medicine; Medicine & Public Health; Original; Original Article; Patients; Peripheral blood mononuclear cells; Polymerase chain reaction; Real-Time Polymerase Chain Reaction - methods; Surgery; Target detection; Technology; Viruses
• ISSN: 1936-0533; 1936-0541
• DOI: 10.1007/s12072-022-10311-0

Background and aims The formation of an intranuclear pool of covalently closed circular DNA (cccDNA) in the liver is the main cause of persistent hepatitis B virus (HBV) infection. Here, we established highly sensitive and specific methods to detect cccDNA based on CRISPR-Cas13a technology. Methods We used plasmid-safe ATP-dependent DNase (PSAD) enzymes and HindIII to digest loose circle rcDNA and double-stranded linear DNA, amplify specific HBV cccDNA fragments by rolling circle amplification (RCA) and PCR, and detect the target gene using CRISPR-Cas13a technology. The CRISPR-Cas13a-based assay for the detection of cccDNA was further clinically validated using HBV-related liver tissues, plasma, whole blood and peripheral blood mononuclear cells (PBMCs). Results Based on the sample pretreatment step, the amplification step and the detection step, we established a new CRISPR-Cas13a-based assay for the detection of cccDNA. After the amplification of RCA and PCR, 1 copy/μl HBV cccDNA could be detected by CRISPR/Cas13-assisted fluorescence readout. We used ddPCR, qPCR, RCA-qPCR, PCR-CRISPR and RCA-PCR-CRISPR methods to detect 20, 4, 18, 14 and 29 positive samples in liver tissue samples from 40 HBV-related patients, respectively. HBV cccDNA was almost completely undetected in the 20 blood samples of HBV patients (including plasma, whole blood and PBMCs) by the above 5 methods. Conclusions We developed a novel CRISPR-based assay for the highly sensitive and specific detection of HBV cccDNA, presenting a promising alternative for accurate detection of HBV infection, antiviral therapy evaluation and treatment guidance.