# Development of a Sensitive and Specific RPA-CRISPR/Cas12a Assay for Intrahepatic Quantification of HBV cccDNA

## TL;DR

A new test using RPA and CRISPR/Cas12a accurately detects and quantifies HBV cccDNA in liver tissue, offering a sensitive and specific tool for HBV research and treatment evaluation.

## Contribution

A novel RPA-CRISPR/Cas12a assay was developed for highly sensitive and specific quantification of HBV cccDNA in liver samples.

## Key findings

- The assay detected as few as 10 cccDNA copies per reaction with no cross-reactivity to non-cccDNA forms.
- It showed 90% sensitivity and strong correlation (R² = 0.9155) with qPCR results in liver tissue samples.
- The system is robust, cost-effective, and suitable for high-throughput therapeutic screening.

## Abstract

Hepatitis B virus (HBV) persists in infected hepatocytes through covalently closed circular DNA (cccDNA), a stable episomal form that serves as the transcriptional template for viral replication. Accurate and sensitive quantification of intrahepatic cccDNA is crucial for evaluating antiviral therapies, particularly those targeting a functional cure. Here, we report the development of a novel, cccDNA-specific detection system combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a-based fluorescence detection. We designed and validated CRISPR RNAs (crRNAs) targeting HBV cccDNA-specific regions conserved across genotypes A–D. Reaction conditions for both RPA and Cas12a detection were optimized to enhance sensitivity, specificity, and accuracy. The system reliably detected as few as 10 copies of cccDNA-containing plasmid per reaction and showed no cross-reactivity with non-cccDNA forms in serum or plasma, indicating assay specificity. When applied to liver tissue samples from 10 HBV-infected and 6 non-HBV patients, the RPA-CRISPR/Cas12a assay exhibited a high sensitivity (90%) and a strong correlation with qPCR results (R2 = 0.9155), confirming its accuracy. In the conclusion, the RPA-CRISPR/Cas12a system provides a robust, cost-effective, and scalable platform for sensitive and specific quantification of intrahepatic HBV cccDNA. This method holds promises for research and high-throughput therapeutic screening applications targeting cccDNA clearance.