The menace of severe adverse events and deaths associated with viral gene therapy and its potential solution.

Over the past decade, in vivo gene replacement therapy has significantly advanced, resulting in market approval of numerous therapeutics predominantly relying on adeno-associated viral vectors (AAV). While viral vectors have undeniably addressed several critical healthcare challenges, their clinical application has unveiled a range of limitations and safety concerns. This review highlights the emerging challenges in the field of gene therapy. At first, we discuss both the role of biological barriers in viral gene therapy with a focus on AAVs, and review current landscape of in vivo human gene therapy. We delineate advantages and disadvantages of AAVs as gene delivery vehicles, mostly from the safety perspective (hepatotoxicity, cardiotoxicity, neurotoxicity, inflammatory responses etc.), and outline the mechanisms of adverse events in response to AAV. Contribution of every aspect of AAV vectors (genomic structure, capsid proteins) and host responses to injected AAV is considered and substantiated by basic, translational and clinical studies. The updated evaluation of recent AAV clinical trials and current medical experience clearly shows the risks of AAVs that sometimes overshadow the hopes for curing a hereditary disease. At last, a set of established and new molecular and nanotechnology tools and approaches are provided as potential solutions for mitigating or eliminating side effects. The increasing number of severe adverse reactions and, sadly deaths, demands decisive actions to resolve the issue of immune responses and extremely high doses of viral vectors used for gene therapy. In response to these challenges, various strategies are under development, including approaches aimed at augmenting characteristics of viral vectors and others focused on creating secure and efficacious non-viral vectors. This comprehensive review offers an overarching perspective on the present state of gene therapy utilizing both viral and non-viral vectors.

Biomimetic approaches for targeting tumor-promoting inflammation.

With the ultimate goal of increasing tumor accumulation of therapeutics, various nanocarriers have been designed to overcome biological barriers encountered at each stage, from drug administration to the cancerous lesion. Stabilizing circulation and functionalization of the targeting surface impart high tumor accumulation properties to nanocarriers. However, various cells can recognize and infiltrate the tumor microenvironment more efficiently than synthetic carriers via overexpression of adhesive ligands, particularly in inflamed stroma of tumors. Thus, a new field of nanomedicine, called biomimicry, has evolved to generate nanoparticles with the same biological characteristics as cells that naturally infiltrate tumors. Revolutionary synthetic processes have been developed to transfer the cell membrane of leukocytes and mesenchymal cells to synthetic carriers. In addition, cells can generate their own "nanocarriers," known as exosomes, to transport molecular messages to distant sites, while biomimicry of viral and bacterial agents allows high targeting efficiency towards inflammatory immune cells. Alterations in the protein expression in cancer cells caused by inflammation can also be exploited for drug delivery. Finally, new developments in biomimetic drug delivery focus on turning the infiltrating cells into microcarriers that can actively perfuse the tumor and eventually release their therapeutic payload. In this review, we summarize recent developments in biomimetic drug delivery with a particular focus on targeting the tumor inflammatory microenvironment.

CRISPR-Cas systems for diagnosing infectious diseases.

Infectious diseases are a global health problem affecting billions of people. Developing rapid and sensitive diagnostic tools is key for successful patient management and curbing disease spread. Currently available diagnostics are very specific and sensitive but time-consuming and require expensive laboratory settings and well-trained personnel; thus, they are not available in resource-limited areas, for the purposes of large-scale screenings and in case of outbreaks and epidemics. Developing new, rapid, and affordable point-of-care diagnostic assays is urgently needed. This review focuses on CRISPR-based technologies and their perspectives to become platforms for point-of-care nucleic acid detection methods and as deployable diagnostic platforms that could help to identify and curb outbreaks and emerging epidemics. We describe the mechanisms and function of different classes and types of CRISPR-Cas systems, including pros and cons for developing molecular diagnostic tests and applications of each type to detect a wide range of infectious agents. Many Cas proteins (Cas3, Cas9, Cas12, Cas13, Cas14 etc.) have been leveraged to create highly accurate and sensitive diagnostic tools combined with technologies of signal amplification and fluorescent, potentiometric, colorimetric, lateral flow assay detection and other. In particular, the most advanced platforms -- SHERLOCK/v2, DETECTR, CARMEN or CRISPR-Chip -- enable detection of attomolar amounts of pathogenic nucleic acids with specificity comparable to that of PCR but with minimal technical settings. Further developing CRISPR-based diagnostic tools promises to dramatically transform molecular diagnostics, making them easily affordable and accessible virtually anywhere in the world. The burden of socially significant diseases, frequent outbreaks, recent epidemics (MERS, SARS and the ongoing COVID-19) and outbreaks of zoonotic viruses (African Swine Fever Virus etc.) urgently need the developing and distribution of express-diagnostic tools. Recently devised CRISPR-technologies represent the unprecedented opportunity to reshape epidemiological surveillance and molecular diagnostics.

Orthologous CRISPR/Cas9 systems for specific and efficient degradation of covalently closed circular DNA of hepatitis B virus.

Covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is the major cause of viral persistence and chronic hepatitis B. CRISPR/Cas9 nucleases can specifically target HBV cccDNA for decay, but off-target effects of nucleases in the human genome limit their clinical utility. CRISPR/Cas9 systems from four different species were co-expressed in cell lines with guide RNAs targeting conserved regions of the HBV genome. CRISPR/Cas9 systems from Streptococcus pyogenes (Sp) and Streptococcus thermophilus (St) targeting conserved regions of the HBV genome blocked HBV replication and, most importantly, resulted in degradation of over 90% of HBV cccDNA by 6 days post-transfection. Degradation of HBV cccDNA was impaired by inhibition of non-homologous end-joining pathway and resulted in an erroneous repair of HBV cccDNA. HBV cccDNA methylation also affected antiviral activity of CRISPR/Cas9. Single-nucleotide HBV genetic variants did not impact anti-HBV activity of St CRISPR/Cas9, suggesting its utility in targeting many HBV variants. However, two or more mismatches impaired or blocked CRISPR/Cas9 activity, indicating that host DNA will not likely be targeted. Deep sequencing revealed that Sp CRISPR/Cas9 induced off-target mutagenesis, whereas St CRISPR/Cas9 had no effect on the host genome. St CRISPR/Cas9 system represents the safest system with high anti-HBV activity.

Gene Editing by Extracellular Vesicles.

CRISPR/Cas technologies have advanced dramatically in recent years. Many different systems with new properties have been characterized and a plethora of hybrid CRISPR/Cas systems able to modify the epigenome, regulate transcription, and correct mutations in DNA and RNA have been devised. However, practical application of CRISPR/Cas systems is severely limited by the lack of effective delivery tools. In this review, recent advances in developing vehicles for the delivery of CRISPR/Cas in the form of ribonucleoprotein complexes are outlined. Most importantly, we emphasize the use of extracellular vesicles (EVs) for CRISPR/Cas delivery and describe their unique properties: biocompatibility, safety, capacity for rational design, and ability to cross biological barriers. Available molecular tools that enable loading of desired protein and/or RNA cargo into the vesicles in a controllable manner and shape the surface of EVs for targeted delivery into specific tissues (e.g., using targeting ligands, peptides, or nanobodies) are discussed. Opportunities for both endogenous (intracellular production of CRISPR/Cas) and exogenous (post-production) loading of EVs are presented.

Clearing of Foreign Episomal DNA from Human Cells by CRISPRa-Mediated Activation of Cytidine Deaminases.

Restriction of foreign DNA is a fundamental defense mechanism required for maintaining genomic stability and proper function of mammalian cells. APOBEC cytidine deaminases are crucial effector molecules involved in clearing pathogenic DNA of viruses and other microorganisms and improperly localized self-DNA (DNA leakages). Mastering the expression of APOBEC provides the crucial means both for developing novel therapeutic approaches for combating infectious and non-infectious diseases and for numerous research purposes. In this study, we report successful application of a CRISPRa approach to effectively and specifically overexpress APOBEC3A and APOBEC3B deaminases and describe their effects on episomal and integrated foreign DNA. This method increased target gene transcription by >6-50-fold in HEK293T cells. Furthermore, CRISPRa-mediated activation of APOBEC3A/APOBEC3B suppressed episomal but not integrated foreign DNA. Episomal GC-rich DNA was rapidly destabilized and destroyed by CRISPRa-induced APOBEC3A/APOBEC3B, while the remaining DNA templates harbored frequent deaminated nucleotides. To conclude, the CRISPRa approach could be readily utilized for manipulating innate immunity and investigating the effects of the key effector molecules on foreign nucleic acids.

Dead Cas Systems: Types, Principles, and Applications.

The gene editing tool CRISPR-Cas has become the foundation for developing numerous molecular systems used in research and, increasingly, in medical practice. In particular, Cas proteins devoid of nucleolytic activity (dead Cas proteins; dCas) can be used to deliver functional cargo to programmed sites in the genome. In this review, we describe current CRISPR systems used for developing different dCas-based molecular approaches and summarize their most significant applications. We conclude with comments on the state-of-art in the CRISPR field and future directions.

m6A Methylation in Regulation of Antiviral Innate Immunity.

The epitranscriptomic modification m6A is a prevalent RNA modification that plays a crucial role in the regulation of various aspects of RNA metabolism. It has been found to be involved in a wide range of physiological processes and disease states. Of particular interest is the role of m6A machinery and modifications in viral infections, serving as an evolutionary marker for distinguishing between self and non-self entities. In this review article, we present a comprehensive overview of the epitranscriptomic modification m6A and its implications for the interplay between viruses and their host, focusing on immune responses and viral replication. We outline future research directions that highlight the role of m6A in viral nucleic acid recognition, initiation of antiviral immune responses, and modulation of antiviral signaling pathways. Additionally, we discuss the potential of m6A as a prognostic biomarker and a target for therapeutic interventions in viral infections.

Swelling, Rupture and Endosomal Escape of Biological Nanoparticles Per Se and Those Fused with Liposomes in Acidic Environment.

Biological nanoparticles (NPs), such as extracellular vesicles (EVs), exosome-mimetic nanovesicles (EMNVs) and nanoghosts (NGs), are perspective non-viral delivery vehicles for all types of therapeutic cargo. Biological NPs are renowned for their exceptional biocompatibility and safety, alongside their ease of functionalization, but a significant challenge arises when attempting to load therapeutic payloads, such as nucleic acids (NAs). One effective strategy involves fusing biological NPs with liposomes loaded with NAs, resulting in hybrid carriers that offer the benefits of both biological NPs and the capacity for high cargo loads. Despite their unique parameters, one of the major issues of virtually any nanoformulation is the ability to escape degradation in the compartment of endosomes and lysosomes which determines the overall efficiency of nanotherapeutics. In this study, we fabricated all major types of biological and hybrid NPs and studied their response to the acidic environment observed in the endolysosomal compartment. In this study, we show that EMNVs display increased protonation and swelling relative to EVs and NGs in an acidic environment. Furthermore, the hybrid NPs exhibit an even greater response compared to EMNVs. Short-term incubation of EMNVs in acidic pH corresponding to late endosomes and lysosomes again induces protonation and swelling, whereas hybrid NPs are ruptured, resulting in the decline in their quantities. Our findings demonstrate that in an acidic environment, there is enhanced rupture and release of vesicular cargo observed in hybrid EMNVs that are fused with liposomes compared to EMNVs alone. This was confirmed through PAGE electrophoresis analysis of mCherry protein loaded into nanoparticles. In vitro analysis of NPs colocalization with lysosomes in HepG2 cells demonstrated that EMNVs mostly avoid the endolysosomal compartment, whereas hybrid NPs escape it over time. To conclude, (1) hybrid biological NPs fused with liposomes appear more efficient in the endolysosomal escape via the mechanism of proton sponge-associated scavenging of protons by NPs, influx of counterions and water, and rupture of endo/lysosomes, but (2) EMNVs are much more efficient than hybrid NPs in actually avoiding the endolysosomal compartment in human cells. These results reveal biochemical differences across four major types of biological and hybrid NPs and indicate that EMNVs are more efficient in escaping or avoiding the endolysosomal compartment.

Hydroxychloroquine Enhances Cytotoxic Properties of Extracellular Vesicles and Extracellular Vesicle-Mimetic Nanovesicles Loaded with Chemotherapeutics.

Because of their high biocompatibility, biological barrier negotiation, and functionalization properties, biological nanoparticles have been actively investigated for many medical applications. Biological nanoparticles, including natural extracellular vesicles (EVs) and synthetic extracellular vesicle-mimetic nanovesicles (EMNVs), represent novel drug delivery vehicles that can accommodate different payloads. In this study, we investigated the physical, biological, and delivery properties of EVs and EMNVs and analyzed their ability to deliver the chemotherapeutic drug doxorubicin. EMNVs and EVs exhibit similar properties, but EMNVs are more effectively internalized, while EVs show higher intracellular doxorubicin release activity. In addition, these nanotherapeutics were investigated in combination with the FDA-approved drug hydroxychloroquine (HCQ). We demonstrate that HCQ-induced lysosome destabilization and could significantly increase nanoparticle internalization, doxorubicin release, and cytotoxicity. Altogether, these data demonstrate that, from the delivery standpoint in vitro, the internalization of EMNVs and EVs and their payload release were slightly different and both nanotherapeutics had comparable cytotoxic performance. However, the synthesis of EMNVs was significantly faster and cost-effective. In addition, we highlight the benefits of combining biological nanoparticles with the lysosome-destabilizing agent HCQ that increased both the internalization and the cytotoxic properties of the particles.

Technological aspects of manufacturing and analytical control of biological nanoparticles.

Extracellular vesicles (EVs) are cell-derived biological nanoparticles that gained great interest for drug delivery. EVs have numerous advantages compared to synthetic nanoparticles, such as ideal biocompatibility, safety, ability to cross biological barriers and surface modification via genetic or chemical methods. On the other hand, the translation and the study of these carriers resulted difficult, mostly because of significant issues in up-scaling, synthesis and impractical methods of quality control. However, current manufacturing advances enable EV packaging with any therapeutic cargo, including DNA, RNA (for RNA vaccines and RNA therapeutics), proteins, peptides, RNA-protein complexes (including gene-editing complexes) and small molecules drugs. To date, an array of new and upgraded technologies have been introduced, substantially improving EV production, isolation, characterization and standardization. The used-to-be "gold standards" of EV manufacturing are now outdated, and the state-of-art requires extensive revision. This review re-evaluates the pipeline for EV industrial production and provides a critical overview of the modern technologies required for their synthesis and characterization.

Transient and tunable CRISPRa regulation of APOBEC/AID genes for targeting hepatitis B virus.

APOBEC/AID cytidine deaminases play an important role in innate immunity and antiviral defenses and were shown to suppress hepatitis B virus (HBV) replication by deaminating and destroying the major form of HBV genome, covalently closed circular DNA (cccDNA), without toxicity to the infected cells. However, developing anti-HBV therapeutics based on APOBEC/AID is complicated by the lack of tools for activating and controlling their expression. Here, we developed a CRISPR-activation-based approach (CRISPRa) to induce APOBEC/AID transient overexpression (>4-800,000-fold increase in mRNA levels). Using this new strategy, we were able to control APOBEC/AID expression and monitor their effects on HBV replication, mutation, and cellular toxicity. CRISPRa prominently reduced HBV replication (∼90%-99% decline of viral intermediates), deaminated and destroyed cccDNA, but induced mutagenesis in cancer-related genes. By coupling CRISPRa with attenuated sgRNA technology, we demonstrate that APOBEC/AID activation can be precisely controlled, eliminating off-site mutagenesis in virus-containing cells while preserving prominent antiviral activity. This study untangles the differences in the effects of physiologically expressed APOBEC/AID on HBV replication and cellular genome, provides insights into the molecular mechanisms of HBV cccDNA mutagenesis, repair, and degradation, and, finally, presents a strategy for a tunable control of APOBEC/AID expression and for suppressing HBV replication without toxicity.

Depleting hepatitis B virus relaxed circular DNA is necessary for resolution of infection by CRISPR-Cas9.

CRISPR-Cas9 systems can directly target the hepatitis B virus (HBV) major genomic form, covalently closed circular DNA (cccDNA), for decay and demonstrate remarkable anti-HBV activity. Here, we demonstrate that CRISPR-Cas9-mediated inactivation of HBV cccDNA, frequently regarded as the "holy grail" of viral persistence, is not sufficient for curing infection. Instead, HBV replication rapidly rebounds because of de novo formation of HBV cccDNA from its precursor, HBV relaxed circular DNA (rcDNA). However, depleting HBV rcDNA before CRISPR-Cas9 ribonucleoprotein (RNP) delivery prevents viral rebound and promotes resolution of HBV infection. These findings provide the groundwork for developing approaches for a virological cure of HBV infection by a single dose of short-lived CRISPR-Cas9 RNPs. Blocking cccDNA replenishment and re-establishment from rcDNA conversion is critical for completely clearing the virus from infected cells by site-specific nucleases. The latter can be achieved by widely used reverse transcriptase inhibitors.

CRISPR/Cas and Hepatitis B Therapy: Technological Advances and Practical Barriers.

After almost a decade of using CRISPR/Cas9 systems to edit target genes, CRISPR/Cas9 and related technologies are rapidly moving to clinical trials. Hepatitis B virus (HBV), which causes severe liver disease, cannot be cleared by modern antivirals, but represents an ideal target for CRISPR/Cas9 systems. Early studies demonstrated very high antiviral potency of CRISPR/Cas9 and supported its use for developing a cure against chronic HBV infection. This review discusses the key issues that must be solved to make CRISPR/Cas9 an anti-HBV therapy.

Previous hepatitis B viral infection-an underestimated cause of pancreatic cancer.

BACKGROUND: The etiology of pancreatic cancer remains unclear. This limits the possibility of prevention and effective treatment. Hepatitis B virus (HBV) is responsible for the development of different types of cancer, but its role in pancreatic cancer is still being discussed. AIM: To assess the prevalence of previous HBV infection and to identify viral biomarkers in patients with pancreatic ductal adenocarcinoma (PDAC) to support the role of the virus in etiology of this cancer. METHODS: The data of 130 hepatitis B surface antigen-negative subjects were available for the final analysis, including 60 patients with PDAC confirmed by cytology or histology and 70 sex- and age-matched controls. All the participants were tested for HBV biomarkers in blood [antibody to hepatitis B core antigen (anti-HBc), antibody to hepatitis B surface antigen (anti-HBs) and HBV DNA], and for those with PDAC, biomarkers in resected pancreatic tissues were tested (HBV DNA, HBV pregenomic RNA and covalently closed circular DNA). We performed immunohistochemistry staining of pancreatic tissues for hepatitis B virus X antigen and Ki-67 protein. Non-parametric statistics were used for the analysis. RESULTS: Anti-HBc was detected in 18/60 (30%) patients with PDAC and in 9/70 (13%) participants in the control group (P = 0.029). Accordingly, the odds of PDAC in anti-HBc-positive subjects were higher compared to those with no previous HBV infection (odds ratio: 2.905, 95% confidence interval: 1.191-7.084, standard error 0.455). HBV DNA was detected in 8 cases of PDAC and in 6 of them in the pancreatic tumor tissue samples only (all patients were anti-HBc positive). Blood HBV DNA was negative in all subjects of the control group with positive results of the serum anti-HBc test. Among 9 patients with PDAC, 5 revealed signs of replicative competence of the virus (covalently closed circular DNA with or without pregenomic RNA) in the pancreatic tumor tissue samples. Hepatitis B virus X antigen expression and active cell proliferation was revealed by immunohistochemistry in 4 patients with PDAC in the pancreatic tumor tissue samples. CONCLUSION: We found significantly higher risks of PDAC in anti-HBc-positive patients. Detection of viral replication and hepatitis B virus X protein expression in the tumor tissue prove involvement of HBV infection in pancreatic cancer development.

May Previous Hepatitis B Virus Infection Be Involved in Etiology and Pathogenesis of Autoimmune Liver Diseases?

INTRODUCTION: Viral infections, especially with hepatotropic viruses, may trigger autoimmune liver diseases (AILDs) and deteriorate their course. However, association of previous hepatitis B virus (HBV) infection (presence of anti-HBc with or without anti-HBs or HBV DNA in serum) with AILDs is poorly studied so far. The aim of the study was to assess the prevalence of previous hepatitis B virus infection markers and its clinical significance in patients with autoimmune liver diseases. METHODS: The study was based on the data obtained from 234 consecutive HBsAg-negative patients with AILDs [81 with autoimmune hepatitis (AIH), 122 with primary biliary cholangitis (PBC) and 31 with primary sclerosing cholangitis (PSC)] and 131 subjects of the control group without liver diseases. Blood samples of the enrolled patients were tested for anti-HBc and HBV DNA. Samples of liver tissue were examined by standard morphologic protocol and, in anti-HBc positive subjects, for HBV DNA. We assessed estimated risks of AILDs according to anti-HBc positivity and association of anti-HBc positivity with stage of liver fibrosis. RESULTS: Anti-HBc was detected in 14.5% participants in the control group vs 26.1% (p = 0.016) in patients with AILDs (including 27.1% subjects with PBC (p = 0.021 vs control group), in 29% of PSC and 23.5% in AIH. HBV DNA was detected in three patients with PBC and in one with AIH. Positive anti-HBc test result was associated with higher risk of AILDs-odds ratio (OR) = 2.078 [95% confidence interval (CI) 1.179-3.665], especially in PBC: OR (95% CI) 2.186 (1.165-4.101). Odds of advanced stage of liver fibrosis (F3-F4 by METAVIR) in anti-HBc-positive subjects with PBC were also higher compared to those who had no previous HBV infection: OR (95% CI) 2.614 (1.153-5.926). CONCLUSIONS: Significant proportions of patients with AILDs are anti-HBc positive, and some of them have OBI. Among patients with AILDs, anti-HBc-positivity is most widespread in the PBC group and in subjects with advanced stage of liver fibrosis. Our data may support the idea of an important role of previous HBV infection in the etiology and pathogenesis of AILDs (namely PBC).

Host-cell interactions in HBV infection and pathogenesis: the emerging role of m6A modification.

Hepatitis B virus (HBV) is a DNA virus with a complex life cycle that includes a reverse transcription step. HBV is poorly sensed by the immune system and frequently establishes persistent infection that can cause chronic infection, the leading cause of liver cancer and cirrhosis worldwide. Recent mounting evidence has indicated the growing importance of RNA methylation (m6A modification) in viral replication, immune escape, and carcinogenesis. The value of m6A RNA modification for the prediction and clinical management of chronic HBV infection remains to be assessed. However, a number of studies indicate the important role of m6A-marked transcripts and factors of m6A machinery in managing HBV-related pathologies. In this review, we discuss the fundamental and potential clinical impact of m6A modifications on HBV infection and pathogenesis, as well as highlight the important molecular techniques and tools that can be used for studying RNA m6A methylome.

Immunity and Viral Infections: Modulating Antiviral Response via CRISPR-Cas Systems.

Viral infections cause a variety of acute and chronic human diseases, sometimes resulting in small local outbreaks, or in some cases spreading across the globe and leading to global pandemics. Understanding and exploiting virus-host interactions is instrumental for identifying host factors involved in viral replication, developing effective antiviral agents, and mitigating the severity of virus-borne infectious diseases. The diversity of CRISPR systems and CRISPR-based tools enables the specific modulation of innate immune responses and has contributed impressively to the fields of virology and immunology in a very short time. In this review, we describe the most recent advances in the use of CRISPR systems for basic and translational studies of virus-host interactions.

CRISPR Screening: Molecular Tools for Studying Virus-Host Interactions.

CRISPR/Cas is a powerful tool for studying the role of genes in viral infections. The invention of CRISPR screening technologies has made it possible to untangle complex interactions between the host and viral agents. Moreover, whole-genome and pathway-specific CRISPR screens have facilitated identification of novel drug candidates for treating viral infections. In this review, we highlight recent developments in the fields of CRISPR/Cas with a focus on the use of CRISPR screens for studying viral infections and identifying new candidate genes to aid development of antivirals.

Suppressing the NHEJ pathway by DNA-PKcs inhibitor NU7026 prevents degradation of HBV cccDNA cleaved by CRISPR/Cas9.

Chronic hepatitis B is a severe liver disease caused by hepatitis B virus (HBV) infection. Covalently closed circular DNA (cccDNA), a super-spiralized, double-stranded form of the HBV genome, is the major determinant of viral persistence. CRISPR/Cas9 nucleases have been recently shown to introduce double-stranded DNA breaks into HBV cccDNA. The inflicted damage results predominantly in erroneous repair of cccDNA by non-homologous end-joining (NHEJ). NHEJ has been suggested to enhance anti-HBV activity of CRISPR/Cas9 and increase cccDNA mutation. In this study, we assessed anti-HBV activity of CRISPR/Cas9 and cccDNA repair outcomes in an altered NHEJ/HR environment. NU7026, a strong inhibitor of NHEJ, prevented CRISPR/Cas9-mediated degradation of cccDNA and resulted in frequent on-target deletions. We conclude that CRISPR/Cas9 is a highly effective tool to degrade cccDNA and first demonstrate that inhibiting NHEJ impairs cccDNA degradation.