CRISPR/Cas9 cleavage of viral DNA efficiently

CRISPR/Cas9 cleavage of viral DNA efficiently Welcome to a Vacuum Cleaner Battery specialist of the Agilent Battery

Chronic hepatitis B virus (HBV) infection is prevalent, deadly, and seldom cured due to the persistence of viral episomal DNA (cccDNA) in infected cells. Newly developed genome engineering tools may offer the ability to directly cleave viral DNA, thereby promoting viral clearance. Here, we show that the CRISPR/Cas9 system can specifically target and cleave conserved regions in the HBV genome, resulting in robust suppression of viral gene expression and replication. Upon sustained expression of Cas9 and appropriately chosen guide RNAs, we demonstrate cleavage of cccDNA by Cas9 and a dramatic reduction in both cccDNA and other parameters of viral gene expression and replication. Thus, we show that directly targeting viral episomal DNA is a novel therapeutic approach to control the virus and possibly cure patients.

Hepatitis B virus (HBV) chronically infects over 250 million people worldwide with battery like Agilent N9330 Battery, Agilent N9330B Battery, Agilent N9340B Battery, Agilent N9330B-BAT Battery, Agilent N9330B-BCG Battery, Agilent TY 3CGR18650D-2 Battery, IAI AV6413 Battery, Unipower B11588 Battery, Alpha Source AS30139 Battery, Interstate Batteries AMED2160, Interstate Batteries ACAM0300, Alpha Source AS36011 Battery. Chronically infected individuals are at an increased risk for deadly complications, including cirrhosis, end-stage liver disease and hepatocellular carcinoma, resulting in approximately 600,000 deaths per year1. HBV is a member of the Hepadnaviridae family and its life cycle involves both DNA and RNA intermediates. The HBV genome exists in the nuclei of infected hepatocytes as a 3.2kb double-stranded episomal DNA species called covalently closed circular DNA (cccDNA). cccDNA is a key component in the HBV life cycle, since it is the template for all viral genomic and subgenomic transcripts2. Currently approved HBV therapies act post-transcriptionally to inhibit viral replication and thus fail to target or eliminate the cccDNA pool, which exhibits extraordinary stability and persistence3. Consequently, these drugs must often be taken indefinitely to prevent viral rebound. Agents that act directly on viral DNA to deplete this reservoir may represent more desirable and possibly curative therapeutic alternatives4.

To this end, targeted nucleases may provide an efficient and specific way to damage the HBV genome while sparing host genomic DNA5,6,7. Targeted nucleases catalyze double-stranded DNA break (DSB) formation, which leads to the formation of mutagenic insertions and deletions (indels) through error-prone nonhomologous end-joining (NHEJ) at the target DNA locus. Recently, the type II CRISPR-Cas system of Streptococcus pyogenes SF370 has been adapted as an RNA-guided, sequence-specific DNA nuclease for use in mammalian cells8,9. CRISPR/Cas9 and other genome engineering technologies have been employed to design candidate therapeutics via gene targeting, knockout of beneficial host genes, and mutation of integrated viruses10, and we sought to further study the application of CRISPR/Cas9 to direct targeting and cleavage of HBV cccDNA. We hypothesized that by directly targeting the HBV genome for cleavage using CRISPR/Cas9, we could suppress HBV by mutagenizing critical genomic elements or decreasing the stability of cccDNA and other viral intermediates through repeated linearization of the circular genomes (Fig. 1a).