Inhibition Of Hepatitis B Virus By The CRISPR/Cas9 System Via Targeting The Conserved Regions Of The Viral Genome

The infection of Hepatitis B virus(HBV)is really a global public health problem,there are approximately 350 million people are chronically infected by HBV,including 100 million Chinese.Therefore,the HBV infection is a much severer trouble for Chinese.Chronic HBV infection usually cause liver diseases such as hepatitis,fibrosis,cirrhosis,and these desaeses might lead to hepatocyte cancer at last.HBV is a kind of small DNA virues,the genomic size is approximately 3.2kb.With such a short genome,the viral protein ORFs must be overlapped to encode all proteins.Even though the HBV is not retrovirus,reverse transcription which is likely to cause high mutation efficiency consists in the viral life cycle.So,the divergency of HBV genome is very high,and can be defined into many different genotype and subgenotype.HBV exists in a quasispecies status for different patients,which shows a highly complexity of the viral genomes.After entering into the host cells,the HBV transports it's rcDNA into the nucleus.Then the rcDNA is completed into the cccDNA,which will serve as the template for transcription of viral pgRNA and mRNAs.The cccDNA exhibits staggering stability and declines slowly under antiviral therapy.The antiviral drugs used in the clinical treatment of HBV infection can only inhibit the viral replication but can't clear the virus and can't cure the infection.Once the drug treatment is breaked,the HBV will be produced on a large scale with the existence of cccDNA.Therefore,the efficient and nontoxic clearance of cccDNA is the main goal for HBV therapy.Some other groups have reported the use of ZFN and TALEN in the genetherapy of HBV infection.Our team tried to research if the CRISPR/Cas9 system could inhibit the viral replication or clear the viral infection.Comparing with the ZFN and TALEN,the CRISPR/Cas9 system cost less,is the simplest as we only need to design a 20/21 nucleotide guide RNA,and has some other advantages.Considering the high mutation efficiency of HBV,we aligned the genomic sequences of 26 representative HBV genotypes and subgenotypes to identify the relatively conserved genomic regions among different HBV genotypes,which also implied the low variable regions of the viral template.Then we designed 8 spgRNAs targeting the conserved regions of the viral genome,and the gRNAs target the different ORF of the HBV.We tested the HBV inhibition efficiency of the HBV-specific CRISPR/spCas9 systems in vivo and in vitro,the spgRNAs performed very well.The HBV-specific CRISPR/spCas9 systems can also inhibit HBV with different genotypes,including genotypes A,B,C and D.Moreover,we demonstrated the CRISPR/Cas9 system inhibit the HBV by cleaving the viral DNA,which would lead to the mutation or degradation of the viral genome.What's more,we found the multiple spgRNA/Cas9 systems can cleave the HBV DNA into fragments,and showed the higher inhibition efficiency compared with single gRNA.At last,considering the clinical dose delivery system,we plan to chose the AAV8 as the carrier to delivery the CRISPR/Cas9 system into the hepatocytes.Because the restrictive cargo size of AAV is too small to package the normal used spCas9,we changed the spCas9 with the saCas9,and designed 7 sagRNAs targeting the conserved region of the HBV genome.Then,we detected the HBV inhibition efficiency of the HBV-specific CRISPR/saCas9 system,and compared the efficiency of the two different CRISPR/Cas9 systems.