Construct A New TALEN Toolbox For Gene Editing And Use CRISPR/Cas9 Specifically Knock Out HBV CccDNA

Mouse and human share similar genome size,number of genes and genomic organization in a large part.Many genes of mouse and human appear to be conserved,so these give the precondition of using mouse knockout models to investigate the functions of human genes for disease curing.In mice,traditional gene targeting needs homologous targeting vector,electroporation as the means of introducing targeting vector into mouse embryonic stem cells.The next step involes the isolation of a clonal embryonic stem(ES)cell line that contain the desired mutation.Positive-negative selection can be used to enrich for ES cell lines that contain the desired modifified gene.The last step is to use these ES cells to generate chimeric mice that are able to transmit the mutant gene to their progeny.The whole process cost at least 1-2 years and labor consuming and money consuming.As a result,a new gene targeting strategy without relying on ES cells,safer and more effective needs to be developed eagerly.Transcription activator-like effector nucleases(TALEN)is fused of transcription activator-like(TAL)effectors and Fokl nucleases.TAL effectors are bacterial proteins that are injected into infected plant cells via a type ? secretion system by pathogens of the genus Xanthomonas.Their targeting specificity is determined by a central domain of tandem,33-35 amino acid(aa)repeats,followed by a single truncated repeat of 20 amino acids.A polymorphic pair of adjacent residues at positions 12 and 13 in each repeat,the repeat-variable di-residue(RVD),specifies the target,one RVD to one nucleotide,with the four most common RVDs each preferentially associating with one of the four bases.Fusions of transcription activator-like(TAL)effectors of plant pathogenic Xanthomonas spp.to the FokI nuclease,TALENs bind and cleave DNA in pairs,resulting DNA double strainds break(DSB).Conventional methods for constructing TALEN vectors are not perfect.Golden Gate is one of the most widely used methods for constructing TALEN vectors.It will cost at least 5 days to obtain the correct plasmid,which is time and labor consuming.Here,we constructed a new TALEN toolbox for gene engineering.Compared to Golden Gate,the new TALEN toolbox is easier to operate,time-saving and high-effective.With the new toolbox,we got Cntn4 knock out mice successfully.Also,the clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated(Cas)system is a novel genome editing tool derived from the adaptive immune system of bacteria and archaea in which it provides the host with resistance to invading foreign viruses or plasmids.The type ? CRISPR/Cas system consists of two short RNAs,CRISPR RNAs(crRNAs)and trans-activating crRNAs(tracrRNAs),and the DNA endonuclease Cas9.The mature crRNA::tracrRNA complex directs Cas9 DNA endonuclease to the target DNA sequence called the protospacer on the target DNA next to the protospacer adjacent motif(PAM)for site-specific cleavage.This system is further simplified by fusing the crRNA and tracrRNA into a single chimeric guide RNA(sgRNA).Compared with ZFNs and TALENs,the CRISPR/Cas system can be easily reprogrammed to cleave virtually any DNA sequence by simply redesigning the sgRNAs.Recently,the CRISPR/Cas system has been successfully applied to human cells and several model organisms for genome editing.Here,we evaluated the potential of using the CRISPR/Cas9 system to disrupt the HBV genome.To improve the specificity of the system,we were therefore interested in exploring the use of CRISPR/Cas9 nickase for genome editing.Because nicked genomic DNA is corrected by the endogenous base-excision repair pathway,Cas9 nickase would be expected to induce little or no damage to the genome.However,if two stranded nicks are located close together on opposite DNA strands,then the resulting double stranded break may be imprecisely repaired by non-homologous end joining(NHEJ).Thus,co-expression of Cas9 nickase and sgRNAs to closely paired sites on opposing DNA strands provides a general strategy for genome modification with minimal off-target damage.Here,we designed four pairs of sgRNAs that target four HBV-specific sites within the viral genome.With the HBV-specific sgRNAs,the CRISPR/Cas9 system significantly resulted in reduction of serum surface antigen levels in both 293T cells and HepG2 cells transfected with an HBV-expression vector.To assess CRISPR/Cas9 system toxicity,we utilized MTT assay as an indicator of cell viability following 48 hours after transfection.We verified that CRISPR/Cas9 system take effects without evidence for cellular toxicity.These date suggest that the CRISPR/Cas9 system could disrupt the HBV-expressing templates in vitro,indicating its potential in eradicating persistence HBV infection.