SgRNA-shRNA Tandem Expression Technology And Its Application In Genome Editing

As the 3rd generation of the artificial endonucleases,the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9(CRISPR/Cas9)technology,with its properties of effective,economical,easy to customize,has been broadly applied to various of organisms for genome editing,which has extremely accelerated the researches in biological,medical and animal breeding fields.The CRISPR/Cas9 system consists of a single guide RNA(sgRNA)in charging of recognizing specific targets and the Cas9 protein performing the cleavage.After CRISPR/Cas9 system introduced double strand breaks(DSBs)into the genome,cells can repair the DSBs through the non-homologous end joining(NHEJ)or the homology-directed recombination(HDR)pathways,leading to gene knock-out,knock-in or point mutation(precise edting),which is significant for the study of gene function,gene theraphy and molecular breeding.At present,the multiplex genome targeting and precise genome editing are two major research directions in genome editing field.Focused on these two issues,this study proposed the sgRNA-shRNA tandem expression techonoly,which was based on the tandem transcript of multiple short RNAs(msRNAs,sgRNAs and shRNAs)driven by a single promoter.The sgRNAs can be used to guide Cas9 for multiplex genome targeting.The shRNA can be used for LIG4 mRNA interference,which is a key gene in the NHEJ pathway,leading to the improvement of the HDR efficiency.Making use of the SSA-RG and the SSA-RPG surrogate reporter systems that we developed previously,the multiplex genome targeting activities of sgRNAs transcribed from the msRNA structure were firstly examined on both reporter plasmid and genome level.Secondly,shRNA against LIG4 gene was introduced into the msRNAs structure to improve the HDR efficiency for precise genome editing study.What's more,we further modified the msRNAs structure and desgined the msRNAs/Cas9 co-expressing vector,which was applied successfully for multiplex genome targeting in chicken DF-1 cells.In this study,the novel msRNAs/Cas9 system enriched the “CRISPR/Cas9 toolkit”,which is especially suitable for multiplex genome targeting and precise genome editing,providing more choices for genome editng studies in the fields of gene therapy and animal breeding,etc.The main results of this research were as follows:1.Firstly,the msRNAs tandemly expressing system driven by a single promoter was developed.Employing CMV and U6 promoters respectively,the msRNAs expression plasmids with CMV-sgRNA-shRNA-sgRNA or U6-sgRNA-shRNA-sgRNA structure were constructed.In the transfection assay,the msRNAs expression plasmids,Cas9 expression plasmid and SSA-RG surrogate reporter plasmids were co-transfected into HEK293 T cells,and the activities of sgRNAs transcribed from the ms RNAs structures were examined.The results demonstrated that the U6 promoter performed much better than the CMV promoter did.2.An msRNAs plasmid(ms RNA-3)targeting human AAVS1,VEGF and CCR5 genes was constructed.The msRNA-3,Cas9 expression plasmid and the SSA-RPG reporter were used to co-transfect HEK293 T cells.Positive cell clones were randomly picked after the puromycin resistant screening for genotyping.The results indicated that the targeting efficiencies at AAVS1,VEGF and CCR5 loci were 81%,19% and 34% respectively,while the duplex and triplex targeting efficientcies were 22% and 9%.The results indicated that the msRNAs strategy had the potential of guiding Cas9 for multiplex genome targeting.3.To apply the shRNA transcribed from the msRNAs structure for improving the HDR-based precise genome editing,two msRNAs plasmids were constructed,namely msRNA-ALA and msRNA-ACA.The msRNA-ALA construct was designed to transcribe the sgRNA targeting AAVS1 locus and the shRNA against LIG4 gene,while the msRNA-ACA was the negative control with a non-functional shRNA.In the transfection assay,the LIG4 expression was firstly proved to be knocked down significantly by msRNA-ALA.Then,plasmids msRNA-ALA and msRNA-ACA were individually co-transfected with the Cas9 expression plasmid,the SSA-RPG reporter and the HDR donor plasmid into HEK293 T cells.Positive cell clones were randomly picked after the puromycin resistant screening for genotyping.The HDR-based precise editing efficiencies at the AAVS1 locus were 29% and 13%,respectively.4.The msRNAs structure was further modified and an msRNAs/Cas9 co-expression vector was designed.Next,the msRNAs/Cas9 vector targeting chicken ATP5 E,PPAR? and OVA genes was constructed.By using this msRNAs/Cas9 vector and corresponding SSA-RPG reporters,high efficient multiplex genome targeting in chicken DF-1 cells was achieved.The targeting efficiencies at ATP5 E,PPAR? and OVA loci were 75%,17% and 42% respectively,and the duplex and triplex targeting efficientcies were 25% and 17%.