Construction And Application Of MiRNA And RISC-mediated SgRNA Expression System

CRISPR/Cas9,which consists of sgRNA and Cas9 protein,is the most powerful gene editing tool currently.The Cas9 nuclease can be recruited to the target gene by base pairing between the sgRNA and its target gene in site-specific gene editing.In conventional eukaryotic CRISPR/Cas9 expression system,transcription of Cas9 and sgRNA were initiated by RNA polymerase II and III promoter,respectively.Compared to the RNA polymerase III promoter,transcription level of the RNA polymerase II promoter is easily regulated and it can transcribe longer RNA.Given the inherent differences between the RNA polymerase II and III promoters,we propose that there are two advantages in expressing sgRNAs with RNA polymerase II promoter rather than with RNA polymerase III promoter.On the one hand,RNA polymerase II promoter can express sgRNA in a controllable manner.On the other hand,transcription of longer sgRNA polycistronic can achieve multiple gene editing at the same time.Though there was no significant differences in the expression levels of sgRNA transcribed by CMV(a widely used RNA polymerase II promoter)and the U6 promoter(a widely used RNA polymerase III promoter),sgRNAs directly transcribed by CMV promoter lack the ability of gene editing,whereas the TLR1.1 sgRNA expressed by U6 promoter resulted in a frameshift mutation in 90.3% of the reporter plasmid.We hypothesized that special structures such as caps and polyA tails in RNA polymerase II transcripts may lead to inactivation of sgRNAs.In order to achieve RNA polymerase II-mediated functional sgRNA expression,we designed two strategies that rely on RNA polymerase II promoter to express sgRNA,namely miRNA based sgRNA and RISC based sgRNA,respectively.Both strategies utilize endogenous RNase III to cleave RNA polymerase II transcripts and remove special structures such as caps and tails,to guarantee the proper structure and activity of sgRNA.The miRNA-based sgRNA expression strategy inserts sgRNAs between miRNA polycistronic,thus releasing mature sgRNAs from cap and tail structures during pri-and/or pre-miRNA processing.The RISC based sgRNA expression strategy uses RISC complex(an RNA-directed RNase III)to release mature sgRNA from primary transcript transcribed by RNA polymerase II.The study confirmed that both strategies can effectively express functional sgRNAs by CMV promoter for gene editing.Furthermore,most of miRNA-based sgRNA vectors expressed functional TLR1.1 sgRNA,and the inhibitory rate of luciferase is between 40~75%.Nuclease digestion analysis showed that miRNA-based sgRNA driven by CMV caused frame-shift mutations in 68~72% of the reporter genes,which is comparable to the efficiency of gene editing mediated by U6(87.7%).Our research also elucidated that sgRNA located upstream of the first miRNA was deficient in genome editing though it slightly inhibited the expression of reporter gene,indicating that though it could bind to the target gene and interfere with gene transcription,the nuclease activity was destroyed.While the insertion of sgRNA at other sites among the miRNA polycistronic did not affect the expression or activity of both miRNA and sgRNA.Moreover,northern blot analysis showed that the molecular weight of the sgRNA containing the cap structure was relatively larger.Based on those above and the three-dimensional structure of the Cas9: sgRNA: DNA complex,we proposed the “scissor repression model” to explain the phenomenon that the sgRNA containing the cap structure failed to perform gene editing.The use of the nerve-specific promoter hSynapsin to express sgRNA successfully edited the reporter gene in neurons,and there was no significant difference in the luciferase inhibitory rate(~30%)compared to the sgRNA transcribed by U6.However,sgRNA driven by h Synapsin did not edit genes in kidney-derived cells,indicating the tissue specificity of miRNA based sgRNA.Using miRNA based sgRNA,we also edited the reporter gene and the endogenous p53 gene simultaneously.The editing rates of the reporter vector and the endogenous p53 gene were 100% and 30.3%,respectively.The editing efficiency of the gene is also at the same level as that in the sgRNA driven by U6 promoter reported in previous researches.This shows that multiple sgRNAs can be stimultaneously transcribed by a single promoter with the miRNA based sgRNA strategy in multiple gene editing.Finally,this research also used mi RNA based sgRNA to express Lig4 shRNA and sgRNA simultaneously.QRT-PCR experiments confirmed that 80% of endogenous Lig4 gene expression was suppressed in HEK293 T cells transfected with this vector,and the ratio of homologous recombination gene repair was increased by about 3 times,which provides a feasible strategy for more accurate gene editing.In conclusion,this study successfully developed two strategies for expressing sgRNA using RNA polymerase II,named miRNA and RISC based sgRNA,respectively.Besides,with miRNA based sgRNA,we have achieved a series of applications including tissue-specific gene editing,multi-gene editing,and improved homologous recombination gene editing efficiency.In these applications,the advantages of RNA polymerase II promoter were fully reflected.Beside,we found that sgRNA processing mature in the cytoplasm can be export into nucleus for gene editing,implying that “The Subcelluar Distribution Theory” has certain flaws.In addition,this study also proposed a “scissor repression model” to explain how sgRNA's cap structure and redundant sequences result in the loss of nuclease activity,which laid a theoretical foundation for further understanding of the working principle of the CRISPR/Cas9 system and improving it.