Method Exploration On Construction Of Aptamer Plasmid Expression Vector Using Crispr/Cas9 Technique

Objective: This project based on the principle that the CRISPR/Cas9 mutant Cas9 D10 A nickase can shear the specific position of the single chain in the DNA double chain.By two single-nickase cutting two specific location in the complementary chain of including aptamer sequence chain from recombinant vector substrate,and the part of aptamer sequence maintain completely connected with vector,to explore a construction medthod of a new“targeted expression vector”.This kind of aptamer-plasmid targeted expression vector has the characteristics of both aptamer targeting and plasmid carrying functional expression genes.In order to provide early technical and methodological guidance for the realization of safe,efficient and economical non-viral vector biological acid chain autonomous targeting therapeutic function in the future.Methods: 1.Two recombinant vectors were constructed.Firstly,the aptamer extension sequence with a specific sequence at both ends and its complementary sequence are designed and synthesized,and the double chain is formed by annealing.The double strands with PX461 linear vector or PX461-sg RNA1+2 linear vector were linked by T4 DNA ligase.The recombinant vector of PX461-END and PX461-sg RNA1+2-END were successfully constructed.2.Extracellular shear verification with RNP1(Cas9D10A + sg RNA1,RNP1)and RNP2(Cas9 D10 A + sg RNA2,RNP2)complex.3.PX461-END recombinant vector and double RNP complexes were successively transferred into 293 T cells in order to intracellular shear verification.4.The PX461-sg RNA1+2-END co-expression vector was used to transfect 293 T cells for self-shear validation.The cut products was verified by agarose gel electrophoresis,polymerase chain reaction(PCR),sequencing and using nano-DNA probe to detect the cut short chain indirectly.Results: 1.Two recombinant vectors of PX461-sg RNA1+2-END and PX461-END were successfully constructed.2.The product of recombinant vector PX461-END cleaved by the extracellular double RNP complexes was visible in agarose gel imaging.The fragment containing the target site was further amplified by PCR.The results showed that the control group was a single band with the expected size.The experimental group was a double band,but the brightness of smaller band is weak.It is speculated that there is a possibility the RNP complex has an effective target cleavage outside the cell.After annealing and purification of shear products,T4 PNK and T4 DNA ligase were added.The target fragment amplified by PCR showed that the bands of each group were single.The short single chain DNA cut off was detected by the nano probe.The results showed that the fluorescence signal of the shear product group was the same as that of the blank group,and the positive control group had a strong fluorescence signal.3.Plasmid DNA was extracted by plasmid extraction kit after the recombinant plasmid PX461-END was cut by double RNP complexes in 293 T cells.And the target fragment was amplified by PCR,the results showed that the experimental group and the control group had double bands,and the brightness of the smaller band was also weak.The target shear fragment was amplified by PCR.In the experimental group,there were double bands,and the brightness of the smaller bands was also weak.4.After the PX461-sg RNA1+2-END was transfected into 293 T cells,the target fragment was also amplified by PCR.There was no obvious double band in agarose gel imaging in the experimental group,which was consistent with that in the positive control group.Shear products of double RNP complexes outside cells and the products of PCR target fragment were sent to Guangzhou sequencing department for sequencing,the forward primer sequencing results included the aptamer sequence,and the reverse primer sequencing results contained the complementary sequence.There is no expected shear result.Conclusion: In this study,a double Cas9 D10 A nickase splicing substrate recombinant vector was successfully constructed.The method of constructing plasmid(PX461)and aptamer(endoglin)“targeted expression vector” was explored by using efficient site-directed gene editing technique.The methods and results obtained can provide experimental basis and guidance for the further study of targeted expression vector.