Establishment And Study Of A CRISPR/Cas9 Delivery System Based On Chitosan And Its Derivative

The rapid development of more vigorous gene therapy urgently requires efficient and low-toxicity delivery platforms for transmitting gene-editing tools,which strongly motivates further research of gene carrier.In this thesis,a kind of non-viral vectors for CRISPR/Cas9 gene-editing system was studied.A model CRISPR All-In-One gene-editing plasmid was constructed as the template.Besides,TMC,a kind of chitosan modified derivative,was prepared.Both chitosan and TMC were used as gene carriers loaded with CRISPR/Cas9 gene-editing tools to form nanocomplexes for achieving effective transfection in vitro.Gene therapy is an emerging treatment strategy to deal with difficult and severe diseases from the perspective of genetic material.In gene therapy based on CRISPR/Cas9 gene editing technology,treatment can be achieved by constructing an All-In-One expression plasmid that co-loads sgRNA and Cas9 protein.Numerous studies have shown that ApoE gene is a potential therapeutic target.Therefore,in this study,a CRISPR All-In-One expression plasmid targeting it was constructed as a complete gene-editing system model by gene recombination technology.The sequencing results confirmed that the recombinant plasmid was successfully constructed,and the Nanodrop results showed that the extracted plasmid had high concentration and good purity.Chitosan is a natural positively charged linear polymer,which has many advantages such as strong gene-loading capacity,high biocompatibility,low cytotoxicity,easy modification and modification,etc.It has attracted much attention as a non-viral vector material.However,its water-insolubility and low transfection efficiency make its application extremely limited.Quaternization modification can solve this problem.Therefore,N,N,N-Trimethyl chitosan(TMC)was prepared by one step method in this study.1H-NMR spectroscopy and FT-IR spectroscopy confirmed that the structure of the synthesized product was correct.Chitosan-CRISPR/Cas9 nanocomplexes and TMC-CRISPR/Cas9 nanocomplexes were formed at different N/P ratios by complex agglomeration and characterized in terms of size and zeta potential by dynamic light scattering analysis.Besides,the agarose gel electrophoresis was used to confirm the capacity of chitosan and TMC to bind to CRISPR All-In-One plasmid.By examining the single factor variables,the preparation process was optimized.The average particle size of these nanocomposite samples prepared under optimal conditions was less than 200 nm,the PDI was no more than 0.3,and they all were positively charged.HEK-293T human embryonic kidney cells were used to evaluate the in vitro gene delivery capability and biosafety.The transfection efficiency was assessed by observing fluorescence under an inverted fluorescence microscope and counting the percentage of GFP-positive cells by flow cytometry.The results of transfection experiments show that chitosan materials can effectively transfer exogenous CRISPR All-In-One expression plasmids into cells.The TMC delivery group has higher transfection efficiency than the chitosan delivery group.The biosafety evaluation was performed by MTT assay,and the results showed that the chitosan-based CRISPR/Cas9 nanocomplexes had high biological safety.