Construction Of CRISPR/Cas Gene Editing System And Its Study Of Highly Selective And Sensitive Detection Of KRAS Gene Mutations

Objective: In order to detect the mutation of single base KRAS-G12 D,we first selected isothermal amplification technology to amplify the target fragment,and then further amplified the mutation signal by using rolling ring amplification technology and gold nanoparticles.A colorimetric biosensor based on CRISPR/Cas9 gene editing system was constructed for accurate diagnosis of KRAS-G12 D gene mutation,in order to meet the demand of point-of-care diagnosis(POCT).Methods: The KRAS-G12 D mutant template was amplified by RPA,and the PAM recognition sequence which is required for Cas9 protein cleavage was introduced into the template at the same time.Then,we constructed a vector with the capable of expressing Cas9 protein in prokaryotic cells under the induction of IPTG and purified with gradient concentrations of imidazole.After optimizing the experimental conditions such as SgRNA sequence and Padlock sequence in RCA reaction,colloidal gold nanoparticles were prepared by sodium citrate reduction method as signal amplifiers,and streptavidinmodified horseradish peroxidase was adsorbed on the surface of gold nanoparticles(hGNPs)by electrostatic adsorption interaction.The hGNPs were characterized by transmission electron microscopy and ultraviolet absorption spectroscopy.After the sensitivity and specificity analysis of the biosensor,the potential application of the colorimetric biosensor in clinical laboratory diagnosis was further evaluated by simulating biological samples.Results: Firstly,we successfully amplified the target fragment according to the RPA reaction,and the PAM site was introduced into the amplified fragment to ensure the accurate recognition and specific cleavage of the target by Cas9 protein;Secondly,by combining the optimized CRISPR/Cas cleavage system with the rolling circle amplification reaction,the colorimetric sensor greatly improved the detection specificity and reduced the generation of non-specific signals.In addition,after multiple signal amplification,the detection system not only ensured the specificity of KRAS wild-type and other mutations,but also greatly improved the sensitivity of KRAS-G12 D detection,so that the detection limitation could be as low as 0.2 f M.Finally,on simulated clinical samples,the colorimetric biosensor could detect the mutation probability of 0.01% by naked eye without the aid of any analytical instruments.