A New Method Based On QPCR High Resolution Melting System To Detect CRISPR/Cas System Inducedmammal Genomic Mutation

The main task of biology science is to explore more functions of large amounts of newgene, reveal the importance of a few genes invovled in the development and disease in humanand animal, on the train of science development who born bioinformatics,in post-genomicera.As a ideal way to modify genome, genome editing can locate and alter chromosomestablely,not only allows people to establish a stable transgenetic model following their ownwishes, but also provides solution to solve miscellaneous disease.It plays an improtant roleboth in basic scientific research and practical application.It is impossbile to alter and modify genome efficiently and precisely before genomeediting spreads around. Genome editing now is consist of three routes: Zinc-Finger Nucleases,Transcription Activator-Like Effector Nucleases and Clustered Regularly Interspaced ShortPalindromic Repeats.In contrast to CRISPR， ZFN and TALEN seems rewarding lowrecognition efficiency, shooting effect and complex operation.Cas9system has a highlyefficient targeting efficiency and identify the shooting sites accurately. In this experiment,weconstructed a targeting vector to knock out gene in situ in human293T cell through Cas9system efficiently. Then a variety of means,including improved AFLP detection, Surveyorassay detection kit, high resolution melting curve analysis to verify gene targeting efficiencyof Cas9system, on the other hand looking forward a quick and convenient means of mutationdetection.The result shows that HRM detection techniques seem to be more efficient and accurate,compared with the classical mutation detection techniques, coupled with all the advantages ofthe PCR experiment,high efficiency,high throughput, high stability. Comparing to ZFN andTALEN technology has obvious advantages, CRISPR/Cas system efficiency approaches to40%, in site-specific mutation, which indicates that a fast and efficient operating system ismore conducive to our knock out genome editing and essential to experiment genome editingtechnology. Meanwhile, the construction of gRNA is also unique to the classical methodslisted in recent paper. We conduct a pair of oligo matching with target site in genome and anneal them in PCR protocol. The product will be used in the next PCR along with other twofragment amplified from plasmid gRNA_AAVS1_T1, amplified by a pair of primers,resulting in455bp fragment which is similar to mature gRNA. In this way, we have improvedthe efficiency of the construction of gRNA, reduced the cost of this experiment. GRNAassembled this got the same result through the validation of the detection in this experiment.