The Evaluation And Application Of CRISPR/Cas9 Gene Editing Efficiency With White-to-Blue Colony Formation Assay

Part ?.The application and validation of CRISPR/Cas9 AHI1 gene editing efficiency with white-to-blue colony formation assayAim: Gene editing efficiency of the CRISPR/Cas9 system is influenced by many factors,among which the level of specificity between different sg RNA sequences is particularly important.The editing efficiency is relatively high when specificity is well.Nowadays,there are many ways to evaluate the level of editing efficiency between different sg RNAs.The purpose of this project is to test whether the prokaryotic evaluation system of white-to-blue colony formation assay can also be used to evaluate the editing efficiency of different sg RNA in CRISPR/Cas9 systems and to screen sg RNA with the optimal editing efficiency of AHI1 gene.The study could provide a reference for possible future gene therapy.Methods: In this study,the white-to-blue colony formation assay was performed using the classical blue-white screening principle.First,the partial sequence encoding the ?-gal region on the p MD-19 T plasmid was replaced by a long sequence containing the target sequence,resulting in a frameshift mutation.The replaced plasmid is called p MD-repeat plasmid.When transformed into solid culture medium containing X-gal and IPTG,it cannot form blue colonies.When co-transformed with the p Cas9 plasmid loaded with sg RNA corresponding to the target,the target sequence of p MD-repeat plasmid is cleaved by the Cas9 protease guided by sg RNA and homologous recombination occurs between two repeats.The sequence of the gene encoding ?-gal is restored from frameshift status to in-frame status,blue colonies form induced by X-gal and IPTG.Multiple sg RNAs of AHI1 were designed using online tools to construct p Cas9 plasmid containing sg RNA sequences and p MD-repeat plasmid containing the corresponding targetsequences.Equal quantities of two types of plasmids were co-transformed into DH5 a competent cells using X-gal –TPTG –chloramphenicol –ampicillin plates and observe the proportion of blue colonies to total colonies.The plasmid p Sp Cas9(BB)-2A-GFP containing the sg RNA sequence were established and transfected into He La cells.After gene editing,the cell genomes of the control and experimental groups were extracted and primers were designed on the upstream and downstream regions of the sg RNA.Then PCR reaction using Q5 high-fidelity enzyme,product purification,T7E1 enzyme digestion,agarose gel electrophoresis,observed the results after electrophoresis.Sequencing primers were designed,purified products were sequenced.Whether there was a peak near the Cas9 enzyme cleavage site or not was observed.Results: After the CRISPR plasmid was transfected into cells,the genome was extracted,and primers were designed near the editing site for PCR.The T7E1 enzyme could recognize the mismatched bases in the product and arouse the double strands break(DSB).The result of the electrophoresis band can reflect editing efficiency of specific sg RNA.After the sequencing of the product,the level of peak in the sequencing chromatogram can also reflect the level of editing efficiency of sg RNA.The screening results of the sg RNA editing efficiency of AHI1 in the white-to-blue colony formation assay are consistent with the results of the above two methods and the sg RNA with the optimal editing efficiency of the AHI1 gene was screened.Conclusion: The white-to-blue colony formation assay can be used to evaluate the editing efficiency of different sg RNAs in the CRISPR/Cas9 system.The new method is economical,easy to operate,time-saving,and low-demand for equipment.At the same time,the screened optimal sg RNA of AHI1 gene provides a reference for future gene therapy.Part ?.CRISPR/Cas9-mediated generation of HeLa DJ-1 knockout cell lineAims: Using the white-to-blue colony formation assay to screen out the sg RNA with optimal editing efficiency of DJ-1.Established CRISPR plasmid transfect He La cells,monoclonal He La cell line with DJ-1 knocked out would be obtained and to study theeffect of knockout DJ-1 on the proliferation of He La cells.Methods: Using white-to-blue colony formation assay,which was used in part one,multiple sg RNAs of DJ-1 with high specificity were designed by online tools to construct p Cas9 plasmid containing sg RNA sequences and p MD-repeat plasmid containing the corresponding target sequences.Equal quantities of two types of plasmids were co-transformed into DH5 a competent cells using X-gal –TPTG –chloramphenicol–ampicillin plates and to screen out specific sg RNA with optimal editing efficiency.The p Sp Cas9(BB)-2A-GFP plasmid containing optimal sg RNA sequence was constructed and transfected into He La cells.The single cell culture with fluorescence was selected by flow cytometry,and DJ-1 gene knockout was identified by Western blot,immunofluorescence and TA clone sequencing.The effect of the DJ-1 knockout cell line on cell proliferation was examined by CCK-8 assay.Results: The optimal editing efficiency of sg RNA against DJ-1 gene was screened by white-to-blue colony formation assay,and the constructed CRISPR knockout plasmids were transfected into cells.After sorting and culture,Western blot,immunofluorescence and TA clone sequencing were used to identify DJ-1 gene knockouts and obtained monoclonal human He La knockout cell line.Compared with the control group,knocked out cell lines slowed their cell proliferation.Conclusion: With the optimal sg RNA screened by white-to-blue colony formation assay,monoclonal He La cell line with DJ-1 knocked out was successfully obtained and the proliferation of He La cells was affected by the expression of the DJ-1 gene.The feasibility and effectiveness of the white-to-blue colony formation assay in constructing a monoclonal knockout cell line were verified and constructed monoclonal knockout He La cell line provides an effective tool for the further study of DJ-1 mechanism.