动物造模,CRISPR/Cas9 z-bio 2022-04-26 944

　　Objective: To establish a method system for gene editing of the second exon of mouse MAD2L1 gene using CRISPR/Cas9 system, and to analyze the off-target effect of CRISPR/Cas9 on MAD2L1 gene editing.

　　Methods: The target sequence located in the second exon of the mouse MAD2L1 gene was designed through the CHOPCHOP website, and the Cas9-MAD2L1 vector was constructed. The vector was transfected into NIH/3T3 cells, screened by puromycin and observed with fluorescence microscopy. After GFP, the genomic DNA of the transfected cells was extracted, and the PCR method, combined with T7E1 analysis and Sanger sequencing, were used to identify the gene editing of mouse MAD2L1, and the transfected NIH/3T3 cells were subjected to CRISPR/Cas9 off-target effect. analyze.

　　Results: After the Cas9-MAD2L1 vector was transfected into NIH/3T3 cells and screened by puromycin, a large number of GFP-expressing cells were observed under the fluorescence microscope. The results of PCR combined with T7E1 showed that the transfected cell DNA was used as the template for amplification The resulting 228 bp MAD2L1 PCR product can be enzymatically cut into 166 bp and 62 bp fragments. The sequencing results show that the gene editing at the second exon target of the MAD2L1 gene was successfully performed, and the off-target effect analysis did not detect CRISPR/Cas9. Gene editing at off-target sites.

　　Conclusion: A method system for gene editing of the second exon of the mouse MAD2L1 gene was successfully established, and no off-target effects were detected for the designed MAD2L1 gene editing target.