动物造模,基因编辑技术 z-bio 2022-08-26 332

　　Objective: To use CRISPR/Cas9 gene editing technology to knock out the mouse FcγR2b, FcγR3, FcγR4 gene clusters with a length of about 90 kb, so as to lay the foundation for the construction of FcγR gene humanized mice.

　　Methods: Using online prediction software to design sgRNAs in FcγR2b and FcγR3 exons, and select five candidate sgRNAs with low off-target effects at each site. CRISPR/Cas9 activity detection kits were used to detect the in vitro activities of sgRNAs. Select the activities The higher sgRNA was transcribed in vitro, and injected into fertilized eggs together with Cas9 mRNA. Through PCR detection and sequencing, a genetically modified mouse with a knockout fragment of 89711 bp was obtained, and the 5' end of FcγR2b gene and the 3' end of FcγR3 gene were simultaneously knocked out. and FcγR4 gene. The software also predicted 8 sites with the highest off-target possibility, and confirmed all the above-mentioned 8 off-target sites in the first mouse genome by sequencing.

　　RESULTS: The results showed that no small insertions or deletions were found near the predicted off-target sites.

　　CONCLUSION: The technology of knocking out large segments of the genome using CRISPR/Cas9 gene editing technology has been established. This technology combined with BAC transgenic technology will provide a new way to establish humanized mice with complex gene families.