动物造模,CRISPR/Cas9基因编辑技术 z-bio 2021-10-27 354

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

　　 Method: Use the online prediction software with FcγR2b and FcγR3Design sgRNA in the exon region, and select 5 candidate sgRNAs with low off-target effects at each site? Do you want to use CRISPR/Cas9 activity detection kit to detect sgRNA activity in vitro? Choose sgRNA with higher activity to have the same in vitro transcription function as Cas9 mRNA. Have you injected fertilized eggs? Genetically modified mice were obtained by PCR detection and sequencing. The knockout fragment was 89,711 bp, and the 5'end of the FcγR2b gene, the 3'end of the FcγR3 gene and the FcγR4 gene could be knocked out at the same time, which was predicted by software. Have you sequenced and confirmed the eight most likely off-target sites and all the off-target sites in the first mouse genome?

　　Result: Does the result show that no small fragment insertion or deletion is found near the predicted off-target site?

　　 Conclusion: We have established a technology that uses CRISPR/Cas9 gene editing technology to knock out very large genome fragments. Will the combination of this technology and BAC transgenic technology provide a new way to establish humanized mice with complex genetic families?