Construction of CRISPR/Cas9 system targeting mouse Tsc1 and Tsc2 gene and verification of targeting effectiveness
10.13200/j.cnki.cjb.003875
- VernacularTitle:靶向小鼠Tsc1和Tsc2基因的CRISPR/Cas9系统的构建及其打靶效力验证
- Author:
YANG Lingling
- Publication Type:Journal Article
- Keywords:
CRISPR/Cas9 system;
Gene editing;
Tuberous sclerosis complex(TSC);
Tsc1;
Tsc2
- From:
Chinese Journal of Biologicals
2023;36(4):400-
- CountryChina
- Language:Chinese
-
Abstract:
ObjectiveTo design and construct CRISPR/Cas9 gene editing system targeting Tsc1 and Tsc2 genes,and verify the effectiveness of gene editing at cellular level.MethodsThree sgRNA guide sequences were designed for mouse Tsc1 and Tsc2 genes respectively. The sgRNA expression vector was constructed and co-transfected with the Cas9 expression plasmid into mouse N2a cells. After the positive cells were obtained through drug screening,the DNA fragments at the targeting site were amplified by PCR,and the targeting efficiency was verified by TA clone sequencing.ResultsThe five targets of Tsc1-M-sgRNA2 and Tsc1-M-sgRNA3 of Tsc1 gene and Tsc2-M-sgRNA1,Tsc2-M-sgRNA2 and Tsc2-M-sgRNA3 of Tsc2 gene were all edited,and the editing efficiency was 40%,80%,30%,30% and 20%,respectively.ConclusionA CRISPR-Cas9 gene editing system with editing efficiency targeting mouse Tsc1 and Tsc2 genes was successfully constructed.
