CRISPR-Cas9 gene-editing technique for repair of antithrombin gene SERPINC1 c.318_319insT mutation
10.13602/j.cnki.jcls.2025.06.02
- VernacularTitle:CRISPR-Cas9基因编辑技术修复抗凝血酶基因SERPINC1 c.318_319insT突变
- Author:
Haixiao XIE
1
;
Xingxing ZHOU
1
;
Qiyu XU
1
;
Ke ZHANG
1
;
Siqi LIU
1
;
Mingshan WANG
1
Author Information
1. 温州医科大学附属第一医院医学检验中心,浙江温州 325035
- Publication Type:Journal Article
- Keywords:
thrombotic disease;
antithrombin;
mutation;
CRISPR-Cas9;
gene-editing technique
- From:
Chinese Journal of Clinical Laboratory Science
2025;43(6):405-409
- CountryChina
- Language:Chinese
-
Abstract:
Objective To discuss the preliminary application of CRISPR-Cas9 gene editing technology in repair of antithrombin gene(SERPINC1)c.318_319insT mutation.Methods The single guide RNA(sgRNA)was designed by CRISPR online design website,and AT c.318_319 insT mutant and CRISPR-Cas9 repairsome were constructed.The gene fragments from the wild-type gene,AT c.318_319 insT mutant and CRISPR-Cas9 repairsome were transferred into lentiviral expression vectors,and then PCR sequencing was performed for verification.The successfully constructed lentiviral recombinant plasmids were transfected into the human embryonic kid-ney cells(HEK293T).After cell culture,HEK293T cells were lysed.The AT:Ag levels in the cell lysing reagents from wild-type gene,CRISPR-Cas9 repairsome and mutant were compared by ELISA and Western blot,respectively.The recombinant AT protein was characterized in vitro by cellular immunofluorescence assay.Results Both the AT c.318_319insT mutant and CRISPR-Cas9 repair-some were successfully constructed.The results of experiments with HEK293T cells in vitro showed that the wild-type AT:Ag in the cell lysing reagents was set as 100％,the AT:Ag of CRISPR-Cas9 repairsome was 47％,and the AT:Ag of AT c.318_319insT was 22％,which were consistent with the results of western blot and cellular immunofluorescence assay.Conclusion The cellular experiments in vitro verified that CRISPR-Cas9 gene editing technology could effectively repair the SERPINC1 c.318_319 insT mutation in situ,which might provide the experimental support for the application of CRISPR-Cas9 gene editing technology in the gene therapy of hereditary thrombotic diseases.
