| Autor: |
Shi Y; Provincial Key Laboratory for Transfusion-Transmitted Infectious Diseases, Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu, China., Tan Q; Provincial Key Laboratory for Transfusion-Transmitted Infectious Diseases, Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu, China., Yang C; Provincial Key Laboratory for Transfusion-Transmitted Infectious Diseases, Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu, China., Li S; Provincial Key Laboratory for Transfusion-Transmitted Infectious Diseases, Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu, China., Li Y; Provincial Key Laboratory for Transfusion-Transmitted Infectious Diseases, Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu, China., He B; The Joint Laboratory on Transfusion-Transmitted Diseases (TTDs) Between Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Nanning Blood Center, Nanning Blood Center, Nanning, China., Xie H; The Hospital of Xidian Group, Xi'an, China., Duan X; Provincial Key Laboratory for Transfusion-Transmitted Infectious Diseases, Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu, China., Chen L; Provincial Key Laboratory for Transfusion-Transmitted Infectious Diseases, Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu, China.; The Joint Laboratory on Transfusion-Transmitted Diseases (TTDs) Between Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Nanning Blood Center, Nanning Blood Center, Nanning, China.; The Hospital of Xidian Group, Xi'an, China. |
| Abstrakt: |
CRISPR-Cas technology is a widely utilized gene-editing tool that involves gRNA-guided sequence recognition and Cas nuclease-mediated cleavage. The design and evaluation of gRNA are essential for enhancing CRISPR/Cas editing efficiency. Various assays such as single-strand annealing, in vitro cleavage, and T7 endonuclease I (T7EI) are commonly used to assess gRNA-mediated Cas protein cleavage activity. In this study, a firefly luciferase and Renilla luciferase co-expressed and a cleavage-based single-plasmid dual-luciferase surrogate reporter was built to evaluate the gRNA-mediated Cas12a cleavage efficiency. The cleavage activities of CRISPR-Cas12a can be quantitatively determined by the recovery degree of firefly luciferase activity. The cleavage efficiency of CRISPR-Cas12a can be quantitatively measured by the recovery of firefly luciferase activity. By using this system, the cleavage efficiency of CRISPR-Cas12a on hepatitis B virus (HBV)/D expression plasmid was evaluated, revealing a negative correlation between gRNA cleavage efficiency and HBV gene expression measured using an enzyme-linked immunosorbent assay. This simple, efficient, and quantifiable system only requires the dual-luciferase vector and CRISPR-Cas12a vector, making it a valuable tool for selecting effective gRNAs for gene editing. |
