RESUMO
Functional genetic screening as an important method for exploring biological processes, diseases development research and functional annotation of genetic elements, has been widely used in pharmaceutical research, new therapeutic targets identifying and screening, and tumor resistance. CRISPR-Cas9 (Clustered regularly interspaced short palindromic repeat sequences/CRISPR-associated protein 9) is the newest tool in the geneticist's toolbox, allowing researchers to edit genome with unprecedented ease, accuracy and high-throughput. CRISPR-Cas9 system provides a high-throughput, practical and efficient tool for the discovery of functionally important genes responsible for certain phenotypes. In this review, we summarize the characterization of CRISPR/Cas9 system and applications of this new genetic toolkit in functional genetic screening.
RESUMO
Objective:To prepare and screen monoclonal antibodies against Herpes simplex virus-1(HSV-1),and develop a double antibody sandwich quantitative enzyme-linked immunosorbent assay( Q-ELISA) for detection of HSV-1 particle. This method was used to control the quality of viral particle in the developing and manufacturing process of HSV-1. Methods: BALB/c mice was immunized with HSV-1 to prepare monoclonal antibodies. A double antibody sandwich Q-ELISA was developed to determine concentration of HSV-1 particle,which was based on the neutralizing monoclonal antibody 1F6 as capture antibody,and 2B1 as HRP-conjugated antibody. The performance of the reagent was evaluated,including specificity,sensitivity,precision,accuracy and linear. And the relation between the amount of virus detected by this method and the virus titer was analyzed by regression analysis method. Results: The Q-ELISA for HSV-1 particle was developed. The quantitation scope was 0. 125-2 μg/ml, the coefficient correlation was 0. 995 5, the limit of detection was 0. 125 μg/ml, the recovery was between 85. 6% and 107. 1%, the variation coefficient was lower than 10%, and the reagent does not react with other samples except HSV-1 antigen. This method has a good correlation with virus titer. Conclusion:The Q-ELISA for HSV-1 particle was successfully developed,which provide a new approach for rapid and quantitative detection of HSV-1 antigen.