Gene knock-in in mammalian cells usually uses homology-directed repair(HDR)mechanism to integrate exogenous DNA template into the target genome site.However,HDR efficiency is often low,and the co-localization of exogenous DNA template and target genome site is one of the key limiting factors.To improve the efficiency of HDR mediated by CRISPR/Cas9 system,our team and previous studies fused different adaptor proteins with SpCas9 protein and expressed them.By using their characteristics of binding to specific DNA sequences,many different CRISPR/SpCas9 donor adapter gene editing systems were constructed.In this study,we used them to knock-in eGFP gene at the 3′-end of the terminal exon of GAPDH and ACTB genes in HEK293T cells to facilitate a comparison and optimization of these systems.We utilized an optimized donor DNA template design method,validated the knock-in accuracy via PCR and Sanger sequencing,and assessed the efficiency using flow cytometry.The results showed that the fusion of yGal4BD,hGal4BD,hLacI,hTHAP11 as well as N57 and other adaptor proteins with the C-terminus of SpCas9 protein had no significant effect on its activity.At the GAPDH site,the donor adapter systems of SpCas9 fused with yGal4BD,hGal4BD,hLacI and hTHAP11 significantly improved the knock-in efficiency.At the ACTB site,SpCas9 fused with yGal4BD and hGal4BD significantly improved the knock-in efficiency.Furthermore,increasing the number of BS in the donor DNA template was beneficial to enhance the knock-in efficiency mediated by SpCas9-hTHAP11 system.In conclusion,this study compares and optimizes multiple CRISPR/Cas9 donor adapter gene editing systems,providing valuable insights for future gene editing applications.
维普
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