A-T to G-C base editing efficiency at targeted gene sites in HEK293T cell line
A-T to G-C base editing efficiency at targeted gene sites in HEK293T cells using the dCas12f-ABE design or the Cas12f-ABE design. Found that the total A-T to G-C conversion efficiency of Circular gRNAs exhibited about two-fold increase compared with U6 gRNAs. We further analyzed the pattern for A-T to G-C conversion on the target site, and observed that the most efficient base editing occurred in a narrow window A3 (3bp downstream of the PAM) similar to U6 gRNAs. In summary, Circular gRNAs with dCas12f-ABE design could enhance A-T to G-C base editing efficiency in a narrow window. Overall design: To test whether Cas12f/cgRNA system could improve the efficiency of cas12f mediated base editing, we transiently transfected different plasmids encoding gRNAs and dCas12f-ABE or the Cas12f-ABE. We constructed the library with two rounds of PCR using indexed and universal primers to amplify on-target sites. The library was performed Deep-sequencing, and then our sequenced reads were trimmed and aligned to the reference.