Reconstructed glycosylase base editors GBE2.0 with enhanced C-to-G base editing efficiency and purity.

ABSTRACT

Base editing techniques were developed for precise base conversion on cellular genomic DNA, which has great potential for the treatment of human genetic diseases. The glycosylase base editor (GBE) recently developed in our lab was used to perform C-to-G transversions in mammalian cells. To improve the application prospects of GBE, it is necessary to further increase its performance. With this aim, we replaced the human Ung in GBE with Ung1 from Saccharomyces cerevisiae. The resulting editor APOBEC-nCas9-Ung1 was tested at 17 chromosomal loci and was found to have an increased C-to-G editing efficiency ranging from 2.63% to 52.3%, with an average of 23.48%, which was a significant improvement over GBE, with an average efficiency of 15.54%, but with a decreased purity. For further improvement, we constructed APOBEC(R33A)-nCas9-Rad51-Ung1 with two beneficial modifications adapted from previous reports. This base editor was able to achieve even higher editing efficiency ranging from 8.70% to 72.1%, averaging 30.88%, while also exhibiting high C-to-G purity ranging from 35.57% to 92.92%, and was designated GBE2.0. GBE2.0 provides high C-to-G editing efficiency and purity in mammalian cells, making it a powerful genetic tool for scientific research or potential genetic therapies for disease-causing G/C mutations.