With the development of CRISPR/Cas9 technology, a new generation of editing methods that convert specific bases has enabled precise single-base mutations. To date, conversion of cytosine to thymidine and adenine to guanine has been achieved using the cytidine deaminaseand adenosine deaminase (TadA), respectively. However, the base editing efficiency can be unacceptably low in some cell types or at certain target loci. One reason might be the lack of a selective pressure against the survival of nonedited cells. Few studies on ABE in prokaryotes have been reported, probably due to the relatively low editing efficiency of TadA. Improving the editing efficiency is the key for establishing base editing techniques and especially the ABE technologies. In this work, a selective pressure against nonedited cells was implemented to increase the base editing efficiency. First, we fused nCas9 or dCas9 with TadA to compare the editing efficiency of nCas9-TadA and dCas9-TadA fusion complexes in the model prokaryote. While nCas9-TadA was able to achieve A to G base editing (ABE) with a moderate efficiency, dCas9-TadA had a very low efficiency. To enrich for edited cells and increase the base-editing efficiency, we utilized the induction of double-strand breaks by active Cas9, which leads to the death of prokaryotic cells. By introducing an inducible active Cas9 with the same editing gRNA as the nCas9-TadA in the base editing process, the cells with nonedited target bases remained vulnerable to Cas9 and were eliminated. Thus, a double-check base editing (DBE) method was established, which significantly improved the editing efficiency of ABE in, reaching 99.0% for some sites. By placing a selective pressure against nonedited cells, the DBE strategy might also be applied to various scenarios to increase the efficiency of many different base editing targets or even for epigenetic DNA modification techniques. APOBEC1Escherichia coli E. coli
