Double-Check Base Editing for Efficient A to G Conversions.

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• CRISPR/Cas9 technology enables precise single-base mutations through base editing.
• Adenine to guanine (ABE) base editing efficiency can be low in certain contexts, hindering its application.
• Lack of selective pressure against non-edited cells contributes to low base editing efficiency.

Purpose of the Study:

• To improve the efficiency of adenine to guanine (ABE) base editing in prokaryotes.
• To establish a method that increases base editing efficiency by applying selective pressure.
• To develop a novel base editing strategy for enhanced genetic modification.

Main Methods:

• Fusion of nCas9 or dCas9 with TadA to create base editors.
• Comparison of editing efficiency between nCas9-TadA and dCas9-TadA fusion complexes in *Escherichia coli*.
• Implementation of double-check base editing (DBE) using inducible active Cas9 to eliminate non-edited cells.

Main Results:

• nCas9-TadA demonstrated moderate ABE efficiency, while dCas9-TadA showed very low efficiency.
• The double-check base editing (DBE) method significantly enhanced ABE efficiency in *E. coli*.
• DBE achieved up to 99.0% editing efficiency at specific target sites.

Conclusions:

• The developed double-check base editing (DBE) strategy effectively increases ABE efficiency in *E. coli*.
• Applying selective pressure against non-edited cells is a viable approach to boost base editing outcomes.
• The DBE strategy holds potential for various base editing targets and epigenetic DNA modification techniques.