How does base-editing work?

When the base-editing complex identifies the target DNA sequence complementary to the guide RNA spacer, Cas9 facilitates the separation of the double-stranded DNA to form an R-loop structure. This displays a short segment of single-stranded DNA in the non-complementary strand, permitting the deaminase enzyme to chemically modify bases within the region where modifications occur (base editing window). This window, typically 5-10 base pairs long, is located some distance from the protospacer adjacent motif (PAM) within the target site.

Second-generation CBEs incorporate a uracil glycosylase inhibitor (UGI) fused with Cas9 and the cytosine deaminase. UGI prevents the removal of uracil by uracil DNA N-glycosylase (UNG), leading to significantly higher purity and editing efficiency. Furthermore, the cell's DNA mismatch repair mechanisms aim to correct incorrect nucleotide pairings.The nCas9 enzyme cleaves the unedited DNA strand, guiding the mismatch repair process to use the edited strand as a template for correction.

In ABEs, the deamination of adenine results in the generation of an inosine residue, which is recognized by DNA polymerases as a guanosine. Consequently, an A-to-G edit is achieved. Unlike CBEs, ABEs do not require a uracil glycosylase inhibitor (UGI) or a similar inhibitor, as inosine residues are not removed like uracil residues are.