Engineering Adenine Base Editors
Alexandra Faragher, Biochemistry Discipline, Eckerd College
Emily King, Harvard University, Center for Genomic Medicine, Massachusetts General Hospital
Ben Kleinstiver, Harvard University, Center for Genomic Medicine, Massachusetts General Hospital
Adenine base editors (ABEs) are gene editing tools capable of making a single A to G base change without causing double-stranded DNA breaks. ABEs have significant clinical potential because they could be used to reverse almost half of the pathogenic point mutations that cause diseases. However, a challenge associated with base editors is their propensity to act on bases other than the target nucleotide if they fall within its editing window, known as bystander editing. Editing the wrong nucleotide could alter a patient’s protein sequence, posing therapeutic implementation problems. In an effort to reduce inaccurate editing, amino acid side chain interactions between the Cas9 portion of ABE8e and the DNA strand were removed via alanine substitutions. It was hypothesized that this would reduce the ability of the base editor to mutate bystander bases because the R-loop of DNA would be destabilized, therefore decreasing the amount of time the ABE would have to act on the non-target bases. Of the twelve different alanine mutations tested, none increased the precision of ABE8e. One mutation (M1021A) widened the edit window, which may provide insight into relevant residues for further ABE engineering.
For more information: alfaragh@eckerd.edu
