Chapter 3 —— 33 —— of dystrophic traits at the cellular and organismal levels upon intramuscular or systemic administrations. This study identifies ABE:gRNA complexes compatible with ~30% of DMD-causing genotypes and, notwithstanding its inherent complexity, establishes dual AAV ABE trans-splicing as a DSB-free DMD gene correction option. Figure 1. Adenine base editing. Adenine base editors (ABEs) catalyze A·T-to-G·C substitutions and consist of a fusion product between a disabled or nicking Cas9, or ortholog protein, and an evolved deoxyadenosine deaminase, e.g., Escherichia coli tRNA adenosine deaminase (TadA) derivatives. Upon PAM binding, ABE:gRNA complexes form an R-loop at a gRNA-defined target sequence exposing a region of single-stranded DNA. A nucleotides in this single-stranded protospacer “bubble” become targets for the ABE effector domain that converts A nucleotides to inosine (I) intermediates preferentially within an “activity window.” Subsequently, nicking of the unedited strand induces DNA repair that installs C nucleotides opposite I intermediates with additional DNA repair events (or replication) establishing the final A·T-to-G·C transitions. Chai and colleagues start by testing in HEK293T cells and DMD iPSCs, ABE:gRNA complexes that, depending on their ABE component, i.e., ABE8e [6] or ABEe-NG, recognize, respectively, canonical NGG or NG PAMs. DNA sequencing assays in DMD iPSCs identified ABE:gRNA complexes yielding high-frequency target-base editing at DMD exons 51 and 45 splice acceptor (SA) motifs (71.6% and 79.3%–83.3%, respectively). As a result,
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