Engineered transformer base editor with unconstrained PAM requirements

Canonical base editors (BEs) enable programmable nucleotide conversions without inducing DNA double-strand breaks (DSBs), yet the significant off-target (OT) effects restrict their applications. The transformer BE (tBE), a BE derivative, minimizes DNA and RNA OT mutations, but its dual single-guide RNA (sgRNAs) requirement limits its targeting scope. To address this, we integrated SpRY, a protospacer-adjacent motif (PAM)-relaxed Streptococcus pyogenes Cas9 variant, into tBE. The resulting tBE-SpRY achieved robust C-to-T editing across various cell lines in a PAM-unlimited manner, with minimal OT effects at the tested genomic loci and negligible transcriptome-wide RNA editing. We further demonstrated its versatility ex vivo and in vivo. Microinjection of tBE-SpRY components into zygotes efficiently generated albino C57BL/6J mice, while dual adeno-associated virus (AAV)-mediated delivery in mice resulted in modest C-to-T modifications (up to 2.96%) in ocular tissues. Thus, tBE-SpRY provides a safe and PAM-flexible platform for generating genetic mouse models and exploring potential therapeutic applications.