Nicole Gaudelli, PhD
Director, Head of Gene Editing Platform Technologies
Beam Therapeutics
Directed evolution and engineering of CBE-T: next-generation cytosine base editors utilizing TadA
Cytosine base editors (CBEs) are gene editing tools that create researcher-defined, programmable C to T transition mutations in the human genome without causing double-stranded DNA (dsDNA) breaks. CBEs have therapeutic potential for applications requiring gene correction, gene modification, and gene silencing. To date, CBEs typically consist of a naturally occurring cytidine deaminase from the APOBEC family of enzymes, which are known to induce unguided cytosine deamination in genomic DNA and this enzyme class may suffer from sequence bias. For therapeutic use, genome-wide off-target outcomes may not be tolerated, and intrinsic sequence preference can limit an editor’s utility for applications requiring editing efficiencies at multiple targets within a cell (e.g. T-cell engineering for allogenic CAR-T therapies). Mitigation strategies toward lowering guide-independent off-targets have been reported with APOBEC-based CBEs, but these editors typically result in lower on-target editing tradeoffs. Here, we report a new class of cytosine base editors, CBE-Ts, that utilize an engineered and evolved TadA enzyme that is capable of catalyzing C to U deamination at levels comparable to APOBEC-based CBEs (e.g. BE4) with high product purity, and with low off targets. This new class of TadA-based CBEs was developed via iterative rounds of directed evolution and x-ray structure-guided mutagenesis of select TadA variants. En route to accessing our CBE-Ts, we also developed a new class of cytosine and adenine base editors that utilize a TadA variant (CABE-T), capable of catalyzing both A-to-I and C-to-U editing. Together with ABEs, CBE-T and CABE-Ts enable the programmable installation of all transition mutations in genomic DNA via synthetic TadAs.
