Fig. 1: ABE8e introduces A > G conversion at targeted nucleotide with up to 95% efficiency and negligible indels.
From: Multiplex base editing to protect from CD33 directed drugs for immune and gene therapy
a Top Possible splicing outcomes at CD33 exon 1–3, ag: Splicing acceptor site (SA). gt: Splicing donor site. (Ab icon was created in BioRender. Du, X. (2025) https://BioRender.com/y35d204). Bottom Intron 1 (lower case)/exon2 (upper case) junction DNA sequence with highlight of exon 2 SA (red) and Exon Splicing Enhancer site (ESE in yellow). b CD33Δ2 lacks exon 2 due to the polymorphism rs12459419 that results in an altered ESE site. c Sequences of the protospacers designed to either edit the ESE with BE4max (sgCB1 and CB2) or the SA with ABE8e (sgABE). Protospacer adjacent motif (PAM) is in red. d Sanger sequencing profiles of edited CD34+ cells compared to the wild-type sequence (top) 7 days post electroporation. Edited nucleotides are indicated by arrows. e Editing efficiency (HTS analysis) at the targeted nucleotides and bystanders, as well as indels. f FACS analysis of the edited CD34+ cells 7 days post electroporation with antibody clone P67.6 which recognizes an epitope located in exon 2. g PCR on cDNA with sets of primers, specific to CD33Δ2 (spanning exon junction 1–3), or common to all isoforms (in exons 1, 5 and 7), (L: Ladder in bp). Sanger sequencing of PCR products confirm the absence of exon 2 in edited cells while all other exons are intact. 1 independent experiment. h ML-1 cells were mock electroporated (ML-1mock), or electroporated with ABE8e and CD33 monoclonal antibody (mAb) staining compared to parental ML-1 cells or ML-1 cells in which both alleles of CD33 had been disrupted via CRISPR targeting of exon 1 (ML-1CD33KO). P67 mAb (i), 9G2 (ii) binds to the C2-set domain of CD33, whether V-set is present or not, and 11D5 (iii and iv) binds to the C2-set domain of CD33 when the V-set is absent, e.g., CD33 lacking exon 2. Specificity of 11D5 to CD33Δ2 is shown (iv.) using mouse 3T3 cells that lack human CD33 expression are shown (m3T3) with forced expression of either CD33Δ2 (m3T3+CD33Δ2) or full length CD33 (m3T3+CD33FL). Source data are provided as a Source Data file.
