Fig. 3: In vitro selection of the optimal ABE system for correcting the MPZL2 c.220 C > T mutation. | Nature Communications

Fig. 3: In vitro selection of the optimal ABE system for correcting the MPZL2 c.220 C > T mutation.

From: PAM-flexible adenine base editing rescues hearing loss in a humanized MPZL2 mouse model harboring an East Asian founder mutation

Fig. 3: In vitro selection of the optimal ABE system for correcting the MPZL2 c.220 C > T mutation.The alternative text for this image may have been generated using AI.

a Schematic representation of a correction strategy using PAM-flexible ABEs in HEK293T monoclonal cells harboring the homozygous C > T nonsense mutation (c.220 C > T: p.Q74X) at the MPZL2 locus. b Design of sgRNAs targeting the MPZL2 c.220 C > T mutation. For more effective editing, the target adenine A needs to be within ABE’s preferred targeting window. In the table below, 6 sgRNA candidates featuring different PAMs are listed, each placing the target A (red) at different positions on the protospacer: A2, A4, A5, A6, and A7. c Comparison of the A-to-G editing efficiencies in vitro at the target adenine and bystander adenines for all ABEs used in this study (n = 3 for all conditions, except for ABE8eWQ:sgRNA3, for which n = 4). d C-to-other editing efficiencies at either AC*N and TC*N motifs within the targetable window of the selected SpRY-based ABEs. The data is from c (Mean values, same as in c, n = 3, except for ABE8eWQ:sgRNA3, for which n = 4). e Potential off-target sites identified by Cas-OFFinder and GUIDE-seq. The venn diagram shows the degree of off-target site overlap between the two methods (f) Heatmap showing A-to-G conversion rates at off-target sites predicted using Cas-OFFinder following ABE (sgRNA3 + ) treatment. g Validation of A-to-G conversion rates at GUIDE-seq-detected off-target sites after ABE (sgRNA3 + ) treatment. Sites that showed differences between control (untreated) and ABE (sg3 + ) samples are marked with an asterisk (*) and labeled by genomic locus, and coding region is shown in purple text. A purple asterisk (*) indicates overlapping site detected by both methods. h Targeted RNA off-target effects in  HEK293T cells mimicking the MPZL2 c.220 C > T variant with three SpRY ABEs that were expressed using NAN PAM sgRNA. The average frequency of A-to-I transitions in three mRNA transcripts (CCNB1IP1, AARS1, and TOPORS) with each of the SpRY-mediated ABE variants. Source data for all relevant panels are provided within the Source Data file.

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