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. 2021 Jun 4;20(7):e13388. doi: 10.1111/acel.13388

FIGURE 6.

FIGURE 6

Correction of HGPS causing mutation by ABE in HGPS iPSC‐ECs. (a) Scheme of ABE‐mediated genome‐editing strategy. ABE targets the pathogenic adenosine (A) nucleobase in the human LMNA (c. 1824C>T) allele. A catalytically impaired Cas9 nickase–deoxyadenosine deaminase complex localizes to the LMNA gene locus by an allele‐specific sgRNA, generating a single‐stranded R‐loop of target genomic sequence. Exposed adenosines within the R‐loop can be deaminated by the fused evolved ssDNA‐specific deoxyadenosine deaminase domain. (b) Sanger sequence traces of normal, HGPS, and ABE‐edited HGPS iPSC‐ECs (top); sequence alignment of part of LMNA exon 11 from normal, HGPS, and ABE‐corrected HGPS ECs by DNAMAN software (bottom). (c) DNA sequence at the LMNA c.1824 nucleotide in normal, HGPS, and ABE7.10max‐VRQR lentivirus‐treated HGPS iPSC‐ECs after 20 days. (d) Western blotting analysis with indicated antibodies on the lysates of normal and HGPS iPSC‐ECs. Data are presented as mean ±SEM, ****p < 0.0001; n, three independent experiments