Fig. 1: Synonymous cytosine base editing of CAG repeats in vitro.
a, An overview of the base editing approach to reduce triplet-repeat expansions. b, Schematic of the CAG-CBE base editing strategy. c, An illustration of cytosine base editing at CAG repeats. The smaller cartoon illustrates the multiple binding opportunities for the Cas9-sgCTG complex at CAG repeats. The magnified snippet shows a singular binding event. d, Optimization of cytosine base editing strategies in HEK293T cells. Data are mean ± s.d. of biological triplicates. e, Optimization of the ‘GS’ linker of EA-evoA-Cas9-NG in HEK293T cells. Data are mean ± s.d. of biological triplicates. f, CAG repeat base editing at HTT alleles in human fibroblasts. Numbers below the bars indicate the number of CAG repeats (CAG size) in HTT alleles. Data are mean ± s.d. of biological replicates (n = 2 for HD cell lines with 20/48 and 17/71 CAGs, n = 3 for HD cell lines with 15/16 and 18/180 CAGs). g, Distribution of HTT CAG allele sizes in CBE-treated (CBE) and untreated HD fibroblasts with 18/180 CAG repeats in Rep1, 5 d (P1) and 30 d (P5) after electroporation, as measured by fragment analysis. h, CAG repeat base editing in HD fibroblasts with 18/180 CAG repeats measured across 30 d and five cell passages. P1–P5 refer to cell passages 1–5. Rep1 and Rep2 refer to two independent biological replicates. Illustrations in a, c and e were created using BioRender.com. CBE, CBE-treated; UGI, uracil DNA glycosylase inhibitor ___domain; UT, untreated cells.
