Editing repeats in Huntington's:fewer somatic repeat expansions in patient cells

Summary

Synonymous cytosine base editing of CAG repeats in vitroIn patients suffering from poly-Q disorders including HD and SCAs, naturally occurring synonymous CAA interruptions in pathogenic CAG repeats are associated with a delayed or lack of disease onset13,22,36,42,50. These interruptions are proposed to stabilize repeats and suppress somatic repeat expansion17,18,22. In HD knock-in mice, long stretches of alternating CAG and CAA codons do not undergo somatic expansion, unlike pure CAG repeats of similar length22. We hypothesized that introducing CAA interruptions throughout CAG repeat tracts by cytosine base editing might reduce the expansion of long pathogenic TNR alleles (Fig. 1a).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....