Fig. 3: Heteroplasmic shifting operates on nonsynonymous but not silent mtDNA variants.
From: Single-cell analysis reveals context-dependent, cell-level selection of mtDNA
293T cells were transfected with LHON or SILENT DdCBEs, and sorted based on the expression of the editors reflected by the fluorescence intensity of the eGFP and mCherry reporters. a, Alleles and their frequency introduced by LHON and SILENT DdCBEs. Missense indicates an on-target missense G11696A edit, and Silent indicates an on-target silent C11698T edit. b, Single-cell heteroplasmy in edited cells interrogated using SCI-LITE or by amplicon sequencing of bulk mtDNA and bulk mtRNA. Dots represent single cells. c,d, Heteroplasmy levels assessed by next-generation sequencing of MT-ND4 amplicon in high heteroplasmic cells treated with the LHON (c) or the SILENT (d) editor cultured in media containing glucose (GLU) or galactose (GAL). e,f, Population doublings and viability in LHON-edited and SILENT-edited low heteroplasmic (e) and high heteroplasmic (f) cells cultured in media containing either glucose or galactose. For c–f, n = 3 independent biological replicates. Error bars reflect the mean ± s.d. ****P ≤ 0.0001, ***P ≤ 0.001 and NS > 0.05, by Student’s unpaired two-tailed t-test. g, Joint distribution of missense and silent heteroplasmy changes over time. The graphs are sorted on the x axis by missense heteroplasmy and show the missense and silent heteroplasmy introduced by the LHON DdCBE in single cells measured by SCI-LITE. Each column represents one cell, with the stacked colours representing the percent missense, silent and wild-type heteroplasmy in red, yellow and blue, respectively. The dashed lines are for reference and indicate the midpoints of the x and y axes. h,i, Single-cell heteroplasmy in LHON-edited (h) and SILENT-edited (i) cells. Cells were grown for 5, 10 and 15 days and were subjected to SCI-LITE. The graphs on the left show the binned relative frequency and the graphs on the right show cumulative distributions of MT-ND4 missense and silent heteroplasmy for n = 3 independent biological replicates. The Kolmogorov–Smirnov test was used to calculate D statistics and P values.
