American Society of Hematology

Figure 2.

$Longitudinal tracking of mutant clones in engineered CH macaques. (A) Schematic outline of RM HSPC gene editing for CH loci and subsequent autologous transplantation. (B) Summary of the gRNAs used for gene editing in each animal (n = 3; ZL26, ZL39, and ZH63). (C) Mutation frequencies at target sites are quantified from PB granulocytes collected at different time points from the 3 macaques using targeted deep sequencing. The percentage of reads containing indel mutations for each target site by individual macaque is plotted over time. (D) Indel frequencies at the TET2 target site in granulocytes collected over time are combined into a single graph for comparison. (E) Heatmaps on the right show the fractional contribution of each indel to total sequencing reads in granulocyte samples over time. Indels in HSPC measured before editing (Precut) and in infusion product (IP) are also shown in the first 2 columns. The most abundant indel types for the TET2 target site in each animal are listed on the left, with the WT sequence at the bottom. Indels are highlighted in red font, and the predicted Cas9 cut site is indicated by a red arrow. Changes in percent contribution of unique clones over time are shown stacked on the left, with each indel type marked with a distinct color across the 3 RMs. Td, doubling time; PAM, protospacer adjacent motif.$

Longitudinal tracking of mutant clones in engineered CH macaques. (A) Schematic outline of RM HSPC gene editing for CH loci and subsequent autologous transplantation. (B) Summary of the gRNAs used for gene editing in each animal (n = 3; ZL26, ZL39, and ZH63). (C) Mutation frequencies at target sites are quantified from PB granulocytes collected at different time points from the 3 macaques using targeted deep sequencing. The percentage of reads containing indel mutations for each target site by individual macaque is plotted over time. (D) Indel frequencies at the TET2 target site in granulocytes collected over time are combined into a single graph for comparison. (E) Heatmaps on the right show the fractional contribution of each indel to total sequencing reads in granulocyte samples over time. Indels in HSPC measured before editing (Precut) and in infusion product (IP) are also shown in the first 2 columns. The most abundant indel types for the TET2 target site in each animal are listed on the left, with the WT sequence at the bottom. Indels are highlighted in red font, and the predicted Cas9 cut site is indicated by a red arrow. Changes in percent contribution of unique clones over time are shown stacked on the left, with each indel type marked with a distinct color across the 3 RMs. Td, doubling time; PAM, protospacer adjacent motif.

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