Extended Data Fig. 5: Dual-sgRNA editing of UMPS to increase full gene knockout in T cells and analysis strategy for in vivo samples. | Nature Biotechnology

Extended Data Fig. 5: Dual-sgRNA editing of UMPS to increase full gene knockout in T cells and analysis strategy for in vivo samples.

From: Metabolic engineering generates a transgene-free safety switch for cell therapy

Extended Data Fig. 5

a, Illustration of the genomic region with UMPS exon 1. The sgRNA binding sites in exon 1 are indicated, with dashed lines illustrating the cleavage sites. The cut sites of the two sgRNAs UMPS-1 and UMPS-7 are 89 bp apart in order to create a frameshift deletion, as UMPS is an enzyme consisting of 2 separate enzymatic functions and InDels in exon 1 that keep the open reading frame intact will not disrupt the function of ODC, which is encoded by the downstream part of the gene. b, InDel frequencies in human T cells electroporated with RNPs using sgRNA UMPS-7 (blue) or both UMPS-7 and UMPS-1 (red). The graph and error bars represent means and standard deviations. Statistical significance by comparison using two-sided t tests is annotated. Data from n = 4 (UMPS-7) and n = 3 (UMPS-7 + UMPS-1) biological replicates. c, InDel spectrum of T cells edited with one or two RNPs. The relatively low frequency of frameshift-mutations using only 1 sgRNA is due to the high frequency of 6 bp deletions. The increase in frameshift InDels is explained primarily by the high frequency of deletions of the 89-bp fragment between the two cut sites. Shown is 1 representative sample per condition. d, Gating strategy used to detect and quantify human T cells in mouse peripheral blood. Plot 1 shows gating of beads to be used as counting reference. Plot 2 shows all events excluding beads and gates on cells without debris. Plot 3 excludes dead cells. Plot 4 excludes events not expressing CD45. Plots 5 and 6 show the same population (CD45+ viable cells) with different axes combinations. huCD45, human CD45. mCD45, murine CD45. huTCR, human T cell receptor.

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