Fig. 4.

Detection of CsCRUC alleles with CRISPR/Cas9-mediated mutations in the T₃, T₄, and T₅ generations using a ddPCR drop-off assay. a Fluorescence intensity plots of drop-off assays representing occurrence of single- and double-positive droplets for both the drop-off probe (FAM) and reference probe (HEX). Plant_gRNA512–1–69-8, plant_gRNA512–1–69-8-7, and plant_gRNA512–1–69-8-7-11 are from the T₃, T₄ and T₅ generation, respectively. b Quantification of drop-off probe events and reference probe events from a. Probe targets are normalized to 100 reference probe events. Values are an average of three replicates ± S.D. Differences between expected reference probe events and observed reference probe events were tested using a chi-square test. c Fractional abundance (%) of mutated alleles from b. Plant_gRNA512–1–69-1-8, plant_gRNA512–1–69-8-7, and plant_gRNA512–1–69-8-7-11 contain 4, 5 and 6 mutated alleles, respectively