Schematic description of the single-molecule telomere length assay. In both telomere labeling schemes, the Cas9 D10A (Cas9n) directed by the telomere gRNA nicks the telomere repeats (TTAGGG)n and Nt.BspQI globally nicks the GCTCTTC motif. In the two-color scheme, all nicks are labeled with green fluorescently labeled nucleotides. In the three-color scheme, the Nt.BspQI-generated nicks are labeled with green fluorescently labeled nucleotides, and the telomeres are labeled with red fluorescently labeled nucleotides. All samples are linearized in the nanochannels and optically imaged. De novo assembly is performed, and the telomere label intensities are used for calculating the telomere length.
