Figure 5.

Locus-specific and variant-specific barcodes based on Cas9n labeling. Cas9n fluorescent nick-labeling creates locus-specific and variant-specific barcodes. The expected labeling pattern and distances between labels is shown above corresponding fluorescent microscopy images. (A) For each of the tested subtelomere regions specific gRNAs were designed and pooled in the in vitro nicking reaction mix in order to produce a unique pattern. In each instance, the expected pattern of the red fluorophores in the Cas9n fluorescent nick-labeling system was obtained. (B) Human genomic DNA was labeled with Alu gRNAs, followed by conventional sequence-motif labeling with Nt.BspQI. Conventional sequence-motif dependent labeling sites (green labels) are found infrequently within these genomic DNA regions. However, Alu-specific gRNAs direct Cas9n D10A to generate information-rich long-range barcoding patterns (red labels).