We have developed a novel method that enables global subtelomere and haplotype-resolved analysis of telomere lengths at the single-molecule level. An in vitro CRISPR/Cas9 RNA-directed nickase system directs the specific labeling of human (TTAGGG)n DNA tracts in genomes that have also been barcoded using a separate nickase enzyme that recognizes a 7-bp motif genome-wide. High-throughput imaging and analysis of large DNA single molecules from genomes labeled in this fashion using a nanochannel array system permits mapping through subtelomere repeat element (SRE) regions to unique chromosomal DNA while simultaneously measuring the (TTAGGG)n tract length at the end of each large telomere-terminal DNA segment. The methodology also permits subtelomere and haplotype-resolved analyses of SRE organization and variation, providing a window into the population dynamics and potential functions of these complex and structurally variant telomere-adjacent DNA regions. At its current stage of development, the assay can be used to identify and characterize telomere length distributions of 30–35 discrete telomeres simultaneously and accurately. The assay's utility is demonstrated using early versus late passage and senescent human diploid fibroblasts, documenting the anticipated telomere attrition on a global telomere-by-telomere basis as well as identifying subtelomere-specific biases for critically short telomeres. Similarly, we present the first global single-telomere-resolved analyses of two cancer cell lines.

我们已经开发出一种新颖的方法，可以在单分子水平上对端粒长度进行全局亚端粒和单体型解析分析。体外CRISPR / Cas9 RNA定向切口酶系统指导基因组中人（TTAGGG）n DNA片段的特异性标记，该基因组也已使用可识别全基因组7 bp基序的单独切口酶进行了条形码编码。使用纳米通道阵列系统以这种方式标记的基因组中的大型DNA单分子进行高通量成像和分析，可通过端粒亚重复序列（SRE）区域映射到独特的染色体DNA，同时在序列末端测量（TTAGGG）n道长度每个大的端粒末端DNA片段。该方法还可以对SRE的组织和变异进行亚端粒和单倍型解析分析，提供了一个窗口，可以了解这些复杂且结构变异的邻近端粒的DNA区域的种群动态和潜在功能。在目前的发展阶段，该测定法可用于同时准确地鉴定和表征30-35个离散端粒的端粒长度分布。该方法的实用性通过使用早期与晚期传代和衰老的人类二倍体成纤维细胞进行了证明，证明了端粒在全球各个端粒基础上的端粒磨损，并鉴定了关键性短端粒的端粒特异性偏倚。同样，我们提出了两种癌细胞系的第一个全球单端粒解析的分析。该测定法可用于同时准确地鉴定和表征30–35个离散端粒的端粒长度分布。该方法的实用性通过使用早期与晚期传代和衰老的人类二倍体成纤维细胞进行了证明，证明了端粒在全球各个端粒基础上的端粒磨损，并鉴定了关键性短端粒的端粒特异性偏倚。同样，我们提出了两种癌细胞系的第一个全球单端粒解析的分析。该测定法可用于同时准确地鉴定和表征30–35个离散端粒的端粒长度分布。该方法的实用性通过使用早期与晚期传代和衰老的人类二倍体成纤维细胞进行了证明，证明了端粒在全球各个端粒基础上的端粒磨损，并鉴定了关键性短端粒的端粒特异性偏倚。同样，我们提出了两种癌细胞系的第一个全球单端粒解析的分析。在全球端粒逐个端基的基础上，记录预期的端粒磨损，并确定临界短端粒的亚端粒特异性偏倚。同样，我们提出了两种癌细胞系的第一个全球单端粒解析的分析。在全球端粒逐个端基的基础上，记录预期的端粒磨损，并确定临界短端粒的亚端粒特异性偏倚。同样，我们提出了两种癌细胞系的第一个全球单端粒解析的分析。