We developed a generally applicable method, CRISPR/Cas9-targeted long-read sequencing (CTLR-Seq), to resolve, haplotype-specifically, the large and complex regions in the human genome that had been previously impenetrable to sequencing analysis, such as large segmental duplications (SegDups) and their associated genome rearrangements. CTLR-Seq combines in vitro Cas9-mediated cutting of the genome and pulse-field gel electrophoresis to isolate intact large (i.e., up to 2,000 kb) genomic regions that encompass previously unresolvable genomic sequences. These targets are then sequenced (amplification-free) at high on-target coverage using long-read sequencing, allowing for their complete sequence assembly. We applied CTLR-Seq to the SegDup-mediated rearrangements that constitute the boundaries of, and give rise to, the 22q11.2 Deletion Syndrome (22q11DS), the most common human microdeletion disorder. We then performed de novo assembly to resolve, at base-pair resolution, the full sequence rearrangements and exact chromosomal breakpoints of 22q11.2DS (including all common subtypes). Across multiple patients, we found a high degree of variability for both the rearranged SegDup sequences and the exact chromosomal breakpoint locations, which coincide with various transposons within the 22q11.2 SegDups, suggesting that 22q11DS can be driven by transposon-mediated genome recombination. Guided by CTLR-Seq results from two 22q11DS patients, we performed three-dimensional chromosomal folding analysis for the 22q11.2 SegDups from patient-derived neurons and astrocytes and found chromosome interactions anchored within the SegDups to be both cell type-specific and patient-specific. Lastly, we demonstrated that CTLR-Seq enables cell-type specific analysis of DNA methylation patterns within the deletion haplotype of 22q11DS.

中文翻译：

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使用 CTLR-Seq 解析 22q11.2 缺失揭示了染色体重排机制和断点的个体差异


我们开发了一种普遍适用的方法，即 CRISPR/Cas9 靶向长读长测序 (CTLR-Seq)，以特定于单倍型的方式解析人类基因组中以前无法进行测序分析的大型且复杂的区域，例如大型区域。片段重复（SegDups）及其相关的基因组重排。 CTLR-Seq 结合了体外 Cas9 介导的基因组切割和脉冲场凝胶电泳，以分离完整的大（即高达 2,000 kb）基因组区域，这些区域包含以前无法解析的基因组序列。然后使用长读长测序以高目标覆盖率对这些靶标进行测序（无扩增），从而实现完整的序列组装。我们将 CTLR-Seq 应用于 SegDup 介导的重排，这些重排构成了 22q11.2 缺失综合征 (22q11DS)（最常见的人类微缺失疾病）的边界，并引发了 22q11.2 缺失综合征 (22q11DS)。然后，我们进行从头组装，以碱基对分辨率解析 22q11.2DS（包括所有常见亚型）的完整序列重排和精确染色体断点。在多个患者中，我们发现重排的 SegDup 序列和确切的染色体断点位置都存在高度的变异性，这与 22q11.2 SegDups 内的各种转座子一致，表明 22q11DS 可以由转座子介导的基因组重组驱动。在两名 22q11DS 患者的 CTLR-Seq 结果的指导下，我们对来自患者来源的神经元和星形胶质细胞的 22q11.2 SegDups 进行了三维染色体折叠分析，发现 SegDups 中锚定的染色体相互作用既具有细胞类型特异性，又具有患者特异性。具体的。 最后，我们证明 CTLR-Seq 能够对 22q11DS 缺失单倍型内的 DNA 甲基化模式进行细胞类型特异性分析。