Automated, high-throughput identification of genetic structural variants for gene editing and undiagnosed genetic diseases screening

摘要 一个简单的方法，全面发现，表征和识别结构变异所产生的正常 代谢过程以及细胞操纵将对基因编辑，肿瘤学和罕见的 疾病研究，以及其他应用。De Novo定向基因组杂交（dGH ™）已被广泛应用。 开发用于有效筛选数千个细胞中存在的简单，复杂和异质性的细胞。 结构变体。在这个项目中，遗传结构变异的自动化，高通量鉴定， 基因编辑和未诊断的遗传疾病筛查，我们提出了K-Band ™ dGH，一种扩展的dGH 法 K-Band dGH是一种原位杂交方法，利用高密度染色单体涂料，具有不同的条带， 谱正常的染色体具有确定的带型、光谱和探针密度。结构变体 通过信号模式的变化来检测和识别。建议的K-Band ™ dGH方法将提供 从头发现涉及相同基因座处的断点的平衡等位基因易位的方法， 倒位，姐妹染色单体重组和交换事件，这是不可见的现有方法 例如测序和aCGH。K带dGH还将表征缺失、重复 易位、非整倍性、多倍性和更复杂的重排。 结构变异会导致从罕见疾病到癌症的各种疾病，并且可以是精确的， 明确的生物标志物。此外，由于变异是由DNA双链断裂的错误修复引起的， 非预期的结构损伤是基因组编辑的不可避免且潜在高风险的副产品。的 基因组编辑方法如CRISPR-Cas9在疾病治疗中的潜力被广泛应用。 认识和实现这种治疗方法的承诺将依赖于准确的确认 存在或不存在潜在风险的结构变异。因此，全面 结构变异的检测和表征是理解、诊断、 并最终精确治疗遗传疾病。从同质或异质的细胞群体， 在一个实验中，K带dGH将识别具有结构正常表型的细胞，检测所有 类的结构变体，并定位在每个简单和复杂的结构变体的断点 cell. K-Band dGH的检测限低于5Kb，是一种理想的基因检测方法 编辑，发现未诊断的罕见疾病的原因，分析基因组结构不稳定性， 变异性，发现和验证以前未知的疾病结构遗传驱动因素。

ABSTRACT A simple method to comprehensively discover, characterize and identify structural variants arising from normal metabolic processes, as well as cell manipulations, would have great utility for gene editing, oncology, and rare disease research, among other applications. De Novo Directional Genomic Hybridization (dGH™) has been developed to efficiently screen thousands of cells for the presence of simple, complex, and heterogenous structural variants. In this project, Automated, High-Throughput Identification of Genetic Structural Variants for Gene Editing and Undiagnosed Genetic Diseases Screening, we propose K-Band™ dGH, an expanded dGH method. K-Band dGH is an in-situ hybridization method that utilizes high-density chromatid paints with bands of distinct spectra. A normal chromosome has a definitive pattern of bands, spectra and probe density. Structural variants are detected and identified via changes to the signal pattern. The proposed K-Band™ dGH method will provide the means for de novo discovery of balanced allelic translocations involving breakpoints at the same loci, inversions, and sister chromatid recombination and exchange events that are invisible to existing methods such as sequencing and aCGH. K-Band dGH will additionally characterize deletions, duplications, translocations, aneuploidy, polyploidy and more complex rearrangements. Structural variations cause a wide range of disorders, from rare diseases to cancers, and can be precise and definitive biomarkers. Also, because variations arise from the mis-repair of DNA double-strand breaks, unintended structural damage is an inevitable and potentially high-risk byproduct of genome editing. The potential of genome editing approaches such as CRISPR-Cas9 in the treatment of diseases is widely recognized and the realization of the promise of such therapeutic approaches will rely on accurate confirmation of the presence and absence of potentially risky structural variants. For these reasons, comprehensive detection and characterization of structural variations is a necessary step toward understanding, diagnosing and ultimately precisely treating genetic diseases. From a homogeneous or heterogenous population of cells, and in a single experiment, K-Band dGH will identify cells with a structurally normal phenotype, detect all classes of structural variants, and locate the breakpoints of all simple and complex structural variants in each cell. With a limit of detection below 5Kb, K-Band dGH is an ideal method for determining the outcomes of gene editing, discovering the causes of undiagnosed rare diseases, profiling genomic structural instability and variability, and discovering and validating previously unknown structural genetic drivers of disease.