Contributions of repair outcome choice to establishing the recombination landscape during meiosis

 描述（由申请人提供）：生物体的基因组必须准确复制并包装到新细胞中，以忠实地将遗传物质从一代繁殖到下一代。非整倍性，即染色体过多或过少的状态，是发育缺陷（例如唐氏综合征）、流产和使用辅助生殖技术的妊娠失败的主要原因;因此，生殖成功完全取决于准确的染色体分离。在减数分裂期间，通过使用重组在同源染色体之间产生CO（CO）来确保准确的染色体分离。DNA双链断裂（DSB）可通过一系列结构中间体以CO或非交换（NCO）的形式修复。修复途径的分叉被认为发生在早期，可能在DSB形成时或之前，并且修复结果的选择是建立跨基因组的交叉分布的关键调节点。目前的建议旨在了解修复结果是如何决定的，以及它在通过三种不同的方法确定重组景观中的作用。首先，不同类型的重组事件（异源双链DNA）的签名将被映射到整个后生动物基因组中，使用遗传技巧，允许仅对母本单倍体基因组进行测序。其次，描述了基于CRISPR-Cas9的全基因组DSB修复测定，其将允许容易地监测和操纵修复结果。最后，将测试几个因素在决定和传达修复结果中的作用，包括染色体环境（位置，异染色质，染色质标记和染色体压实），DSB之间的距离和重组监视机制。这三种方法中的每一种都需要定量和计算技能，这些技能需要候选人在指导阶段发展，以便作为独立科学家取得成功。建议开展一系列培训活动 包括计算生物学和统计学课程，参加生物信息学和计算生物学课程的研讨会，与共同导师的个人培训，专家指导小组，以及在赞助机构的计算生物学研究小组中的沉浸。

 DESCRIPTION (provided by applicant): An organism's genome must be accurately replicated and packaged into a new cell to faithfully propagate genetic material from one generation to the next. Aneuploidy, the state of having too many or too few chromosomes, is a leading cause of developmental defects (e.g. Down's syndrome), miscarriages, and failed pregnancies using assisted reproductive technology; therefore, reproductive success is completely dependent on accurate chromosome segregation. During meiosis, accurate chromosome segregation is ensured by using recombination to create COs (COs) between homologous chromosomes. Recombination is initiated by a DNA double- stranded break (DSB) that can be repaired either as a CO or a noncrossover (NCO) through a series of structural intermediates. The bifurcation in repair pathways is thought to occur early, likely at or before the time DSBs are formed, and the choice in repair outcome is a critical regulatory point in establishing the distribution of crossovers across the genome. The current proposal seeks to understand how repair outcome is decided and its role in determining the recombination landscape through three different approaches. First, the signatures of different types of recombination events (heteroduplex DNA) will be mapped across an entire metazoan genome using a genetic trick that allows the sequencing of only the maternal haploid genome. Secondly, a CRISPR-Cas9 based whole genome DSB repair assay is described that will allow repair outcome to be easily monitored and manipulated. And lastly, the roles of several factors in deciding and communicating repair outcome will be tested, including chromosome environment (location, heterochromatin, chromatin marks and chromosome compaction), the distance between DSBs, and recombination surveillance mechanisms. Each of these three approaches requires quantitative and computational skill sets that need to be developed by the candidate during the mentored phase in order for success as an independent scientist. A series of training activities is proposed included courses in computational biology and statistics, seminar attendance in the Curriculum for Bioinformatics and Computational Biology, individual training with the co-mentor, an expert mentoring panel, and immersion in the Computational Biology research groups at the sponsoring institution.