Using nanobodies to increase the sensitivity and resolution of chromatin profiling through uliCUT&RUN

项目摘要/摘要 DNA结合蛋白在所有DNA模板化过程中起着至关重要的作用，如转录、剪接、 复制和DNA修复。DNA结合蛋白包括优先结合某些特定基因的转录因子 DNA序列和构成核小体核心的组蛋白。重要的是，基因组定位 仅靠DNA序列不能预测细胞类型中的因子或组蛋白。因此，蛋白质图谱 技术被用来识别功能结合的细胞特定特征。 DNA结合蛋白的重要性推动了实验和 更好地识别和描述这些相互作用的分析方法。基因芯片全基因组图谱分析 这是一种广泛使用的技术，已经帮助确定了无数染色质结合蛋白的特征。 然而，该技术在表征具有小单元的样本中的因子占有率的能力方面受到限制 数字和可获得的特定和强大的抗体。这些限制使得有必要 开发互补的方法和扩展芯片序列以提供更完整的生物学 细胞内的进程。最近，我们对一种新的定位方法Cut&Run进行了优化，以获得轮廓系数 在极低的细胞群中的占有率，低至单细胞和单个小鼠囊胚 (命名为uliCUT&Run)。这一技术进步为以下方面提供了分析因素占用情况的机会 罕见的细胞群，例如病人的活组织检查。此外，它还允许测试出现的细胞异质性 在细胞群体中。然而，这项技术的实际限制仍然包括抗体的开发和 效率。 骆驼单链VHH抗体或纳米抗体(NBS)是一类引人注目的新抗体 具有极高的溶解度和热稳定性的特点。我们最近开发了一条强大的管道 用于发现和鉴定高质量的抗原特异性NB曲目。这条管道已经 对十几种不同结构和免疫反应的抗原进行了广泛的测试和优化。使用 通过这种方法，可以识别大量高质量的构象Nb结合体。 在这里，我们建议将我们在Nb开发方面的专业知识与ulcut&Run结合起来开发纳米体 针对低细胞群体的特定切割和运行，并将这项技术应用于单细胞和稀有细胞群体。 基于NB的LULCUT&RUN的开发和应用将在社会上得到广泛的应用，我们正在 鉴于我们对Cut&Run的优化是第一次单细胞转录，我们已经做好了开发这项技术的准备 因素分析已经完成，我们在NB开发新领域的专业知识。此外，结果 将这项技术应用于样本将继续加深我们对正常细胞生物学的理解，但 还提供有助于确定异常原因和后果的关键信息 与疾病相关的细胞状态。

PROJECT SUMMARY/ABSTRACT DNA-binding proteins play crucial roles in all DNA templated processes, such as transcription, splicing, replication, and DNA repair. DNA binding proteins include transcription factors that bind preferentially to certain DNA sequences, and histone proteins that form the core of nucleosomes. Importantly, genomic location of factors or histone proteins cannot be predicted in cell types by DNA sequence alone. Therefore, protein profiling technologies are used to identify cell specific characteristics of functional binding. The importance of DNA-binding proteins has motivated the continued development of experimental and analytical methods to better identify and characterize these interactions. Genome-wide profiling by ChIP-seq is a widely-used technique that has assisted in the characterization of countless chromatin binding proteins. However, this technique is limited in its ability to characterize factor occupancy in samples with small cell numbers and by the availability of specific and robust antibodies. These limitations have necessitated the development of complementary methods and extensions of ChIP-seq to provide a more complete of biological processes in the cell. Very recently, we optimized CUT&RUN, a new localization method, to profile factor occupancy in extremely low cell populations, down to single cells and individual mouse blastocyst embryos (termed uliCUT&RUN). This technical advancement has opened the opportunity to profile factor occupancy in rare cell populations, such as patient biopsies. Furthermore, it permits for testing cell heterogeneity that occurs in cell populations. However, practical limitations of this technology still include antibody development and efficiency. Camelid single-chain VHH antibodies or Nanobodies (Nbs) are a compelling new class of antibodies characterized by exceptionally high solubility and thermostability. We have recently developed a robust pipeline for the discovery and characterization of high-quality antigen-specific Nb repertoires. This pipeline has been extensively tested and optimized for a dozen of antigens with different structures and immune responses. With this approach, a large cohort of high-quality conformational Nb binders can be identified. Here we propose to couple our expertise on Nb development and uliCUT&RUN to develop nanobody specific CUT&RUN for low cell populations and apply this technology to single cells and rare cell populations. The development and application of Nb-based uliCUT&RUN will be of wide use to the community and we are well poised to develop this technology given our optimization of CUT&RUN is the first time single cell transcription factor profiling has been accomplished and our expertise in the new field of Nb development. Further, results from applying this technology to samples will continue to further our understanding of normal cell biology, but also provide crucial information that will benefit efforts to determine the causes and consequences of abnormal cellular states that are associated with disease.