In situ functional genomics to understand transcriptional regulation

原位功能基因组学用于理解转录调控 在过去的三年里，新的基因编辑技术彻底改变了我们操纵人类的能力。 基因组用于基础研究和疾病建模。靶向基因敲除和精确基因修复- 以前在人类细胞中费力或不可能完成的任务-现在是常规的。尽管基因组研究取得了这些进展 在外科手术中，基因组如何转化为表型仍然是一个谜，也是最神秘的 非编码区是非编码基因组中的区域。与蛋白质编码基因不同， 框架来破译非编码基因组的功能。该提案的目标是解决 转录调控中的一个基本问题：我们如何识别控制转录的序列和蛋白质？ 任何基因的表达，以一种无偏见的方式？ 像ENCODE和表观基因组学路线图这样的联盟努力已经产生了大量的生化 与非编码功能相关的标志。然而，相关性并不等于因果关系。证明 基因组的某些区域调节基因表达或充当DNA结合蛋白的着陆垫 需要无偏见的诱变和审问在某种程度上，这个问题与规模有关。非编码 基因组是一个广阔的领域：非编码区占人类30亿DNA碱基的98%以上。 基因组我们迫切需要能够放大功能性的高通量分子显微镜， 并记录蛋白质如何在这些位点相互作用。鉴于基因组工程的新进展， 高通量测序，我们处于了解功能，基因调控， 非编码基因组的结构。 在这里，我们将运用我们的专业知识来研究非编码基因组的功能区域， 内生语境使用人类癌症和干细胞系作为模型系统，我们将开发五个广泛的- 通过利用可扩展的DNA合成、基因组 工程、液滴细胞捕获、深度测序和定量蛋白质组学，从而实现：1）更高的 使用Cas9直系同源物的分辨率非编码CRISPR筛选和2）增加的跨度（染色体规模） 使用新的Cas酶Cpf1进行非编码CRISPR筛选; 3）整个基因的多维读数 通过将合并的诱变与单细胞RNA-seq相结合的网络; 4）所有转录的无偏标记 通过一种新的Cas9激活的蛋白质组学在CRISPR筛选中鉴定的功能元件中定位的因子 技术;以及5）联合应用这一系列新技术，以揭示功能元素的动态， 神经分化和癌症耐药性。

In situ functional genomics to understand transcriptional regulation In the last 3 years, new gene editing technologies have revolutionized our ability to manipulate the human genome for basic research and for disease modeling. Targeted gene knock-out and precision gene repair — previously laborious or impossible tasks in human cells — are now routine. Despite these advances in genome surgery, much of how the genome is translated into phenotype remains a mystery and the most mysterious regions are those in the noncoding genome. Unlike with protein-coding genes, there is no Central Dogma-like framework to decipher how the noncoding genome functions. The goal of this proposal is to address a fundamental problem in transcriptional regulation: How can we identify the sequences and proteins that govern the expression of any gene, in an unbiased way? Consortium efforts like ENCODE and the Epigenomics Roadmap have produced large catalogs of biochemical hallmarks that correlate with noncoding function. However, correlation does not equal causation. Proving that certain regions of the genome regulate gene expression or act as landing pads for DNA-binding proteins requires unbiased mutagenesis and interrogation. In part, the problem has to do with size. The noncoding genome is a vast expanse: Noncoding regions constitute >98% of the 3 billion DNA bases in the human genome. We urgently need high-throughput, molecular microscopes capable of zooming in on functional regions and recording how proteins interact at these loci. Given new advances in genome engineering and high-throughput sequencing, we are in a prime position to understand the functional, gene-regulatory architecture of the noncoding genome. Here, we will apply our established expertise to examine functional regions of the noncoding genome in their endogenous context. Using human cancer and stem cell lines as model systems, we will develop five broadly- applicable cross-disciplinary platforms by harnessing recent advances in scalable DNA synthesis, genome engineering, droplet cell capture, deep sequencing and quantitative proteomics and thereby enable: 1) higher resolution noncoding CRISPR screens using Cas9 orthologs and 2) increased span (chromosome-scale) noncoding CRISPR screens using the new Cas enzyme Cpf1; 3) multidimensional readouts of entire gene networks by combining pooled mutagenesis with single-cell RNA-seq; 4) unbiased labeling of all transcription factors stationed at functional elements identified in CRISPR screens via a novel Cas9-enabled proteomic technology; and 5) applying this fleet of new technologies jointly to reveal dynamics of functional elements in neural differentiation and cancer drug resistance.