Development of potent and predictable Cas9 gene activation tools through high-throughput screening

项目摘要 操纵细胞和生物体中基因表达的能力是基因工程的基础。 遗传学的研究。CRISPR-Cas9基因组编辑工具包彻底改变了我们 精确地修改基因组和表观基因组。虽然CRISPR工具已经过优化， 对于基因表达的鲁棒的、可调节的和可预测的抑制和失活， 诱导基因表达（CRISPR活化，或CRISPRa）的方法不太稳健和可重复。 通过结合创新的高通量筛选和尖端的计算 通过建模，我们将开发一组简单，稳健，可调和可预测的基于CRISPR的 增加任何小鼠或人类基因表达的工具。 我们开发了一个高通量测序分析系统，自我维持肽 Activator Reporter-seq（SPARq），可对数千名候选人进行定量筛选 基因活化肽及其组合以监测和优化它们的基因活化 实力在目标1中，我们将迭代地使用SPARq来系统地评估和优化 基因激活肽的多种特征，包括激活肽的同一性，组合， 连接子和CRISPRa方法。我们将在不同的细胞类型中进行SPARq筛选， 不同的启动子架构，以确定一致工作的工具，设计一套CRISPRa 这些工具比目前最先进的工具更有效、更一致。 在目标2中，我们将通过以下方式提高CRISPRa的可预测性和实用性： 一种新的高通量报告分析和计算工作，以模拟相关的功能， 具有CRISPRa效力。我们设计了一种方法，CRISPR结果和表型 筛选，其将灵敏的报告基因测定与天然基因组表型分析相结合， CRISPRa工具可以在数千个靶位点上进行活性分析。使用数据 通过这个管道收集，我们将开发一个算法，作为输入之一， 在Aim 1中开发的CRISPRa工具，细胞类型，基因和CRISPR指导RNA，并输出一个 准确估计CRISPRa治疗后该基因的表达。我们将验证 该算法的精确性使得能够在广泛的动态范围内进行可调的基因激活 在人类HepG 2和K562细胞中，将其作为网络工具提供给遗传学界。 总而言之，本提案中所述的努力将开创下一代工具包， 更强大的功能获得性基因操作和筛选。

Project Summary The ability to manipulate the expression of genes in cells and organisms is foundational to the study of genetics. The CRISPR-Cas9 genome editing toolkit has revolutionized our ability to modify the genome and epigenome precisely. While CRISPR tools have been optimized to allow for robust, tunable, and predictable repression and inactivation of gene expression, approaches to induce gene expression (CRISPR activation, or CRISPRa) are less robust and reproducible. Through a combination of innovative high-throughput screens and cutting-edge computational modeling, we will develop a cohort of simple, robust, tunable, and predictable CRISPR-based tools to increase the expression of any mouse or human gene. We have developed a high-throughput sequencing-based assay system, Self-sustaining Peptide Activator Reporter-seq (SPARq), which enables quantitative screening of thousands of candidate gene activating peptides and combinations thereof to monitor and optimize their gene activation strength. In Aim 1, we will iteratively employ SPARq to systematically evaluate and optimize multiple features of gene activating peptides, including activation peptide identity, combination, linker, and CRISPRa method. We will perform SPARq screens in distinct cell types and with distinct promoter architectures to identify tools that work consistently, designing a set of CRISPRa tools that are significantly more potent and consistent than the current state-of-the-art. In Aim 2, we will improve the predictability and consequently the utility of CRISPRa through a novel high-throughput reporter assay and a computational effort to model the features associated with CRISPRa potency. We have designed an approach, CRISPR Outcome and Phenotype screening, that combines a sensitive reporter assay with a native genomic phenotypic measurement to profile the activity of a CRISPRa tool at thousands of target sites. Using data collected through this pipeline, we will develop an algorithm that takes as input one of the CRISPRa tools developed in Aim 1, a cell type, gene, and CRISPR guide RNA and outputs an accurate estimate of the expression of that gene following CRISPRa treatment. We will validate the accuracy of this algorithm to enable tunable gene activation over an extensive dynamic range in human HepG2 and K562 cells, providing it to the genetics community as a webtool. Altogether, the efforts described in this proposal will pioneer a next generation toolkit to enable more robust gain-of-function genetic manipulation and screening.