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

通过高通量筛选开发有效且可预测的 Cas9 基因激活工具

• 批准号: 10670807
• 负责人: Richard I Sherwood
• 金额: $ 41.35万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10670807
• 关键词: Algorithms; Architecture; Biological Assay; Budgets; CRISPR screen; CRISPR-mediated transcriptional activation; CRISPR/Cas technology; Candidate Disease Gene; Cell Line; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Collection; Communities; Computer Models; Data; Development; Disease; Engineering; Gene Activation; Gene Expression; Gene Silencing; Generations; Genes; Genetic; Genetic Screening; Genetic Transcription; Genetic study; Genome; Genomics; Guide RNA; HepG2; High-Throughput Nucleotide Sequencing; Human; Individual; K562 Cells; LDL Cholesterol Lipoproteins; Link; Measurement; Measures; Methods; Modeling; Monitor; Mus; Organism; Outcome; Output; Peptides; Phenotype; Productivity; Reporter; Repression; Reproducibility; Research; Site; Stimulus; System; Testing; Tissues; Transactivation; Work; cell type; cohort; data integration; deep learning; deep learning model; design; epigenome; epigenomics; gain of function; gene function; gene therapy; genetic manipulation; genome editing; high throughput screening; improved; innovation; machine learning prediction; next generation; novel; nuclease; phenotypic data; predictive modeling; promoter; recruit; response; screening; therapeutic gene; tool; uptake; user-friendly; web-based tool

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.

项目摘要 在细胞和生物体中操纵基因表达的能力是 遗传学的研究。CRISPR-Cas9基因组编辑工具包彻底改变了我们的能力 精确修改基因组和表观基因组。虽然CRISPR工具已经过优化，以允许 对于稳健、可调和可预测的基因表达抑制和失活，方法 诱导基因表达(CRISPR激活，或CRISPRa)不那么健壮和可重复性。 通过结合创新的高通量屏幕和尖端的计算 建模，我们将开发一组简单、健壮、可调和可预测的基于CRISPR的 增加任何老鼠或人类基因表达的工具。 我们已经开发了一个高通量的基于测序的分析系统，自持性多肽 Actiator Reporter-seq(SPARQ)，支持对数千名候选人进行定量筛选 用于监测和优化其基因激活的基因活化肽及其组合物 力量。在目标1中，我们将迭代地使用SPARQ来系统地评估和优化 基因活化肽的多种特性，包括活化肽的同一性、结合性、 链接器和CRISPRa方法。我们将在不同的细胞类型和 独特的推动者体系结构，以确定一致工作的工具，设计一组CRISPRa 比当前最先进的工具更强大、更一致的工具。 在目标2中，我们将通过一个 新的高通量报告分析和对相关特征进行建模的计算工作 具有CRISPRA效力。我们设计了一种方法，CRISPR结果和表型 筛选，将敏感的报告分析与天然基因组表型相结合 测量以描述CRISPRa工具在数千个目标站点的活动。使用数据 通过此管道收集，我们将开发一种算法，该算法将 在AIM 1中开发的CRISPRa工具，一种细胞类型、基因和CRISPR指导RNA并输出一个 对CRISPRa治疗后该基因表达的准确估计。我们将验证 该算法的准确性能够在广泛的动态范围内实现可调的基因激活 在人类HepG2和K562细胞中，将其作为网络工具提供给遗传学社区。 总之，本提案中描述的努力将开创下一代工具包，以实现 更强大的功能增益基因操作和筛选。