Recombineering-based no-cleavage gene-editing toolkit for large-scale genome engineering and functional screening

基于重组工程的无切割基因编辑工具包，用于大规模基因组工程和功能筛选

• 批准号: 10184864
• 负责人: Le Cong
• 金额: $ 39.14万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-10 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10184864
• 关键词: Area; Auxins; Bacteriophages; Benchmarking; Biological Assay; Biological Models; Biological Process; Bypass; CRISPR screen; CRISPR/Cas technology; Cell model; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Codon Nucleotides; Collection; Custom; DNA; DNA Damage; DNA Repair; DNA Repair Pathway; DNA-PKcs; Data; Dependence; Development; Engineering; Enzymes; Eukaryotic Cell; Event; Exhibits; Gene Expression; Gene Expression Regulation; Generations; Genes; Genetic Engineering; Genetic Screening; Genome; Genome engineering; Genomics; Goals; Gold; Guidelines; Human; Human Genome; Knock-in; Lead; Length; Libraries; Malignant Neoplasms; Mammalian Cell; Measures; Mediating; Metagenomics; Methods; Mining; Mission; Modality; Modeling; Modification; Mus; Mutation; National Institute of General Medical Sciences; Positioning Attribute; Procedures; Protein Engineering; Proteins; Proteome; Protocols documentation; Reagent; Reporter; Research; Ribonucleoproteins; Screening procedure; Single Nucleotide Polymorphism; Site; Source; System; TP53 gene; Technology; Testing; Tissues; Toxic effect; Transgenes; Untranslated RNA; Variant; Viral Vector; Work; arm; base; biological adaptation to stress; cancer cell; cell type; cellular engineering; collaborative environment; deep sequencing; design; experimental study; forward genetics; genome-wide; human disease; improved; insertion/deletion mutation; live cell imaging; mutant; novel; off-target site; plasmid DNA; protein function; repaired; screening; stem; stem cell model; success; technology development; technology validation; tool

Project Abstract Recombineering-based no-cleavage gene-editing toolkit for large-scale genome engineering and functional screening Exemplified by the CRISPR-Cas9 system, gene-editing technology is a powerful collection of tools for probing the hidden mechanisms of human diseases by understanding and controlling the functions of human genome variants. Limitations of existing CRISPR tools stem from two sources: 1) Cas9 cutting causes uncontrollable DNA damage at on/off-target sites, leading to toxicity and stress response. Recent studies confirmed that cutting-induced damages lead to significant gene expression changes and enrichment of p53-mutant cells, thus confounding some CRISPR screens; 2) CRISPR enzymes do not repair the target DNA, thus relying on endogenous DNA repair to complete editing. This results in low efficiency and high variability for Cas9-mediated homology-directed repair (HDR) across cell types and models. To overcome these limitations, we have identified a recombineering-based gene-editing tool, termed RecE/T- induced Editing via Designer-Cas9-Initiated Targeting (REDIT). REDIT uses deactivated Cas9 (dCas9) and generates minimal DNA break and near-zero toxicity. REDIT uses phage recombineering proteins RecE/T for gene-editing, bypassing the dependence on endogenous repair mechanisms. Our proof-of-concept demonstration showed that REDIT achieved efficient kb-scale editing without DNA cutting. We will focus on technology development and validation with well-characterized models using gold-standard assays. The proposed RecE/T-like recombineering proteins present new opportunities as they promote strand invasion/exchange without cleavage when genome sites become transiently accessible via dCas9 DNA-unwinding. Our goal is to develop a safe, scalable toolkit with up to 80% HDR efficiency for kilobase gene- editing and pooled knock-in screening.

项目摘要 基于重组工程的大规模基因组工程无切割基因编辑工具包 和功能筛查 以CRISPR-Cas9系统为例，基因编辑技术是一种强大的 通过理解和控制来探索人类疾病隐藏机制的工具 人类基因组变异的功能。现有CRISPR工具的局限性源于两个 来源：1)Cas9切割会在靶上/靶外部位造成不可控的DNA损伤，导致 毒性和应激反应。最近的研究证实，切割诱导的损害会导致 显著的基因表达变化和p53突变细胞的富集性，从而混淆 一些CRISPR筛选；2)CRISPR酶不修复靶DNA，因此依赖于 内源性DNA修复以完成编辑。这导致了低效率和高可变性 Cas9介导的跨细胞类型和模型的同源定向修复(HDR)。要克服 针对这些局限性，我们已经确定了一种基于重组工程的基因编辑工具，称为Ress/T- 通过Designer-Cas9启动的目标(REDIT)进行诱导编辑。Redit使用停用的Cas9 (DCas9)，并产生最小的DNA断裂和几乎为零的毒性。瑞迪特使用噬菌体 重组工程蛋白Ress/T用于基因编辑，绕过对内源的依赖 修复机制。我们的概念验证演示表明，redit实现了高效 KB级编辑，无需DNA切割。我们将专注于技术开发和验证 使用黄金标准化验的特征很好的模型。建议的Ress/T-like 重组工程蛋白为促进链入侵/交换提供了新的机会 当基因组位置通过dCas9 DNA解开变得瞬时可访问时，没有切割。 我们的目标是开发一个安全、可扩展的工具包，具有高达80%的千碱基基因HDR效率。 编辑和拼接放映。