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

基本信息

批准号：
10622585
负责人：
Le Cong
金额：
$ 41.79万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
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/10622585
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 Code 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 Guidelines Human Human Genome Invaded 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 biological adaptation to stress cancer cell cell type cellular engineering collaborative environment deep sequencing delivery vehicle 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

项目摘要

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.

项目摘要用于大规模基因组工程的基于重组工程的无切割基因编辑工具包和功能筛选以 CRISPR-Cas9 系统为例，基因编辑技术是一个强大的集合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方面来源：1) Cas9 切割会导致靶点/脱靶位点发生无法控制的 DNA 损伤，从而导致毒性和应激反应。最近的研究证实，切割引起的损伤会导致显着的基因表达变化和 p53 突变细胞的富集，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修复完成编辑。这导致低效率和高可变性 Cas9 介导的跨细胞类型和模型的同源定向修复 (HDR)。克服针对这些限制，我们发现了一种基于重组工程的基因编辑工具，称为 RecE/T- 通过 Designer-Cas9-Initiated Targeting (REDIT) 诱导编辑。 REDIT 使用停用的 Cas9 (dCas9) 并产生最小的 DNA 断裂和接近零的毒性。 REDIT 使用噬菌体用于基因编辑的重组工程蛋白 RecE/T，绕过对内源性的依赖修复机制。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具有使用金标准检测的良好表征的模型。拟议的 RecE/T 类重组工程蛋白因促进链入侵/交换而提供了新的机会当基因组位点通过 dCas9 DNA 解旋暂时可访问时，不会发生切割。我们的目标是开发一个安全、可扩展的工具包，对千碱基基因具有高达 80% 的 HDR 效率编辑和汇总敲入筛选。