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.

项目摘要大规模基因组工程的基于重组的无裂解基因编辑工具包和功能筛选基因编辑技术由CRISPR-CAS9系统举例说明通过理解和控制来探测人类疾病的隐藏机制的工具人类基因组变体的功能。现有CRISPR工具的局限性源于两个资料来源：1）CAS9切割会导致无法控制的DNA损伤，靶向位置/脱离靶向地点，导致毒性和压力反应。最近的研究证实，加油引起的损害导致 p53突变细胞的显着基因表达变化和富集，因此混淆一些CRISPR屏幕； 2）CRISPR酶不修复目标DNA，因此依靠内源性DNA修复以完成编辑。这会导致低效率和高可变性 CAS9介导的同源指导修复（HDR）跨细胞类型和模型。克服这些局限性，我们已经确定了一种基于重组的基因编辑工具，称为rece/t- 通过Designer-Cas9引发的靶向（重新）诱导编辑。重新使用停用的cas9 （DCAS9）并产生最小的DNA断裂和接近零的毒性。 REDIT使用噬菌体重组蛋白接受/T用于基因编辑，绕过对内源性的依赖性维修机制。我们的概念验证示范表明，重新提高了有效的效率 KB规模的编辑而无需DNA切割。我们将专注于技术开发和验证使用黄金标准测定法的特征型模型。拟议的接收/T状重新组合蛋白会在促进链入侵/交换时提供新的机会当基因组位点通过DCAS9 DNA毫无风向瞬时访问时，没有切割。我们的目标是开发一个安全，可扩展的工具包，其kilobase基因的HDR效率高达80％。编辑和汇总敲门筛选。