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

项目摘要 一种用于大规模基因组工程的基于测序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， 通过理解和控制人类疾病的隐藏机制来探索人类疾病的工具 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切割在靶上/脱靶位点引起不可控制的DNA损伤，导致 毒性和应激反应。最近的研究证实，切割引起的损害导致 显著的基因表达变化和p53突变细胞的富集，因此混淆 一些CRISPR筛选; 2）CRISPR酶不修复靶DNA，因此依赖于 内源DNA修复以完成编辑。这导致了低效率和高可变性， 跨细胞类型和模型的Cas9介导的同源定向修复（HDR）。克服 这些限制，我们已经确定了一种基于重组的基因编辑工具，称为RecE/T， 通过设计者Cas9启动的靶向（REDIT）诱导编辑。REDIT使用失活的Cas9 它是一种新型的DNA裂解酶（dCas 9），产生最小的DNA断裂和接近零的毒性。REDIT使用噬菌体 重组工程蛋白RecE/T用于基因编辑，绕过对内源性 修复机制我们的概念验证演示表明，REDIT实现了高效的 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 使用金标准分析的良好表征模型。建议的RecE/T-like 重组工程蛋白提供了新的机会，因为它们促进链侵入/交换 当基因组位点通过dCas 9 DNA解旋变得瞬时可接近时，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HDR效率，用于基因工程。 编辑和合并的敲入筛选。

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.