Kanno Seiya, Sato Kota, Nakazawa Toru
Department of Ophthalmology, Tohoku University Graduate School of Medicine, Sendai, Japan.
Department of Advanced Ophthalmic Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan.
Bio Protoc. 2025 Feb 20;15(4):e5191. doi: 10.21769/BioProtoc.5191.
Human induced pluripotent stem (iPS) cell lines harboring mutations in disease-related genes serve as invaluable in vitro models for unraveling disease mechanisms and accelerating drug discovery efforts. Introducing mutations into iPS cells using traditional gene editing approaches based on the CRISPR-Cas9 endonuclease often encounters challenges such as unintended insertions/deletions (indels) and off-target effects. To address these limitations, we present a streamlined protocol for introducing highly accurate gene mutations into human iPS cells using prime editing, a "search-and-replace" genome-editing technology that combines unwanted indel-minimized CRISPR-Cas9 nickase with reverse transcriptase. This protocol encompasses the design of prime editing guide RNAs (pegRNAs) required for binding and replacement at target loci, construction of prime editor and pegRNA expression vectors, gene transfer into iPS cells, and cell line selection. This protocol allows for the efficient establishment of disease-associated gene variants within 6-8 weeks while preserving critical genomic context. Key features • Dramatic improvement in efficiency of In-Fusion cloning using inserts assembled from the three pegRNA components (spacer, spCas9 scaffold, and 3' extension) via overlap extension PCR. • Cost-effective and time-saving selection of pegRNAs for prime editing via bulk Sanger sequencing. • Straightforward gene transfection using polymer-based reagents, which requires no specialized equipment or techniques and offers high reproducibility and broad applicability across different cell lines. • Precise genome editing based on pegRNA/prime editing minimizes off-target effects, enabling a wide range of applications in the study of disease-associated genetic variants. Graphical overview Key steps of generation of human induced pluripotent stem (iPS) cell lines harboring disease-relevant single nucleotide variants (SNVs) using a prime editing system.
携带疾病相关基因突变的人类诱导多能干细胞（iPS）系，是用于阐明疾病机制和加速药物研发的宝贵体外模型。使用基于CRISPR-Cas9核酸内切酶的传统基因编辑方法在iPS细胞中引入突变，常常会遇到诸如意外插入/缺失（indel）和脱靶效应等挑战。为解决这些局限性，我们提出了一种简化方案，利用碱基编辑技术在人类iPS细胞中引入高度精确的基因突变。碱基编辑是一种“搜索并替换”的基因组编辑技术，它将脱靶效应最小化的CRISPR-Cas9切口酶与逆转录酶相结合。该方案包括在目标位点进行结合和替换所需的碱基编辑引导RNA（pegRNA）的设计、碱基编辑器和pegRNA表达载体的构建、基因导入iPS细胞以及细胞系选择。该方案能够在6-8周内高效建立疾病相关基因变体，同时保留关键的基因组背景。关键特性：• 通过重叠延伸PCR，使用由pegRNA的三个组件（间隔区、spCas9支架和3'延伸区）组装而成的插入片段，显著提高In-Fusion克隆效率。• 通过批量Sanger测序，经济高效地选择用于碱基编辑的pegRNA。• 使用基于聚合物的试剂进行直接基因转染，无需专门设备或技术，具有高重现性且广泛适用于不同细胞系。• 基于pegRNA/碱基编辑的精确基因组编辑可将脱靶效应降至最低，在疾病相关遗传变体研究中具有广泛应用。图形概述：使用碱基编辑系统生成携带疾病相关单核苷酸变体（SNV）的人类诱导多能干细胞（iPS）系的关键步骤。
