Characterization of insert fragment.

The clustered regularly interspaced short palindromic repeats (CRISPR) system offers cost-effectiveness, high efficiency, precision, and ease of use compared to traditional gene editing techniques. In this study, we employed findings from prestigious investigations to develop an optimized approach for generating knockout cancer cell lines using a transient transfection method. This protocol introduces a distinctive approach that follows rigorous guidelines for designing gRNA to reduce off-target effects, a major challenge in CRISPR applications. Our step-by-step instructions allow researchers, particularly those with limited laboratory equipment and funding, as well as those undertaking CRISPR projects for the first time, to generate knockout cell lines using CRISPR technology in just ten weeks. This protocol covers all needs for enhancing various yields, such as transfection efficiency, and includes leveraging robust bioinformatics tools, conducting essential assays, isolating monoclonal cells via limiting dilution, validating knockout cells, and providing comprehensive troubleshooting recommendations. Using this method, we successfully created several new generations of colorectal cancer cell lines with monoallelic and biallelic knockouts of the epithelial cell adhesion molecule (EpCAM) gene. Our method, optimized for a wide spectrum of cancer cell lines, makes CRISPR more accessible for applications in personalized and precision medicine. It expands opportunities for novel investigations into cancer mechanisms and paves the way for potential therapeutic interventions.规律间隔成簇短回文重复序列（CRISPR）系统相较于传统基因编辑技术，兼具成本效益高、效率出众、精度优异且操作简便的优势。本研究依托权威研究成果，开发出一套基于瞬时转染法构建敲除癌细胞系的优化方案。本实验方案采用独特设计思路，严格遵循向导RNA（gRNA）的设计规范以降低脱靶效应——脱靶效应正是CRISPR应用领域的核心挑战之一。我们提供的分步操作指南，可帮助研究人员——尤其是实验室设备与经费有限的研究者，以及首次开展CRISPR相关项目的人员——仅用十周时间即可利用CRISPR技术构建敲除细胞系。本方案涵盖提升转染效率等各类产出指标的全流程需求，涉及使用功能强大的生物信息学工具、开展必要检测实验、通过有限稀释法分离单克隆细胞、验证敲除细胞，同时提供详尽的故障排查指导。利用该方法，我们成功构建了多株新一代结直肠癌细胞系，这些细胞系的上皮细胞黏附分子（EpCAM）基因均实现了单等位或双等位敲除。我们针对广谱癌细胞系优化的这套方法，大幅降低了CRISPR技术在个性化医疗与精准医学领域的应用门槛。该方案为癌症机制的创新性研究拓展了空间，也为潜在的治疗干预手段铺平了道路。