Faculty of Advanced Science and Technology, Department of Genetics, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran.
Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran, Microbiology Research Center, Tehran, Iran.
PLoS One. 2024 Nov 14;19(11):e0310368. doi: 10.1371/journal.pone.0310368. eCollection 2024.
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.
与传统的基因编辑技术相比，簇状规律间隔短回文重复序列（CRISPR）系统具有成本效益高、效率高、精确性高和易于使用的特点。在这项研究中，我们利用权威研究的结果，开发了一种使用瞬时转染方法生成敲除癌细胞系的优化方法。该方案采用了一种独特的方法，严格遵循设计 gRNA 的指导方针，以减少 CRISPR 应用中的脱靶效应这一主要挑战。我们的分步说明允许研究人员，特别是那些实验室设备和资金有限的研究人员，以及那些首次进行 CRISPR 项目的研究人员，仅用十周时间即可使用 CRISPR 技术生成敲除细胞系。该方案涵盖了提高各种产量（如转染效率）的所有需求，并利用强大的生物信息学工具，进行必要的测定，通过有限稀释分离单克隆细胞，验证敲除细胞，并提供全面的故障排除建议。使用这种方法，我们成功地创建了几株新的结直肠癌细胞系，这些细胞系中上皮细胞黏附分子（EpCAM）基因的单等位基因和双等位基因敲除。我们的方法针对广泛的癌细胞系进行了优化，使 CRISPR 更易于在个性化和精准医疗中应用。它为癌症机制的新研究提供了机会，并为潜在的治疗干预铺平了道路。
