Yang Jicheng, Guo Fusheng, Chin Hui San, Chen Gao Bin, Zhang Ziyan, Williams Lewis, Kueh Andrew J, Chow Pierce K H, Herold Marco J, Fu Nai Yang
Cancer Biology and Stem Cells Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia.
Department of Medical Biology, The University of Melbourne, Parkville, VIC 3052, Australia.
Cells. 2025 Jul 29;14(15):1165. doi: 10.3390/cells14151165.
Conventional methods for generating knock-out or knock-in mammalian cell models using CRISPR-Cas9 genome editing often require tedious single-cell clone selection and expansion. In this study, we develop and optimise rapid and robust strategies to engineer homozygous fluorescent reporter knock-in cell pools with precise genome editing, circumventing clonal variability inherent to traditional approaches. To reduce false-positive cells associated with random integration, we optimise the design of donor DNA by removing the start codon of the fluorescent reporter and incorporating a self-cleaving T2A peptide system. Using fluorescence-assisted cell sorting (FACS), we efficiently identify and isolate the desired homozygous fluorescent knock-in clones, establishing stable cell pools that preserve parental cell line heterogeneity and faithfully reflect endogenous transcriptional regulation of the target gene. We evaluate the knock-in efficiency and rate of undesired random integration in the electroporation method with either a dual-plasmid system (sgRNA and donor DNA in two separate vectors) or a single-plasmid system (sgRNA and donor DNA combined in one vector). We further demonstrate that coupling our single-plasmid construct with an integrase-deficient lentivirus vector (IDLV) packaging system efficiently generates fluorescent knock-in reporter cell pools, offering flexibility between electroporation and lentivirus transduction methods. Notably, compared to the electroporation methods, the IDLV system significantly minimises random integration. Moreover, the resulting reporter cell lines are compatible with most of the available genome-wide sgRNA libraries, enabling unbiased CRISPR screens to identify key transcriptional regulators of a gene of interest. Overall, our methodologies provide a powerful genetic tool for rapid and robust generation of fluorescent reporter knock-in cell pools with precise genome editing by CRISPR-Cas9 for various research purposes.
使用CRISPR-Cas9基因组编辑技术生成基因敲除或敲入哺乳动物细胞模型的传统方法通常需要繁琐的单细胞克隆筛选和扩增。在本研究中，我们开发并优化了快速且可靠的策略，以通过精确的基因组编辑构建纯合荧光报告基因敲入细胞库，规避传统方法固有的克隆变异性。为减少与随机整合相关的假阳性细胞，我们通过去除荧光报告基因的起始密码子并引入自我切割的T2A肽系统来优化供体DNA的设计。利用荧光激活细胞分选（FACS），我们高效地鉴定并分离出所需的纯合荧光敲入克隆，建立了稳定的细胞库，该细胞库保留了亲本细胞系的异质性，并忠实地反映了靶基因的内源性转录调控。我们评估了双质粒系统（sgRNA和供体DNA分别位于两个不同载体中）或单质粒系统（sgRNA和供体DNA组合在一个载体中）电穿孔方法中的敲入效率和非期望随机整合率。我们进一步证明，将我们的单质粒构建体与整合酶缺陷型慢病毒载体（IDLV）包装系统相结合，能够有效地生成荧光敲入报告细胞库，在电穿孔和慢病毒转导方法之间提供了灵活性。值得注意的是，与电穿孔方法相比，IDLV系统显著减少了随机整合。此外，所得的报告细胞系与大多数可用的全基因组sgRNA文库兼容，能够进行无偏倚的CRISPR筛选，以鉴定感兴趣基因的关键转录调节因子。总体而言，我们的方法提供了一种强大的遗传工具，可通过CRISPR-Cas9进行精确的基因组编辑，快速且可靠地生成荧光报告基因敲入细胞库，用于各种研究目的。
