Ryniawec John M, Amoiroglou Anastasia, Rogers Gregory C
Department of Cellular and Molecular Medicine, University of Arizona Cancer Center, University of Arizona, Tucson, AZ 85724, United States.
Biol Methods Protoc. 2024 Aug 17;9(1):bpae059. doi: 10.1093/biomethods/bpae059. eCollection 2024.
CRISPR/Cas9 genome editing is a pervasive research tool due to its relative ease of use. However, some systems are not amenable to generating edited clones due to genomic complexity and/or difficulty in establishing clonal lines. For example, Schneider 2 (S2) cells possess a segmental aneuploid genome and are challenging to single-cell select. Here, we describe a streamlined CRISPR/Cas9 methodology for knock-in and knock-out experiments in S2 cells, whereby an antibiotic resistance gene is inserted in-frame with the coding region of a gene-of-interest. By using selectable markers, we have improved the ease and efficiency for the positive selection of null cells using antibiotic selection in feeder layers followed by cell expansion to generate clonal lines. Using this method, we generated the first acentrosomal S2 cell lines by knocking-out centriole genes Polo-like Kinase 4/Plk4 or Ana2 as proof of concept. These strategies for generating gene-edited clonal lines will add to the collection of CRISPR tools available for cultured cells by making CRISPR more practical and therefore improving gene function studies.
CRISPR/Cas9基因组编辑因其相对易于使用而成为一种广泛应用的研究工具。然而，由于基因组复杂性和/或建立克隆系的困难，一些系统不适合生成编辑后的克隆。例如，Schneider 2（S2）细胞具有片段非整倍体基因组，单细胞选择具有挑战性。在这里，我们描述了一种简化的CRISPR/Cas9方法，用于S2细胞中的敲入和敲除实验，即将抗生素抗性基因与感兴趣基因的编码区读框内插入。通过使用选择标记，我们提高了在饲养层中使用抗生素选择对空细胞进行阳性选择的简便性和效率，随后进行细胞扩增以生成克隆系。使用这种方法，我们通过敲除中心粒基因Polo样激酶4/Plk4或Ana2，生成了第一个无中心体的S2细胞系，作为概念验证。这些生成基因编辑克隆系的策略将通过使CRISPR更实用，从而改进基因功能研究，增加可用于培养细胞的CRISPR工具的种类。
