用于合成致死基因对组合 CRISPR 筛选的双 gRNA 文库的生成。

College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA.

Department of Biomedical Informatics, College of Medicine, The Ohio State University, Columbus, OH 43210, USA.

STAR Protoc. 2022 Aug 24;3(3):101556. doi: 10.1016/j.xpro.2022.101556. eCollection 2022 Sep 16.

Combinatorial CRISPR screening is useful for investigating synthetic lethality (SL) gene pairs. Here, we detail the steps for dual-gRNA library construction, with the introduction of two backbones, LentiGuide_DKO and LentiCRISPR_DKO. We describe steps for screening with 22Rv1-Cas9 and SaOS2-Cas9 cells followed by sequencing and data analysis. By introducing two backbones, we optimized the library construction process, facilitated standard pair-end sequencing, and provided options of screening on cells with or without modification of Cas9 expression.

组合型 CRISPR 筛选对于研究合成致死（SL）基因对很有用。在这里，我们详细介绍了双 gRNA 文库构建的步骤，引入了两个骨架，LentiGuide_DKO 和 LentiCRISPR_DKO。我们描述了使用 22Rv1-Cas9 和 SaOS2-Cas9 细胞进行筛选的步骤，然后进行测序和数据分析。通过引入两个骨架，我们优化了文库构建过程，方便了标准的双端测序，并提供了在 Cas9 表达修饰或不修饰的细胞上进行筛选的选项。