Generation of dual-gRNA library for combinatorial CRISPR screening of synthetic lethal gene pairs

Authors

• Shan Tang, The Ohio State University
• Xue Wu, The Ohio State University
• Jinghui Liu, University of Kentucky
• Qiongsi Zhang, University of Kentucky
• Xinyi Wang, University of Kentucky
• Shuai Shao, The Ohio State University
• Birkan Gokbag, The Ohio State University
• Kunjie Fan, The Ohio State University
• Xiaoqi Liu, University of Kentucky
• Fuhai Li, Washington University
• Lijun Cheng, The Ohio State University
• Lang Li, The Ohio State University

Abstract

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 in vitro 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.

Document Type

Article

Publication Date

9-16-2022

Digital Object Identifier (DOI)

https://doi.org/10.1016/j.xpro.2022.101556

Repository Citation

Tang, Shan; Wu, Xue; Liu, Jinghui; Zhang, Qiongsi; Wang, Xinyi; Shao, Shuai; Gokbag, Birkan; Fan, Kunjie; Liu, Xiaoqi; Li, Fuhai; Cheng, Lijun; and Li, Lang, "Generation of dual-gRNA library for combinatorial CRISPR screening of synthetic lethal gene pairs" (2022). Toxicology and Cancer Biology Faculty Publications. 114.
https://uknowledge.uky.edu/toxicology_facpub/114