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

Shan Tang; Xue Wu; Jinghui Liu; Qiongsi Zhang; Xinyi Wang; Shuai Shao; Birkan Gokbag; Kunjie Fan; Xiaoqi Liu; Fuhai Li; Lijun Cheng; Lang Li

doi:10.1016/j.xpro.2022.101556

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

Shan Tang, Xue Wu, Jinghui Liu, Qiongsi Zhang, Xinyi Wang, Shuai Shao, Birkan Gokbag, Kunjie Fan, Xiaoqi Liu, Fuhai Li, Lijun Cheng, Lang Li

Research output: Contribution to journal › Article › peer-review

1 Scopus citations

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.

Original language	English (US)
Article number	101556
Pages (from-to)	101556
Journal	STAR Protocols
Volume	3
Issue number	3
DOIs	https://doi.org/10.1016/j.xpro.2022.101556
State	Published - Sep 16 2022

Keywords

Biotechnology and bioengineering
CRISPR
High Throughput Screening
Molecular Biology
Genes, Lethal
CRISPR-Cas Systems/genetics
Gene Library
RNA, Guide, Kinetoplastida/genetics
Synthetic Lethal Mutations

ASJC Scopus subject areas

Biochemistry, Genetics and Molecular Biology(all)
Immunology and Microbiology(all)
Neuroscience(all)

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10.1016/j.xpro.2022.101556

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title = "Generation of dual-gRNA library for combinatorial CRISPR screening of synthetic lethal gene pairs",

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.",

keywords = "Biotechnology and bioengineering, CRISPR, High Throughput Screening, Molecular Biology, Genes, Lethal, CRISPR-Cas Systems/genetics, Gene Library, RNA, Guide, Kinetoplastida/genetics, Synthetic Lethal Mutations",

author = "Shan Tang and Xue Wu and Jinghui Liu and Qiongsi Zhang and Xinyi Wang and Shuai Shao and Birkan Gokbag and Kunjie Fan and Xiaoqi Liu and Fuhai Li and Lijun Cheng and Lang Li",

note = "Funding Information: The work related to cell culture is done by Dr. Liu{\textquoteright}s lab and was supported by NIH R01 CA157429 (X. Liu), R01 CA196634 (X. Liu), R01 CA264652 (X. Liu), R01 CA256893 (X. Liu). Publisher Copyright: {\textcopyright} 2022 {\textcopyright} 2022 The Authors.",

year = "2022",

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doi = "10.1016/j.xpro.2022.101556",

language = "English (US)",

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AU - Tang, Shan

AU - Wu, Xue

AU - Liu, Jinghui

AU - Zhang, Qiongsi

AU - Wang, Xinyi

AU - Shao, Shuai

AU - Gokbag, Birkan

AU - Fan, Kunjie

AU - Liu, Xiaoqi

AU - Li, Fuhai

AU - Cheng, Lijun

AU - Li, Lang

N1 - Funding Information: The work related to cell culture is done by Dr. Liu’s lab and was supported by NIH R01 CA157429 (X. Liu), R01 CA196634 (X. Liu), R01 CA264652 (X. Liu), R01 CA256893 (X. Liu). Publisher Copyright: © 2022 © 2022 The Authors.

PY - 2022/9/16

Y1 - 2022/9/16

N2 - 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.

AB - 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.

KW - Biotechnology and bioengineering

KW - CRISPR

KW - High Throughput Screening

KW - Molecular Biology

KW - Genes, Lethal

KW - CRISPR-Cas Systems/genetics

KW - Gene Library

KW - RNA, Guide, Kinetoplastida/genetics

KW - Synthetic Lethal Mutations

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ER -

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

Abstract

Keywords

ASJC Scopus subject areas

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