Protocol for identification of sgRNA mutants using high-throughput screening technique and multiplex genome editing

Zeyu Liang; Xin Wu; Zhaojin Ye; Lingwei She; Xiaoyan Ma; Yi Xin Huo

doi:10.1016/j.xpro.2025.103690

Protocol for identification of sgRNA mutants using high-throughput screening technique and multiplex genome editing

Zeyu Liang, Xin Wu, Zhaojin Ye, Lingwei She, Xiaoyan Ma, Yi Xin Huo^*

^*Corresponding author for this work

School of Life Science

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

Abstract

In the CRISPR-Cas9 system, tandem expression of multiple identical single-guide RNAs (sgRNAs) often triggers homologous sequences loss, which affects multiplex genome editing efficiencies. Here, we present a protocol for high-throughput screening of functional sgRNAs with nonrepetitive mutants. We describe steps for constructing the screening platform, designing and constructing sgRNA libraries, and screening sgRNA mutants. These mutants can interact with the Cas9 protein, enabling multiplex genome editing. For complete details on the use and execution of this protocol, please refer to Liang et al.¹

Original language	English
Article number	103690
Journal	STAR Protocols
Volume	6
Issue number	2
DOIs	https://doi.org/10.1016/j.xpro.2025.103690
Publication status	Published - 20 Jun 2025

Keywords

CRISPR
biotechnology and bioengineering

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10.1016/j.xpro.2025.103690

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Liang, Z., Wu, X., Ye, Z., She, L., Ma, X., & Huo, Y. X. (2025). Protocol for identification of sgRNA mutants using high-throughput screening technique and multiplex genome editing. STAR Protocols, 6(2), Article 103690. https://doi.org/10.1016/j.xpro.2025.103690

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abstract = "In the CRISPR-Cas9 system, tandem expression of multiple identical single-guide RNAs (sgRNAs) often triggers homologous sequences loss, which affects multiplex genome editing efficiencies. Here, we present a protocol for high-throughput screening of functional sgRNAs with nonrepetitive mutants. We describe steps for constructing the screening platform, designing and constructing sgRNA libraries, and screening sgRNA mutants. These mutants can interact with the Cas9 protein, enabling multiplex genome editing. For complete details on the use and execution of this protocol, please refer to Liang et al.1",

keywords = "CRISPR, biotechnology and bioengineering",

author = "Zeyu Liang and Xin Wu and Zhaojin Ye and Lingwei She and Xiaoyan Ma and Huo, {Yi Xin}",

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AU - Liang, Zeyu

AU - Wu, Xin

AU - Ye, Zhaojin

AU - She, Lingwei

AU - Ma, Xiaoyan

AU - Huo, Yi Xin

PY - 2025/6/20

Y1 - 2025/6/20

N2 - In the CRISPR-Cas9 system, tandem expression of multiple identical single-guide RNAs (sgRNAs) often triggers homologous sequences loss, which affects multiplex genome editing efficiencies. Here, we present a protocol for high-throughput screening of functional sgRNAs with nonrepetitive mutants. We describe steps for constructing the screening platform, designing and constructing sgRNA libraries, and screening sgRNA mutants. These mutants can interact with the Cas9 protein, enabling multiplex genome editing. For complete details on the use and execution of this protocol, please refer to Liang et al.1

AB - In the CRISPR-Cas9 system, tandem expression of multiple identical single-guide RNAs (sgRNAs) often triggers homologous sequences loss, which affects multiplex genome editing efficiencies. Here, we present a protocol for high-throughput screening of functional sgRNAs with nonrepetitive mutants. We describe steps for constructing the screening platform, designing and constructing sgRNA libraries, and screening sgRNA mutants. These mutants can interact with the Cas9 protein, enabling multiplex genome editing. For complete details on the use and execution of this protocol, please refer to Liang et al.1

KW - CRISPR

KW - biotechnology and bioengineering

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VL - 6

JO - STAR Protocols

JF - STAR Protocols

IS - 2

M1 - 103690

ER -

Protocol for identification of sgRNA mutants using high-throughput screening technique and multiplex genome editing

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