Protocol for simplified parallel perturbations using an abridged long non-coding RNA CRISPR library

Joshua M. Hazan; Nisrine Lahoud-Jeries; Assaf C. Bester

doi:10.1016/j.xpro.2025.104110

Protocol for simplified parallel perturbations using an abridged long non-coding RNA CRISPR library

Joshua M. Hazan, Nisrine Lahoud-Jeries, Assaf C. Bester

Biology

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

Abstract

High-throughput CRISPR interference (CRISPRi) screens are invaluable for discovering novel functional genes, but applying such screens to long non-coding RNAs (lncRNAs) is more challenging. Here, we present a protocol for designing and executing pooled CRISPRi screens targeting lncRNAs using an abridged cell-type-specific dual single-guide RNA (sgRNA) library. We describe steps for library design and synthesis, followed by stable lentiviral transduction. We then provide guidelines for performing multiple parallel perturbations tailored to the research question, followed by gRNA amplification and data analysis.

Original language	English
Article number	104110
Journal	STAR Protocols
Volume	6
Issue number	4
DOIs	https://doi.org/10.1016/j.xpro.2025.104110
State	Published - 19 Dec 2025

Keywords

Cell Biology
CRISPR
Gene Expression
Genetics

ASJC Scopus subject areas

General Neuroscience
General Immunology and Microbiology
General Biochemistry, Genetics and Molecular Biology

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10.1016/j.xpro.2025.104110

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abstract = "High-throughput CRISPR interference (CRISPRi) screens are invaluable for discovering novel functional genes, but applying such screens to long non-coding RNAs (lncRNAs) is more challenging. Here, we present a protocol for designing and executing pooled CRISPRi screens targeting lncRNAs using an abridged cell-type-specific dual single-guide RNA (sgRNA) library. We describe steps for library design and synthesis, followed by stable lentiviral transduction. We then provide guidelines for performing multiple parallel perturbations tailored to the research question, followed by gRNA amplification and data analysis.",

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AU - Bester, Assaf C.

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Y1 - 2025/12/19

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AB - High-throughput CRISPR interference (CRISPRi) screens are invaluable for discovering novel functional genes, but applying such screens to long non-coding RNAs (lncRNAs) is more challenging. Here, we present a protocol for designing and executing pooled CRISPRi screens targeting lncRNAs using an abridged cell-type-specific dual single-guide RNA (sgRNA) library. We describe steps for library design and synthesis, followed by stable lentiviral transduction. We then provide guidelines for performing multiple parallel perturbations tailored to the research question, followed by gRNA amplification and data analysis.

KW - Cell Biology

KW - CRISPR

KW - Gene Expression

KW - Genetics

UR - https://www.scopus.com/pages/publications/105016887496

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Protocol for simplified parallel perturbations using an abridged long non-coding RNA CRISPR library

Abstract

Keywords

ASJC Scopus subject areas

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