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

Joshua M Hazan1, Nisrine Lahoud-Jeries1, Assaf C Bester1

  • 1Technion-Israel Institute of Technology, Faculty of Biology, Technion City, Haifa 3200003, Israel.

STAR Protocols
|September 27, 2025
PubMed
Summary

This study introduces a new protocol for CRISPR interference (CRISPRi) screens targeting long non-coding RNAs (lncRNAs). This method enables efficient discovery of functional lncRNAs using a specialized sgRNA library.

Keywords:
CRISPRCell BiologyGene ExpressionGenetics

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Area of Science:

  • Genomics
  • Molecular Biology
  • RNA Biology

Background:

  • High-throughput CRISPR interference (CRISPRi) screens are powerful tools for identifying functional genes.
  • Applying CRISPRi screens to long non-coding RNAs (lncRNAs) presents unique challenges.
  • Novel methodologies are needed to effectively target lncRNAs in functional genomic screens.

Purpose of the Study:

  • To present a detailed protocol for designing and executing pooled CRISPRi screens specifically targeting lncRNAs.
  • To enable researchers to overcome the challenges associated with lncRNA functional screening.
  • To facilitate the discovery of novel functional lncRNAs.

Main Methods:

  • Development of an abridged, cell-type-specific dual single-guide RNA (sgRNA) library for lncRNA targeting.
  • Detailed steps for library design, synthesis, and stable lentiviral transduction.
  • Guidelines for performing parallel perturbations, gRNA amplification, and data analysis.

Main Results:

  • Successful implementation of a pooled CRISPRi screening strategy for lncRNAs.
  • Demonstration of a robust protocol applicable to various research questions.
  • Establishment of a method for efficient lncRNA functional genomics.

Conclusions:

  • The presented protocol offers a robust and efficient approach for lncRNA functional screening using CRISPRi.
  • This methodology expands the utility of CRISPRi screens for exploring the functional roles of lncRNAs.
  • The protocol facilitates the discovery of novel genes and pathways regulated by lncRNAs.