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RNA interference (RNAi) is a cellular mechanism that inhibits gene expression by suppressing its transcription or activating the RNA degradation process. The mechanism was discovered by Andrew Fire and Craig Mello in 1998 in plants. Today, it is observed in almost all eukaryotes, including protozoa, flies, nematodes, insects, parasites, and mammals. This precise cellular mechanism of gene silencing has been developed into a technique that provides an efficient way to identify and determine the...
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Dual CRISPR-Interference Strategy for Targeting Synthetic Lethal Interactions Between Non-Coding RNAs in Cancer Cells
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Genome-Scale Perturbation of Long Noncoding RNA Expression Using CRISPR Interference.

S John Liu1,2, Max A Horlbeck3,4,5,6, Jonathan S Weissman3,4,5,6

  • 1Department of Neurological Surgery, University of California, San Francisco, CA, USA.

Methods in Molecular Biology (Clifton, N.J.)
|December 16, 2020
PubMed
Summary

CRISPR interference (CRISPRi) screens enable large-scale knockdown of long noncoding RNAs (lncRNAs) to study their functions. This method uses pooled lentiviral screens to analyze lncRNA roles in cell growth and proliferation.

Keywords:
CRISPRCRISPRiScreenlncRNA

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Area of Science:

  • Molecular Biology
  • Genomics
  • RNA Biology

Background:

  • Long noncoding RNAs (lncRNAs) play crucial roles in gene regulation but their functions remain largely uncharacterized.
  • Systematic functional characterization of lncRNAs is essential for understanding complex biological processes.
  • CRISPR interference (CRISPRi) offers a powerful tool for targeted gene repression.

Purpose of the Study:

  • To describe the application of CRISPR interference (CRISPRi) for large-scale functional screening of long noncoding RNA (lncRNA) loci.
  • To demonstrate the utility of CRISPRi-based pooled screening for identifying lncRNAs involved in cell growth and proliferation.
  • To discuss considerations for designing custom lncRNA-targeting libraries and alternative screening strategies.

Main Methods:

  • Utilized CRISPR interference (CRISPRi) for programmable transcriptional repression.
  • Employed highly parallel, lentiviral pooled screening approaches to target thousands of lncRNA-expressing loci simultaneously.
  • Assessed cell growth and proliferation as phenotypic readouts to identify functional lncRNAs.

Main Results:

  • Demonstrated the feasibility of using CRISPRi in pooled screens to systematically target lncRNA loci.
  • Successfully identified lncRNAs influencing cell growth and proliferation through high-throughput screening.
  • Provided a framework for future lncRNA functional genomics studies using CRISPRi.

Conclusions:

  • CRISPR interference (CRISPRi) is a versatile and effective tool for large-scale functional genomics of long noncoding RNAs (lncRNAs).
  • Pooled CRISPRi screening enables efficient identification of lncRNAs impacting cellular phenotypes like growth and proliferation.
  • The described methodology facilitates systematic characterization of lncRNA functions and opens avenues for further research.