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Related Concept Videos

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Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced...
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The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...
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Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
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Precise transcript targeting by CRISPR-Csm complexes.

David Colognori1,2, Marena Trinidad1,2, Jennifer A Doudna3,4,5,6,7,8

  • 1Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.

Nature Biotechnology
|January 23, 2023
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Summary

The clustered regularly interspaced short palindromic repeats (CRISPR)-Csm complex precisely targets and degrades RNA in human cells. This novel RNA-targeting tool achieves high-efficiency knockdown and enables live-cell RNA imaging.

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Area of Science:

  • Molecular Biology
  • Gene Regulation
  • CRISPR Technology

Background:

  • Targeting specific RNA transcripts in mammalian cells is challenging due to limitations of current methods.
  • Existing RNA-targeting techniques often suffer from inefficiency, imprecision, and issues with subcellular localization.

Purpose of the Study:

  • To investigate the potential of the CRISPR-Csm complex for precise RNA ablation in eukaryotic cells.
  • To evaluate the efficiency and specificity of CRISPR-Csm compared to existing RNA knockdown technologies.

Main Methods:

  • Delivery of the Streptococcus thermophilus Csm complex via a single vector into human cells.
  • Assessment of RNA knockdown efficiency and off-target effects.
  • Utilizing a catalytically inactivated Csm complex for live-cell RNA imaging.

Main Results:

  • The CRISPR-Csm complex achieved high-efficiency RNA knockdown (90-99%) in human cells.
  • Demonstrated minimal off-target effects, outperforming short hairpin RNA and Cas13-mediated knockdown.
  • Showcased specific and durable RNA binding with catalytically inactivated Csm for live-cell imaging.

Conclusions:

  • The multiprotein CRISPR-Csm complex is an effective tool for surgical RNA ablation in both nuclear and cytoplasmic compartments.
  • CRISPR-Csm offers advantages over CRISPR-Cas13 due to its programmable, RNA-guided mechanism without indiscriminate trans-cleavage.
  • Established the feasibility and efficacy of CRISPR-Csm as a powerful RNA-targeting technology in eukaryotes.