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Related Concept Videos

CRISPR01:59

CRISPR

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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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CRISPR and crRNAs02:53

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Bacteria and archaea are susceptible to viral infections just like eukaryotes; therefore, they have developed a unique adaptive immune system to protect themselves. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) are present in more than 45% of known bacteria and 90% of known archaea.
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The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
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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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Related Experiment Video

Updated: May 28, 2025

Ubiquitous and Tissue-specific RNA Targeting in Drosophila Melanogaster using CRISPR/CasRx
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Ubiquitous and Tissue-specific RNA Targeting in Drosophila Melanogaster using CRISPR/CasRx

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Precise RNA targeting with CRISPR-Cas13d.

Sydney K Hart1,2, Simon Müller1,2, Hans-Hermann Wessels1,2

  • 1New York Genome Center, New York, NY, USA.

Nature Biotechnology
|February 11, 2025
PubMed
Summary
This summary is machine-generated.

CRISPR-Cas13 RNA degradation concerns are mitigated by using low RfxCas13d expression, enabling precise transcriptome editing. A high-fidelity Cas13 variant shows reduced collateral activity, likely due to lower nuclease function.

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Last Updated: May 28, 2025

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Area of Science:

  • Molecular Biology
  • Gene Editing Technologies
  • RNA Biology

Background:

  • CRISPR-Cas13 systems offer precise RNA targeting but raise concerns about unwanted collateral RNA degradation.
  • This collateral activity can perturb transcriptomes and limit therapeutic applications.

Purpose of the Study:

  • To investigate the relationship between RfxCas13d expression levels and collateral RNA degradation.
  • To develop strategies for achieving high on-target knockdown with minimal collateral effects using CRISPR-Cas13.
  • To explore the mechanism behind reduced collateral activity in high-fidelity Cas13 variants.

Main Methods:

  • Transcriptome-scale and combinatorial pooled screening approaches were employed.
  • RfxCas13d expression levels were systematically varied.
  • A high-fidelity Cas13 variant was analyzed for its nuclease activity and collateral effects.

Main Results:

  • Collateral RNA degradation was observed only at high RfxCas13d expression levels.
  • Low-copy RfxCas13d enabled high on-target knockdown without significant collateral activity in screens.
  • The high-fidelity Cas13 variant exhibited reduced collateral activity, correlating with diminished overall nuclease capability.

Conclusions:

  • Optimizing RfxCas13d expression is crucial for minimizing collateral RNA degradation in CRISPR-Cas13 applications.
  • Low-expression RfxCas13d and high-fidelity variants are promising for safe and effective transcriptome-wide RNA editing and therapeutic uses.
  • Reduced nuclease capability appears to be a key factor in mitigating collateral activity in Cas13 systems.