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Related Concept Videos

RNA Interference01:23

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RNA interference (RNAi) is a process in which a small non-coding RNA molecule blocks the post-transcriptional expression of a gene by binding to its messenger RNA (mRNA) and preventing the protein from being translated.
This process occurs naturally in cells, often through the activity of genomically-encoded microRNAs. Researchers can take advantage of this mechanism by introducing synthetic RNAs to deactivate specific genes for research or therapeutic purposes. For example, RNAi could be used...
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CRISPR01:59

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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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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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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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CRISPR/Cas9 Genome Editing01:28

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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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Small interfering RNAs, or siRNAs, are short regulatory RNA molecules that can silence genes post-transcriptionally, as well as the transcriptional level in some cases. siRNAs are important for protecting cells against viral infections and silencing transposable genetic elements.
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RNAi-mediated control of CRISPR functions.

Xinbo Huang1,2,3, Zhicong Chen1,2,3, Yuchen Liu1,2,3

  • 1National and Local Joint Engineering Laboratory of Medical Synthetic Biology, Shenzhen, Second People's Hospital, The First Affiliated Hospital of Shenzhen University Health Science Center, Shenzhen518035, China.

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Summary

Researchers developed a molecular switch using artificial miRNAs and enoxacin to control CRISPR-Cas9 gene editing. This method enhances targeting specificity and efficiency, addressing off-target effects and improving gene regulation in mammalian cells.

Keywords:
CRISPR switchartificial miRNAenoxacinmiRNA sponge

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Area of Science:

  • Molecular Biology
  • Gene Editing Technologies
  • RNA Interference

Background:

  • CRISPR-Cas9 is a powerful tool for genome editing and regulation, but controlling its activity is crucial.
  • RNA interference (RNAi) is a natural gene-regulating mechanism in eukaryotes.
  • Current challenges in CRISPR technology include off-target effects and variable targeting efficiency.

Purpose of the Study:

  • To develop a method for quantitative inhibition of the CRISPR-Cas9 system using artificial miRNAs (amiRNAs).
  • To improve CRISPR targeting specificity by combining amiRNAs with the RNAi enhancer enoxacin.
  • To investigate enhancing CRISPR efficiency by blocking endogenous miRNA effects on single-guide RNAs (sgRNAs).

Main Methods:

  • Designed amiRNAs targeting the sgRNA component of the CRISPR system.
  • Utilized enoxacin, an RNAi enhancer, to potentiate the inhibitory effect of amiRNAs.
  • Assessed CRISPR-mediated gene editing and regulation inhibition both in vitro and in vivo.

Main Results:

  • amiRNAs combined with enoxacin effectively inhibited CRISPR-Cas9 activity, particularly in the presence of Cas9.
  • The amiRNA/enoxacin system demonstrated tunable control over sgRNA targeting specificity.
  • Blocking endogenous miRNA interference with sgRNAs led to increased CRISPR gene editing efficiency.

Conclusions:

  • This study presents an efficient molecular switch for conditional regulation of CRISPR-Cas9 in mammalian cells.
  • The developed approach offers potential solutions for mitigating off-target effects in CRISPR gene editing.
  • The findings provide a strategy to enhance the efficiency of CRISPR-based gene regulation and editing.