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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/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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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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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: Aug 23, 2025

CRISPR-Cas9-Mediated Genome Editing in the Filamentous Ascomycete Huntiella omanensis
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A Simple and Efficient CRISPR/Cas9 System Using a Ribonucleoprotein Method for Flammulina filiformis.

Jianyu Liu1, Haiyang Cui2, Ruijuan Wang1

  • 1National Engineering Research Center of Edible Fungi, Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Shanghai 201403, China.

Journal of Fungi (Basel, Switzerland)
|October 27, 2022
PubMed
Summary

Researchers optimized CRISPR/Cas9 genome editing in Flammulina filiformis using in vitro assembled ribonucleoprotein complexes (RNPs). This novel RNP delivery method achieved 100% targeting efficiency without foreign DNA, advancing fungal gene editing.

Keywords:
CRISPR/Cas9Flammulina filiformisRNPsgenomic editingpyrG

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Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins
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Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins

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Area of Science:

  • Fungal genomics
  • Molecular biology
  • Biotechnology

Background:

  • CRISPR/Cas9 systems in fungi often rely on in vivo expressed components.
  • In vitro assembled Cas9 and sgRNA ribonucleoprotein complexes (RNPs) offer advantages but have limited application and efficiency in fungi.
  • Flammulina filiformis genome editing requires efficient and streamlined methods.

Purpose of the Study:

  • To develop and optimize an efficient CRISPR/Cas9 genome-editing system for Flammulina filiformis using in vitro assembled RNP complexes.
  • To investigate the role of surfactants in enhancing RNP delivery and editing efficiency.
  • To establish a method that avoids foreign DNA for faster and simpler gene editing.

Main Methods:

  • Development of a CRISPR/Cas9 system utilizing in vitro assembled Cas9 and sgRNA ribonucleoprotein complexes (RNPs).
  • Optimization of the RNP delivery method, identifying the critical role of the surfactant Triton X-100.
  • Application of the optimized system in Flammulina filiformis with selection on 5-FOA medium.

Main Results:

  • Achieved 100% targeting efficiency for genomic editing in Flammulina filiformis on a 5-FOA selective medium.
  • Demonstrated the successful establishment of a CRISPR/Cas9 genome-editing system in F. filiformis via RNP complex delivery.
  • The optimized method significantly improved editing efficiency compared to previously reported systems.

Conclusions:

  • The study presents the first RNP complex delivery-based CRISPR/Cas9 genome-editing system for Flammulina filiformis.
  • This method offers a more efficient, time-saving, and labor-saving alternative to traditional plasmid-based methods by eliminating the need for foreign DNA.
  • The optimized protocol using Triton X-100 provides a robust tool for F. filiformis genetic manipulation and research.