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Related Concept Videos

CRISPR/Cas9 Genome Editing01:28

CRISPR/Cas9 Genome Editing

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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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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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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.
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...
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Updated: Jul 19, 2025

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9
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Disrupting Protein Expression with Double-Clicked sgRNA-Cas9 Complexes: A Modular Approach to CRISPR Gene Editing.

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Chemically modified single guide RNA (sgRNA) fragments enhance CRISPR-Cas9 gene editing. This modular approach improves nuclease resistance and enables simultaneous disruption of multiple gene targets.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Synthetic Biology

Background:

  • CRISPR-Cas9 is a powerful gene editing tool, but synthesizing functional single guide RNA (sgRNA) is challenging.
  • A modular approach using copper-catalyzed azide-alkyne cycloaddition has been developed to assemble sgRNAs from smaller fragments.
  • Enhancing sgRNA stability is crucial for efficient and robust gene editing applications.

Purpose of the Study:

  • To improve the stability and efficacy of modular sgRNAs for CRISPR-Cas9 gene editing.
  • To investigate the impact of chemical modifications on sgRNA resistance to ribonucleases.
  • To demonstrate the utility of chemically stabilized sgRNAs for multiplex gene editing.

Main Methods:

  • Chemically synthesized sgRNA fragments were assembled using copper-catalyzed azide-alkyne cycloaddition.
  • Modified nucleotides (2'-O-Me and phosphorothioate) were incorporated at the 5' and 3' ends of the sgRNA.
  • CRISPR-Cas9 mediated gene knockout was assessed, including simultaneous knockout of Siglec-3 and Siglec-7.

Main Results:

  • Incorporation of three modified nucleotides at both ends significantly enhanced sgRNA stability and resistance to ribonucleases.
  • The chemically stabilized sgRNAs demonstrated increased efficiency in Cas9-mediated gene knockout.
  • Successful simultaneous knockout of two target genes (Siglec-3 and Siglec-7) was achieved, enabling protein-level disruption.

Conclusions:

  • Modular sgRNA assembly combined with chemical modifications offers a versatile platform for creating stabilized gene editing tools.
  • This approach overcomes limitations in sgRNA synthesis and enhances CRISPR-Cas9 performance.
  • The developed method facilitates advanced applications like multiplex gene editing for simultaneous protein expression disruption.