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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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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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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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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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A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization
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A Small Molecule-Controlled Cas9 Repressible System.

Youjun Wu1, Lu Yang2, Tammy Chang1

  • 1Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institute, Beckman Research Institute of City of Hope, Duarte, CA 91010, USA.

Molecular Therapy. Nucleic Acids
|January 31, 2020
PubMed
Summary

Researchers developed a new CRISPR-Cas9 control system using a small molecule-assisted shut-off (SMASh) tag. This innovation allows precise control over gene editing dose and duration, enhancing safety and accuracy in biomedical research.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Genomics

Background:

  • CRISPR-Cas9 is a revolutionary genome engineering tool with vast biomedical potential.
  • Precise control over CRISPR-Cas9 dose and exposure time is crucial for expanding its applications.
  • Current methods lack fine-tuned control over Cas9 activity and stability.

Purpose of the Study:

  • To develop a novel method for precise spatiotemporal control of CRISPR-Cas9 activity.
  • To enhance the safety, accuracy, and versatility of CRISPR-Cas9 genome editing.
  • To engineer a Cas9 system responsive to a small molecule inhibitor.

Main Methods:

  • Engineered Cas9 by fusing it with the small molecule-assisted shut-off (SMASh) peptide.
  • SMASh comprises a protease domain and a degron domain from hepatitis C virus (HCV).
  • Utilized the clinically approved HCV protease inhibitor, asunaprevir (ASV), for Cas9 degradation control.

Main Results:

  • Engineered Cas9 rapidly degraded in a dose- and time-dependent manner upon ASV administration.
  • Cas9 degradation was reversible, with gene editing activity restored upon ASV removal.
  • Limiting Cas9 levels significantly increased gene editing specificity.
  • The SMASh tag enabled precise control over Cas9 stability and activity.

Conclusions:

  • The SMASh tag provides an effective tool for controlling Cas9 stability and activity.
  • This system improves the accuracy, safety, and versatility of CRISPR-Cas9 for genome editing.
  • Enables enhanced gene regulation studies and therapeutic applications requiring precise gene editing control.