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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

CRISPR and crRNAs

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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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Homologous Recombination02:31

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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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Related Experiment Video

Updated: Oct 18, 2025

CRISPR/Cas9 Editing of the C. elegans rbm-3.2 Gene using the dpy-10 Co-CRISPR Screening Marker and Assembled Ribonucleoprotein Complexes.
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CRISPR-SCReT (CRISPR-Stop Codon Read Through) method to control Cas9 expression for gene editing.

Pouiré Yaméogo1,2, Benjamin L Duchêne1,2, Nathalie Majeau1,2

  • 1Centre de Recherche du CHU de Québec-Université Laval, Québec City, QC, Canada.

Gene Therapy
|October 1, 2021
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Summary

CRISPR-SCReT enables drug-controlled Cas9 expression by introducing a stop codon. This method limits Cas9 exposure, reducing off-target mutations and immune responses for safer gene editing therapies.

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Area of Science:

  • Molecular Biology
  • Gene Editing Technologies
  • Biotechnology

Background:

  • CRISPR/Cas9 gene editing offers therapeutic potential for genetic disorders.
  • Constitutive Cas9 expression can lead to undesirable off-target mutations and immune reactions.
  • Controlling Cas9 expression duration is crucial for enhancing safety and efficacy.

Purpose of the Study:

  • To develop a drug-inducible CRISPR/Cas9 system for controlled gene expression.
  • To mitigate off-target mutations and immune responses associated with constitutive Cas9 activity.
  • To validate the efficacy of the CRISPR-SCReT system in mammalian cells.

Main Methods:

  • Introduction of a premature termination codon (PTC) into the Cas9 gene.
  • Utilizing aminoglycoside drugs to induce Cas9 readthrough and protein expression.
  • Co-transfection of HEK293T cells with modified Cas9 plasmid and sgRNAs targeting the DMD gene.
  • Treatment with geneticin (G418) and subsequent analysis via Western blot and PCR.

Main Results:

  • Cas9 protein expression, initially blocked by the PTC, was successfully induced by G418 treatment.
  • Targeted gene editing of the DMD gene (exons 50-54 deletion) was confirmed only in drug-treated cells.
  • The CRISPR-SCReT system demonstrated efficacy with both SpCas9 and CjCas9 for gene editing.

Conclusions:

  • The CRISPR-SCReT method provides a viable strategy for regulating Cas9 expression duration.
  • This inducible system enhances the safety profile of CRISPR/Cas9 gene editing by limiting protein exposure.
  • CRISPR-SCReT holds promise for advancing the development of safer and more precise gene therapies.