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Related Concept Videos

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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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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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 stands for Clustered Regularly Interspaced Short Palindromic Repeats is a adaptive immune system found in bacteria and archaea that protects against viral infections. This system enables prokaryotic cells to identify, remember, and neutralize foreign genetic elements, primarily bacteriophages, by storing fragments of the invader’s DNA as a genetic memory.The CRISPR immune response begins during an initial infection. Cas (CRISPR-associated) proteins play a central role in this...
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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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CRISPR-based Shuttle Cloning: A High-throughput Cloning Method
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Self-Cloning CRISPR.

Mandana Arbab1,2,3, Richard I Sherwood1,3

  • 1Hubrecht Institute and UMC Utrecht, Utrecht, The Netherlands.

Current Protocols in Stem Cell Biology
|August 18, 2016
PubMed
Summary
This summary is machine-generated.

Self-cloning CRISPR/Cas9 (scCRISPR) offers rapid and efficient gene editing. This novel method achieves high gene knockout and knock-in rates without complex plasmid construction, streamlining the CRISPR/Cas9 process.

Keywords:
CRISPR/Cas9GFP transgenesisembryonic stem cellsgene editinghomologous recombinationknock-inknockout

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Area of Science:

  • Molecular Biology
  • Gene Editing Technologies
  • Biotechnology

Background:

  • CRISPR/Cas9 gene editing allows precise modification of genomic loci.
  • Traditional methods require constructing specific sgRNA plasmids, which is time-consuming.

Purpose of the Study:

  • To introduce a rapid and efficient CRISPR/Cas9 gene editing system.
  • To circumvent the need for sgRNA plasmid construction.
  • To enable fast and high-efficiency gene knockout and knock-in.

Main Methods:

  • Developed self-cloning CRISPR/Cas9 (scCRISPR) utilizing a self-cleaving palindromic sgRNA plasmid (sgPal).
  • Employed homologous recombination for recombining sgPal with short PCR-amplified sgRNA sequences within target cells.
  • Used PCR-based addition of homology arms for site-specific transgene knock-in.

Main Results:

  • Achieved gene editing within 2 hours of obtaining sgRNA oligos.
  • Demonstrated high gene knockout efficiency (>90%) in mouse and human embryonic stem cells and cancer cell lines.
  • Obtained efficient site-specific knock-in of transgenes (e.g., GFP) with 2% to 4% efficiency, bypassing traditional cloning and selection cassettes.

Conclusions:

  • scCRISPR represents the fastest and most efficient method for CRISPR gene editing currently available.
  • This technology significantly simplifies the CRISPR/Cas9 workflow.
  • Enables rapid and efficient gene knockout and knock-in in various cell types.