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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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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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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 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 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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CRISPR-based Shuttle Cloning: A High-throughput Cloning Method
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Cloning-free CRISPR.

Mandana Arbab1, Sharanya Srinivasan2, Tatsunori Hashimoto3

  • 1Hubrecht Institute and UMC Utrecht, Uppsalalaan 8, 3584 CT Utrecht, the Netherlands; Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine at Utrecht University, Yalelaan 108, 3583 CM Utrecht, the Netherlands.

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Summary
This summary is machine-generated.

Self-cloning CRISPR/Cas9 (scCRISPR) simplifies genome editing by enabling rapid gene knockouts and transgene knockins. This cost-effective technology bypasses the need for cloning, accelerating genetic research in various cell types.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • CRISPR/Cas9 technology enables precise genome editing but often requires extensive cloning of guide RNAs and homology constructs.
  • The process of generating site-specific mutations and knockins can be time-consuming and costly, limiting its accessibility.

Purpose of the Study:

  • To develop a streamlined CRISPR/Cas9 system (scCRISPR) for rapid genomic mutation and site-specific transgene creation.
  • To reduce the cost and preparation time associated with CRISPR/Cas9-mediated genetic modifications.
  • To enable efficient gene knockouts and knockins without the need for traditional cloning methods.

Main Methods:

  • Introduction of a self-cleaving palindromic sgRNA plasmid and a short DNA sequence encoding the locus-specific sgRNA into target cells.
  • Utilizing homologous recombination for the in situ production of locus-specific sgRNA plasmids.
  • Employing PCR-based addition of short homology arms for site-specific transgene integration.

Main Results:

  • Achieved high gene knockout efficiency (approximately 88%) with scCRISPR.
  • Reduced the cost of sgRNA construction by approximately one-sixth compared to traditional plasmid-based methods.
  • Demonstrated efficient site-specific knockin of GFP transgenes (2%-4% rate) in mouse and human embryonic stem cells without selection cassettes.
  • Significantly decreased preparation time to approximately 2 hours per targeted site.

Conclusions:

  • Self-cloning CRISPR/Cas9 (scCRISPR) offers a rapid, cost-effective, and simplified approach for generating gene knockouts and knockins.
  • The technology bypasses the need for extensive cloning of sgRNAs and homology constructs, making transgenesis more accessible.
  • scCRISPR substantially lowers the technical and financial barriers for genome engineering in both mouse and human systems.