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Related Concept Videos

CRISPR01:59

CRISPR

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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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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.
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The Antiviral System of Bacteria and Archaea: CRISPR01:23

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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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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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Conservative Site-specific Recombination and Phase Variation02:53

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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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CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art
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CRISPR-Cas12a nucleases function with structurally engineered crRNAs: SynThetic trAcrRNA.

D J Jedrzejczyk1, L D Poulsen2, M Mohr1

  • 1Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet 220, 2800, Kongens Lyngby, Denmark.

Scientific Reports
|July 16, 2022
PubMed
Summary
This summary is machine-generated.

Engineered CRISPR-Cas12a guide RNAs with modified loops enable precise gene editing. These structural changes, including breaks and gaps, maintain high editing efficiency for diverse applications.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • CRISPR-Cas12a systems offer broader editing capabilities than CRISPR-Cas9.
  • Cas12a lacks trans-activating crRNA (tracrRNA), simplifying the system.
  • CRISPR RNA (crRNA) engineering is a key strategy to enhance Cas12a activity.

Purpose of the Study:

  • To design and validate structurally engineered Cas12a crRNAs.
  • To assess the impact of crRNA modifications on gene-editing efficiency.
  • To explore the utility of engineered crRNAs in various Cas12a systems.

Main Methods:

  • Designed sixteen types of structurally engineered Cas12a crRNAs.
  • Targeted immunologically relevant genetic loci in vitro and in cells.
  • Evaluated gene-cutting activity with different Cas12a nucleases.
  • Assessed insertion rates of short homology-directed repair (HDR) templates.

Main Results:

  • All engineered crRNAs, including STAR-crRNAs and Gap-crRNAs, enabled gene-cutting.
  • Structural modifications in the crRNA loop region did not impede Cas12a nuclease activity.
  • Engineered crRNAs demonstrated comparable insertion efficiencies for HDR templates to wild-type crRNAs.

Conclusions:

  • Cas12a nucleases can effectively utilize structurally engineered crRNAs with modifications in the loop region.
  • Breaks or gaps in the crRNA loop are compatible with Cas12a function.
  • This approach broadens the applicability of CRISPR-Cas12a for genome editing.