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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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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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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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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: Jun 29, 2025

Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution
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Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution

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Eukaryotic-driven directed evolution of Cas9 nucleases.

Giulia Vittoria Ruta1, Matteo Ciciani2,3, Eyemen Kheir2

  • 1Laboratory of Molecular Virology, Department CIBIO, University of Trento, Trento, Italy. giuliavittoria.ruta@unitn.it.

Genome Biology
|March 26, 2024
PubMed
Summary
This summary is machine-generated.

Scientists developed a Eukaryotic Platform to Improve Cas Activity (EPICA) to enhance genome editing tools. This platform successfully improved the Campylobacter jejuni Cas9 (CjCas9) nuclease, creating UltraCjCas9 with significantly higher activity and specificity in mammalian cells.

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Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms
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Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Gene Editing Technologies

Background:

  • Advancements in genome editing necessitate tools with improved eukaryotic compatibility.
  • Many Cas9 variants show potential but require optimization for efficient genome editing.
  • The small size of Campylobacter jejuni Cas9 (CjCas9) is advantageous for delivery in mammalian cells, but its editing activity is limited.

Purpose of the Study:

  • To develop a directed evolution platform for enhancing the activity of weakly active Cas9 nucleases.
  • To create a more active and specific CjCas9 variant for eukaryotic genome editing applications.

Main Methods:

  • Developed the Eukaryotic Platform to Improve Cas Activity (EPICA) using yeast auxotrophic selection and a mammalian cell reporter system.
  • Employed directed evolution to enhance CjCas9 activity through iterative selection and screening.
  • Validated the enhanced Cas9 variant in mammalian endogenous genomic loci.

Main Results:

  • The EPICA platform successfully generated an enhanced CjCas9 variant, named UltraCjCas9.
  • UltraCjCas9 exhibits up to 12-fold higher editing activity in mammalian cells compared to the wild-type CjCas9.
  • The enhanced variant, UltraCjCas9, maintains high specificity across the genome.

Conclusions:

  • A novel eukaryotic pipeline (EPICA) has been established for enhancing Cas9 nuclease activity.
  • This platform facilitates the optimization of naturally occurring RNA-guided nucleases for genome editing.
  • The development of UltraCjCas9 demonstrates the potential of EPICA to unlock new genome editing tools.