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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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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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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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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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Catalytically Enhanced Cas9 through Directed Protein Evolution.

Travis H Hand1, Mitchell O Roth1, Chardasia L Smith2

  • 1Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida, USA; Florida State University, Tallahassee, Florida, USA.

The CRISPR Journal
|April 20, 2021
PubMed
Summary
This summary is machine-generated.

Protein engineering enhances CRISPR-Cas9 by creating catalytically enhanced variants (CECas9). This method improves gene editing efficiency, showing significant gains for both Acidothermus cellulolyticus Cas9 and Streptococcus pyogenes Cas9 in cellular applications.

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Area of Science:

  • Molecular Biology
  • Protein Engineering
  • Gene Editing Technologies

Background:

  • CRISPR-Cas9 systems are powerful tools for gene editing.
  • Protein engineering offers a route to optimize CRISPR-Cas9 for enhanced performance.
  • Directed evolution can yield improved Cas9 variants with altered catalytic properties.

Purpose of the Study:

  • To develop a directed protein evolution method for selecting catalytically enhanced CRISPR-Cas9 variants (CECas9).
  • To improve the catalytic efficiency of Cas9 enzymes through protein engineering.
  • To demonstrate the utility of engineered Cas9 variants in gene editing applications.

Main Methods:

  • A directed protein evolution strategy was employed using a toxic gene with a shortened protospacer.
  • Selection of catalytically enhanced CRISPR-Cas9 variants (CECas9) was performed.
  • Enzyme kinetics were used to quantify improvements in catalytic efficiency.
  • Homology-directed repair (HDR) based gene insertion was assessed in human cells.

Main Results:

  • A catalytically enhanced variant of Acidothermus cellulolyticus Cas9 (AceCECas9) showed up to a fourfold improvement in in vitro catalytic efficiency.
  • Protein engineering successfully improved the performance of Streptococcus pyogenes Cas9 (SpyCas9).
  • The engineered SpyCECas9 demonstrated enhanced efficiency in homology-directed repair-based gene insertion in human colon cancer cells.

Conclusions:

  • Directed protein evolution is an effective strategy for enhancing CRISPR-Cas9 catalytic efficiency.
  • Engineered Cas9 variants exhibit improved performance for gene editing applications.
  • This approach holds promise for developing next-generation CRISPR-Cas9 systems with tailored properties.