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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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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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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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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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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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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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Rationally engineered Cas9 nucleases with improved specificity.

Ian M Slaymaker1, Linyi Gao2, Bernd Zetsche1

  • 1Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Science (New York, N.Y.)
|December 3, 2015
PubMed
Summary
This summary is machine-generated.

Engineered Cas9 variants, called enhanced specificity Cas9 (eSpCas9), significantly reduce unintended DNA cleavage in genome editing. These improved Cas9 tools maintain precise targeting for safer gene editing applications.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Genomics

Background:

  • The RNA-guided endonuclease Cas9 is a key tool for genome editing, enabling targeted DNA modifications.
  • Cas9 enzymes create double-strand breaks (DSBs) at specific genomic locations guided by RNA molecules.
  • A significant limitation of Cas9 is its potential to cleave off-target sites, compromising editing accuracy and safety.

Purpose of the Study:

  • To engineer enhanced specificity Cas9 (eSpCas9) variants derived from Streptococcus pyogenes Cas9 (SpCas9).
  • To rigorously assess the specificity and on-target cleavage activity of eSpCas9 variants in human cells.
  • To address the challenge of off-target cleavage in genome editing applications.

Main Methods:

  • Structure-guided protein engineering was employed to modify SpCas9.
  • Targeted deep sequencing was used to detect Cas9-mediated DNA cleavage at potential off-target sites.
  • Unbiased whole-genome off-target analysis was performed to comprehensively evaluate Cas9 activity across the genome.

Main Results:

  • Engineered eSpCas9 variants demonstrated a significant reduction in off-target DNA cleavage compared to wild-type SpCas9.
  • eSpCas9 variants maintained high on-target cleavage efficiency, ensuring precise editing at desired loci.
  • Both targeted and whole-genome analyses confirmed the enhanced specificity of the eSpCas9 variants.

Conclusions:

  • Enhanced specificity SpCas9 (eSpCas9) variants effectively minimize off-target effects in genome editing.
  • eSpCas9 retains robust on-target cleavage activity, making it a reliable tool for precise gene editing.
  • These engineered Cas9 variants offer improved safety and efficacy for diverse genome-editing applications requiring high specificity.