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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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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.
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...
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Engineered Staphylococcus auricularis Cas9 with high-fidelity.

Nan Wei1,2, Lu Shang1,2, Jing Liu1,2

  • 1Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China.

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology
|June 30, 2023
PubMed
Summary

Engineered CRISPR-Cas9 variants, SauriCas9-HF1 and SauriCas9-HF2, significantly enhance gene editing specificity. These improved tools reduce off-target effects, expanding CRISPR applications in research and therapy.

Keywords:
CRISPR-Cas9Staphylococcus auricularis Cas9 (SauriCas9)high-fidelityoff-target effects

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Area of Science:

  • Molecular Biology
  • Gene Editing Technologies
  • Biotechnology

Background:

  • CRISPR-Cas9 is a powerful gene editing tool, but off-target effects limit its clinical use.
  • Staphylococcus auricularis Cas9 (SauriCas9) is a small, active Cas9 ortholog with a flexible 5'-NNGG-3' PAM recognition.
  • Enhanced-fidelity SaCas9 (efSaCas9) demonstrated improved specificity through a single mutation.

Purpose of the Study:

  • To engineer SauriCas9 variants with improved specificity by applying mutations from efSaCas9.
  • To evaluate the targeting specificity and off-target effects of engineered SauriCas9 variants.

Main Methods:

  • Protein sequence alignment between SauriCas9 and SaCas9.
  • Engineering of SauriCas9 variants (SauriCas9-HF1, SauriCas9-HF2) with specific mutations.
  • Assessment of targeting specificity using targeted deep sequencing and GUIDE-seq.

Main Results:

  • Two engineered SauriCas9 variants, SauriCas9-HF1 (N269D) and SauriCas9-HF2 (D270N), were successfully created.
  • Both variants demonstrated significantly improved targeting specificity compared to wild-type SauriCas9.
  • SauriCas9-HF2 showed substantial reductions in off-target effects (61.6- to 111.9-fold improvement at certain sites).

Conclusions:

  • Engineered SauriCas9 variants, SauriCas9-HF1 and SauriCas9-HF2, enhance CRISPR-Cas9 specificity.
  • These variants reduce off-target mutations, making them valuable for precise genome editing.
  • The developed SauriCas9 variants expand the CRISPR toolkit for research and therapeutic applications.