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An engineered ScCas9 with broad PAM range and high specificity and activity.

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Researchers engineered new CRISPR-Cas9 variants (Sc++ and HiFi-Sc++) from Streptococcus canis Cas9. These variants offer broad PAM compatibility and high fidelity, expanding CRISPR editing capabilities.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Genetics

Background:

  • CRISPR-Cas9 systems are powerful gene editing tools.
  • Protospacer-adjacent motif (PAM) recognition by Cas9 enzymes limits targetable DNA sequences.
  • Existing Cas9 variants have limitations in PAM compatibility and specificity.

Purpose of the Study:

  • To engineer novel Streptococcus canis Cas9 (ScCas9) variants with enhanced properties.
  • To broaden the protospacer-adjacent motif (PAM) compatibility of Cas9 enzymes.
  • To improve the fidelity and reduce off-target activity of CRISPR-Cas9 gene editing.

Main Methods:

  • Protein engineering by combining motifs from different Cas9 orthologs.
  • Development of two ScCas9 variants: Sc++ and HiFi-Sc++.
  • Assays to evaluate PAM compatibility, DNA cleavage activity, and off-target effects.

Main Results:

  • Engineered Sc++ and HiFi-Sc++ variants exhibit broad 5'-NNG-3' PAM compatibility.
  • Both variants demonstrate robust DNA-cleavage activity.
  • HiFi-Sc++ displays minimal off-target DNA cleavage, indicating higher fidelity.

Conclusions:

  • The developed Sc++ and HiFi-Sc++ variants significantly expand the range of sequences amenable to CRISPR editing.
  • These engineered Cas9 enzymes offer improved specificity and versatility for diverse gene editing applications.
  • The enhanced CRISPR tools facilitate broader adoption in research and biotechnology.