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Related Experiment Video

Updated: Apr 6, 2026

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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Efficient fdCas9 Synthetic Endonuclease with Improved Specificity for Precise Genome Engineering.

Mustapha Aouida1, Ayman Eid1, Zahir Ali1

  • 1Laboratory for Genome Engineering, Division of Biological Sciences & Center for Desert Agriculture, 4700 King Abdullah University of Science and Technology, Thuwal, 23955-6900, Kingdom of Saudi Arabia.

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Summary

This study introduces fdCas9, a novel genome editing tool that combines FokI and Cas9. fdCas9 shows improved precision and reduced off-target activity, making it a promising platform for genomic medicine.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Genetics

Background:

  • Cas9 endonuclease is widely used for genome editing but suffers from off-target activity, limiting its use in genomic medicine.
  • Developing precise genome editing tools with minimal off-target effects is crucial for advancing genetic therapies.

Purpose of the Study:

  • To engineer a novel chimeric endonuclease, fdCas9, by fusing the FokI catalytic domain with a catalytically inactive Cas9.
  • To evaluate the genome editing efficiency and specificity of the fdCas9 system.

Main Methods:

  • Generated a synthetic chimeric protein (fdCas9) by combining FokI endonuclease's catalytic domain and catalytically inactive Cas9.
  • Utilized a pair of guide RNAs (gRNAs) to induce dimerization and catalytic activity of fdCas9.
  • Assessed double-strand break (DSB) generation at specific spacer sequences on surrogate reporters and genomic targets.

Main Results:

  • fdCas9 demonstrated enhanced catalytic activity with a spacer range of 15-39 nucleotides.
  • No detectable off-target activity was observed at known Cas9 off-target sites.
  • The fdCas9 system successfully generated DSBs within the targeted spacer sequences.

Conclusions:

  • The fdCas9 endonuclease variant offers improved catalytic activity and specificity compared to traditional Cas9.
  • fdCas9 represents a superior platform for precise genome editing applications in eukaryotic systems, including mammalian cells.
  • This engineered nuclease holds significant potential for advancing genomic medicine and gene therapy.