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Updated: Jan 13, 2026

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The Chimeric Nuclease SpRYc Exhibits Highly Variable Performance Across Biological Systems.

Irina O Deriglazova1, Mikhail V Shepelev2, Natalia A Kruglova2

  • 1Department of the Control of Genetic Processes, Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilova Str., Moscow 119334, Russia.

International Journal of Molecular Sciences
|January 10, 2026
PubMed
Summary

The novel SpRYc CRISPR system offers broader PAM targeting but shows variable editing efficiency across different cell types and organisms. Locus-specific validation is crucial for these PAM-relaxed genome editing tools.

Keywords:
B2MCCR5CRISPR/Cas9CXCR4Drosophila melanogasterSpCas9SpRYcdSpRYc-VPRgenome editingtranscription regulation

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Area of Science:

  • Molecular Biology
  • Genome Engineering
  • Biotechnology

Background:

  • CRISPR-Cas9 genome editing is powerful but limited by protospacer adjacent motif (PAM) requirements.
  • PAM-relaxed nucleases, like SpRYc, aim to expand targeting capabilities.
  • Understanding SpRYc's performance across diverse systems is essential for its application.

Purpose of the Study:

  • To evaluate the genome editing activity and PAM compatibility of the SpRYc nuclease.
  • To compare SpRYc performance in human cell lines and *Drosophila melanogaster*.
  • To investigate the mechanistic basis for SpRYc's context-dependent activity.

Main Methods:

  • Assessed SpRYc editing efficiency in HEK293, CEM-R5 T cells, and *Drosophila* S2 cells and embryos.
  • Utilized CRISPR-Cas9 and SpRYc nucleases for genome editing experiments.
  • Employed transcriptional activator dSpRYc-VPR and analyzed chromatin occupancy.

Main Results:

  • SpRYc demonstrated broad PAM compatibility in HEK293 cells but with reduced efficiency at canonical sites compared to SpCas9.
  • SpRYc activity was context-dependent, primarily targeting NGG PAMs in CEM-R5 T cells.
  • SpRYc showed negligible activity in *Drosophila* embryos, while dSpRYc-VPR was active in S2 cells.
  • Reduced chromatin occupancy of dSpRYc-VPR suggests a trade-off between PAM recognition and DNA-binding stability.

Conclusions:

  • Expanded PAM targeting flexibility with SpRYc comes at the cost of variable editing efficiency.
  • SpRYc's performance is highly dependent on the specific locus and cellular context.
  • Extensive validation is required for PAM-relaxed genome editing tools before application.