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

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.