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

## TL;DR

The SpRYc enzyme allows genome editing at more DNA sites than traditional tools but has variable efficiency depending on the cell type and location.

## Contribution

The study reveals the context-dependent performance of the PAM-relaxed nuclease SpRYc across different biological systems.

## Key findings

- SpRYc shows broad PAM compatibility in HEK293 cells but reduced efficiency at canonical NGG sites.
- SpRYc activity in CEM-R5 T cells is mostly limited to NGG PAMs at endogenous loci.
- dSpRYc-VPR has robust activity in Drosophila S2 cells but not in embryos, suggesting DNA-binding stability influences performance.

## Abstract

The CRISPR–Cas9 system has significantly advanced genome editing but remains constrained by its requirement for specific protospacer adjacent motifs (PAMs). To overcome this limitation, PAM-relaxed nucleases, including the novel near-PAMless chimeric SpRYc, have been developed. Here, we evaluated SpRYc editing activity across multiple experimental systems, including human HEK293 and CEM-R5 cells, as well as Drosophila melanogaster S2 cells and embryos. In HEK293 cells, SpRYc exhibited broad PAM compatibility, enabling editing at non-canonical PAMs, albeit with reduced and variable efficiency at canonical NGG sites compared to SpCas9. This context dependency was more pronounced in CEM-R5 T cells, where SpRYc activity at endogenous CXCR4 and B2M loci was largely restricted to NGG PAMs. In contrast, unlike SpCas9, SpRYc displayed negligible genome-editing activity in Drosophila embryos in vivo. Notably, the transcriptional activator dSpRYc-VPR showed robust activity in Drosophila S2 cells at both canonical and non-canonical PAMs. Reduced chromatin occupancy of dSpRYc-VPR suggests a balance between expanded PAM recognition and DNA-binding stability, providing a mechanistic explanation for context-dependent performance of SpRYc. Overall, our results highlight that expanded targeting flexibility comes at the cost of variable efficiency, underscoring the need for extensive locus- and context-specific validation of PAM-relaxed genome-editing tools.