Large-scale parallel characterization of RNA-guided nuclease activity and specificity.

As systematic comparisons of editing efficiency and specificity seldom keep pace with rapid developments in RNA-guided nucleases (RGNs), the current study examined 50 such editing systems and characterized the off-target effects and genomic structural impacts of a subset of high-efficiency RGNs. Among them, AsCas12a-Ultra, LbCpf1, and AsCas12a-Plus demonstrated similar or higher efficiency compared to SpCas9, while the relatively high efficiency and small size of enOsCas12f1 together support its suitability for in vivo delivery. AsCpf1-YH and FnCpf1 exhibited the lowest single-guide RNA-dependent (sgRNA-dependent) off-target risks, whereas DpFNuc showed the highest. Genomic structural analysis revealed that enCas12f-HKRA frequently introduces chromosomal translocations, while Cas12j-SF05 poses a lower risk of such mutations. Notably, the high-efficiency RGNs were associated with translocation hotspots. Additionally, enRhCas12f1 and SpaCas12f1 had the lowest cytotoxicity, while enAsCpf1-HF strongly inhibited cell proliferation. This study establishes the first multidimensional performance evaluation framework for RGNs, providing a data-driven tool to support precise genome editing.