Chromatin presents a significant obstacle to CRISPR-Cas gene editing, as chromatin restricts nuclease access to DNA. Recent advances have produced a wide range of high-fidelity Cas9 and Cas12a variants with enhanced properties. However, their precision in targeting DNA within different contexts remains poorly understood. This gap limits our ability to predict and optimize Cas performance in the dynamic chromatin landscape. To elucidate how chromatin variability impacts Cas editing accuracy, we utilized GEMiNI-seq to systematically profile wild-type and engineered Cas9 and Cas12a nucleases across a range of nucleosome sequences. All nucleases showed reduced cleavage in nucleosomal DNA relative to naked DNA, with the strongest inhibition at dyad-proximal sites. Cleavage within nucleosomes was highly variable, with wtSpCas9 exhibiting up to 65-fold different activity depending on the nucleosome type. Editors with high catalytic activity (wtSpCas9, HIFIv2, LbCas12a ULTRA) consistently outperformed high-fidelity variants such as evoSpCas9, which displayed excellent specificity on naked DNA but poor performance in nucleosomal contexts. ROC and PRC analyses revealed that nucleosome sequence and orientation shape both sensitivity and specificity, with HIFIv1 emerging as the top-performing nuclease for nucleosomal targets, while evoSpCas9 excelled in exposed contexts. Our findings demonstrate that local nucleosome sequence and structure profoundly influence Cas nuclease accessibility and specificity. Variability in cleavage across nucleosome types underscores the need to consider chromatin context during target selection and nuclease design. These results provide a framework for selecting or engineering Cas editors optimized for therapeutic genome editing within chromatin.
