HypaCas9

HypaCas9 is a high-fidelity variant of the Streptococcus pyogenes Cas9 (SpCas9) enzyme designed to improve genome editing specificity. It was developed to reduce off-target DNA cleavage while preserving on-target activity in many contexts, thereby enabling more precise CRISPR-Cas9 editing in cells and model systems.

Conceptually, HypaCas9 achieves higher specificity by adjusting the catalytic activation of Cas9 in response to guide RNA–DNA pairing. The engineered changes strengthen conformational proofreading of matches between the single-guide RNA and the DNA target, making cleavage more selective for perfectly matched sites. The modifications are generally described as altering the recognition and processing steps that occur after initial target binding, reducing activity at mismatched off-target sites without universally abolishing on-target cleavage.

Performance of HypaCas9 has been reported as substantial reductions in off-target cleavage relative to wild-type Cas9 in various cell types and assay systems, with on-target activity maintained for many guides. Off-target effects are typically evaluated with genome-wide methods such as GUIDE-seq or equivalent sequencing-based assays. HypaCas9 is compatible with standard single-guide RNAs and can be used in conjunction with other specificity-enhancing approaches.

Limitations include the possibility of reduced on-target activity for certain guide sequences or targets, requiring optimization of guide design, expression levels, or delivery methods. As with other high-fidelity Cas9 variants, the degree of improvement in specificity can be context-dependent, varying by target site, cell type, and experimental conditions. Nevertheless, HypaCas9 remains one of several engineered high-fidelity Cas9 options used to minimize unintended edits in genome engineering applications.