SpCas9HF1

SpCas9-HF1, also known as SpCas9HF1, is a high-fidelity variant of the Streptococcus pyogenes Cas9 nuclease designed to reduce off-target DNA cleavage while preserving on-target activity. It was developed by researchers led by James Slaymaker and described in 2016 as part of a set of engineered nucleases with improved specificity for CRISPR genome editing.

SpCas9-HF1 achieves higher specificity through four alanine substitutions that weaken non-specific contacts between Cas9 and the DNA backbone: N497A, R661A, Q695A, and Q926A. These substitutions reduce binding to mismatched DNA without completely disrupting recognition of the correct target, thereby enhancing discrimination against off-target sequences.

In performance terms, SpCas9-HF1 typically shows substantially reduced off-target cleavage in a range of cellular contexts, often by several orders of magnitude, while maintaining comparable on-target activity for many guides. Nevertheless, some target sites or guide RNAs may experience reduced on-target editing efficiency, indicating a need for guide optimization, expression level adjustments, or delivery considerations in certain systems.

The variant retains the same SpCas9 PAM requirement (NGG) and general guide RNA format, enabling use with conventional CRISPR vectors and protocols. It has been applied to gene knockout, knock-in, and genome-wide screens in mammalian cells and model organisms. As with other high-fidelity Cas9 tools, validation of on-target activity and comprehensive off-target assessment are recommended for each experimental context.