A high-fidelity CRISPR-Cas9 variant, SpCas9-HF1, was developed to reduce off-target effects in genome editing. This variant was engineered with substitutions at four residues (N497A, R661A, Q695A, Q926A) to minimize non-specific DNA interactions. SpCas9-HF1 retains on-target activity comparable to wild-type SpCas9, with over 85% of tested single-guide RNAs (sgRNAs) showing similar efficiency. Importantly, SpCas9-HF1 renders most off-target events undetectable using genome-wide break capture and targeted sequencing methods, even for atypical, repetitive target sites. The study demonstrated that SpCas9-HF1 significantly reduces off-target cleavage across a range of human genes, including EMX1, FANCF, RUNX1, and ZSCAN2. GUIDE-seq analysis showed that SpCas9-HF1 eliminated detectable off-target events for six of seven sgRNAs tested, with only one off-target site remaining. Additionally, SpCas9-HF1 showed no new off-target sites compared to wild-type SpCas9. Targeted deep sequencing confirmed that SpCas9-HF1 induced indel mutations at levels indistinguishable from background in most cases, with only two sites showing detectable mutations. SpCas9-HF1 was also effective against sgRNAs targeting atypical homopolymeric or repetitive sequences, such as those in the VEGFA gene. It significantly reduced off-target cleavage sites, with 123/144 and 31/32 sites not detected for VEGFA site 2 and 3, respectively. These results suggest that SpCas9-HF1 can effectively reduce off-target effects for a wide range of target sequences. Further refinements of SpCas9-HF1, such as SpCas9-HF2, SpCas9-HF3, and SpCas9-HF4, were developed to enhance specificity. These variants showed improved off-target reduction, with SpCas9-HF4 reducing indel mutation frequencies to near background levels for one off-target site. The study highlights the potential of SpCas9-HF1 and its derivatives as highly specific tools for genome editing, with applications in research and therapeutic settings. The findings suggest a general strategy for optimizing the specificity of other RNA-guided nucleases.A high-fidelity CRISPR-Cas9 variant, SpCas9-HF1, was developed to reduce off-target effects in genome editing. This variant was engineered with substitutions at four residues (N497A, R661A, Q695A, Q926A) to minimize non-specific DNA interactions. SpCas9-HF1 retains on-target activity comparable to wild-type SpCas9, with over 85% of tested single-guide RNAs (sgRNAs) showing similar efficiency. Importantly, SpCas9-HF1 renders most off-target events undetectable using genome-wide break capture and targeted sequencing methods, even for atypical, repetitive target sites. The study demonstrated that SpCas9-HF1 significantly reduces off-target cleavage across a range of human genes, including EMX1, FANCF, RUNX1, and ZSCAN2. GUIDE-seq analysis showed that SpCas9-HF1 eliminated detectable off-target events for six of seven sgRNAs tested, with only one off-target site remaining. Additionally, SpCas9-HF1 showed no new off-target sites compared to wild-type SpCas9. Targeted deep sequencing confirmed that SpCas9-HF1 induced indel mutations at levels indistinguishable from background in most cases, with only two sites showing detectable mutations. SpCas9-HF1 was also effective against sgRNAs targeting atypical homopolymeric or repetitive sequences, such as those in the VEGFA gene. It significantly reduced off-target cleavage sites, with 123/144 and 31/32 sites not detected for VEGFA site 2 and 3, respectively. These results suggest that SpCas9-HF1 can effectively reduce off-target effects for a wide range of target sequences. Further refinements of SpCas9-HF1, such as SpCas9-HF2, SpCas9-HF3, and SpCas9-HF4, were developed to enhance specificity. These variants showed improved off-target reduction, with SpCas9-HF4 reducing indel mutation frequencies to near background levels for one off-target site. The study highlights the potential of SpCas9-HF1 and its derivatives as highly specific tools for genome editing, with applications in research and therapeutic settings. The findings suggest a general strategy for optimizing the specificity of other RNA-guided nucleases.