This article investigates the DNA targeting specificity of RNA-guided Cas9 nucleases (SpCas9) in human cells. The study evaluates over 700 guide RNA (sgRNA) variants and SpCas9-induced indel mutation levels at more than 100 predicted genomic off-target loci in 293T and 293FT cells. The findings reveal that SpCas9 tolerates mismatches between guide RNA and target DNA in a sequence-dependent manner, with sensitivity to the number, position, and distribution of mismatches. SpCas9 cleavage is unaffected by DNA methylation, and the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. A web-based software tool is provided to guide the selection and validation of target sequences and off-target analyses. The study also explores the architecture of sgRNA, finding that extending the tracrRNA tail improves SpCas9 activity. sgRNA(+67) and sgRNA(+85) were identified as effective guide RNA architectures. The research further demonstrates that SpCas9 can mediate homology-directed repair without detectable indel formation when used with a catalytic mutant (D10A nickase). SpCas9 is shown to efficiently cleave methylated DNA, indicating its insensitivity to genomic CpG methylation. The study systematically investigates the effect of base-pairing mismatches on target modification efficiency, revealing that the PAM-proximal region is more sensitive to mismatches than the PAM-distal region. The results show that the PAM-distal sequences also contribute to the overall specificity of SpCas9-mediated DNA cleavage. The study also demonstrates that the specificity of SpCas9 is sequence- and locus-dependent, with the PAM-proximal 8–12 bp generally defining specificity, while the PAM-distal sequences also contribute to overall specificity. The study provides guidelines for designing sgRNAs to maximize SpCas9 specificity for editing a particular gene. These guidelines include avoiding sequences followed by 5'-NGG or 5'-NAG PAMs, minimizing global sequence similarity to the target sequence, ensuring at least two mismatches within the PAM-proximal region, and maximizing the number of consecutive or spaced mismatches. The study also shows that the amount of SpCas9 and sgRNA can be titrated to optimize the on- to off-target cleavage ratio. The findings suggest that SpCas9 is a versatile tool for genome editing, with the potential to replace ZFNs and TALENs. The study provides a scoring algorithm to integrate and quantify the contributions of mismatch location, density, and identity on SpCas9 on-target and off-target cleavage. The results and tools further extend the SpCas9 system as a versatile alternative to ZFNs and TALENs for genome editing applications.This article investigates the DNA targeting specificity of RNA-guided Cas9 nucleases (SpCas9) in human cells. The study evaluates over 700 guide RNA (sgRNA) variants and SpCas9-induced indel mutation levels at more than 100 predicted genomic off-target loci in 293T and 293FT cells. The findings reveal that SpCas9 tolerates mismatches between guide RNA and target DNA in a sequence-dependent manner, with sensitivity to the number, position, and distribution of mismatches. SpCas9 cleavage is unaffected by DNA methylation, and the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. A web-based software tool is provided to guide the selection and validation of target sequences and off-target analyses. The study also explores the architecture of sgRNA, finding that extending the tracrRNA tail improves SpCas9 activity. sgRNA(+67) and sgRNA(+85) were identified as effective guide RNA architectures. The research further demonstrates that SpCas9 can mediate homology-directed repair without detectable indel formation when used with a catalytic mutant (D10A nickase). SpCas9 is shown to efficiently cleave methylated DNA, indicating its insensitivity to genomic CpG methylation. The study systematically investigates the effect of base-pairing mismatches on target modification efficiency, revealing that the PAM-proximal region is more sensitive to mismatches than the PAM-distal region. The results show that the PAM-distal sequences also contribute to the overall specificity of SpCas9-mediated DNA cleavage. The study also demonstrates that the specificity of SpCas9 is sequence- and locus-dependent, with the PAM-proximal 8–12 bp generally defining specificity, while the PAM-distal sequences also contribute to overall specificity. The study provides guidelines for designing sgRNAs to maximize SpCas9 specificity for editing a particular gene. These guidelines include avoiding sequences followed by 5'-NGG or 5'-NAG PAMs, minimizing global sequence similarity to the target sequence, ensuring at least two mismatches within the PAM-proximal region, and maximizing the number of consecutive or spaced mismatches. The study also shows that the amount of SpCas9 and sgRNA can be titrated to optimize the on- to off-target cleavage ratio. The findings suggest that SpCas9 is a versatile tool for genome editing, with the potential to replace ZFNs and TALENs. The study provides a scoring algorithm to integrate and quantify the contributions of mismatch location, density, and identity on SpCas9 on-target and off-target cleavage. The results and tools further extend the SpCas9 system as a versatile alternative to ZFNs and TALENs for genome editing applications.