The article "DNA targeting specificity of RNA-guided Cas9 nucleases" by Hsu et al. (2013) investigates the DNA targeting specificity of the *Streptococcus pyogenes* Cas9 (SpCas9) nuclease in human cells. The study evaluates over 700 guide RNA variants and SpCas9-induced indel mutation levels at more than 100 predicted genomic off-target loci in 293T and 293FT cells. Key findings include: 1. **Sequence-dependent Mismatches**: SpCas9 tolerates mismatches between guide RNA and target DNA in a sequence-dependent manner, sensitive to the number, position, and distribution of mismatches. 2. **DNA Methylation Resistance**: SpCas9-mediated cleavage is unaffected by DNA methylation, and the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modifications. 3. **Optimal Guide RNA Architecture**: The study identifies the optimal guide RNA architecture for SpCas9, which includes a specific length and sequence of the tracrRNA tail to enhance expression and activity. 4. **Off-Target Analysis**: A web-based software tool is provided to guide the selection and validation of target sequences and off-target analyses, facilitating mammalian genome engineering applications. The research demonstrates that SpCas9-mediated DNA cleavage is sequence- and locus-dependent, governed by the quantity, position, and identity of mismatching bases. The study also provides guidelines for maximizing SpCas9 specificity and efficiency in genome editing applications.The article "DNA targeting specificity of RNA-guided Cas9 nucleases" by Hsu et al. (2013) investigates the DNA targeting specificity of the *Streptococcus pyogenes* Cas9 (SpCas9) nuclease in human cells. The study evaluates over 700 guide RNA variants and SpCas9-induced indel mutation levels at more than 100 predicted genomic off-target loci in 293T and 293FT cells. Key findings include: 1. **Sequence-dependent Mismatches**: SpCas9 tolerates mismatches between guide RNA and target DNA in a sequence-dependent manner, sensitive to the number, position, and distribution of mismatches. 2. **DNA Methylation Resistance**: SpCas9-mediated cleavage is unaffected by DNA methylation, and the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modifications. 3. **Optimal Guide RNA Architecture**: The study identifies the optimal guide RNA architecture for SpCas9, which includes a specific length and sequence of the tracrRNA tail to enhance expression and activity. 4. **Off-Target Analysis**: A web-based software tool is provided to guide the selection and validation of target sequences and off-target analyses, facilitating mammalian genome engineering applications. The research demonstrates that SpCas9-mediated DNA cleavage is sequence- and locus-dependent, governed by the quantity, position, and identity of mismatching bases. The study also provides guidelines for maximizing SpCas9 specificity and efficiency in genome editing applications.