A study published in *Nature Biotechnology* (2014) investigates the genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Researchers mapped the binding sites of a catalytically inactive Cas9 (dCas9) loaded with single guide RNAs (sgRNAs) in mouse embryonic stem cells (mESCs). They found that dCas9 binds to specific genomic regions defined by a 5-nucleotide seed sequence in the sgRNA and a protospacer adjacent motif (PAM) NGG. However, chromatin accessibility significantly influences dCas9 binding, with many off-target sites located near genes. The study also reveals that while dCas9 can bind to many sites, only a small fraction undergoes cleavage, suggesting that the binding and cleavage processes are distinct. The findings highlight the importance of seed sequence and chromatin accessibility in determining Cas9 target specificity. The research provides insights into the mechanisms of Cas9 binding and cleavage, and suggests that off-target binding may be a significant factor in CRISPR-based applications. The study also demonstrates that the seed sequence influences sgRNA abundance and specificity, and that the use of multiple sgRNAs can help reduce off-target effects. Overall, the study underscores the complexity of Cas9 target recognition and the need for careful design of sgRNAs to minimize off-target effects in CRISPR-based applications.A study published in *Nature Biotechnology* (2014) investigates the genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Researchers mapped the binding sites of a catalytically inactive Cas9 (dCas9) loaded with single guide RNAs (sgRNAs) in mouse embryonic stem cells (mESCs). They found that dCas9 binds to specific genomic regions defined by a 5-nucleotide seed sequence in the sgRNA and a protospacer adjacent motif (PAM) NGG. However, chromatin accessibility significantly influences dCas9 binding, with many off-target sites located near genes. The study also reveals that while dCas9 can bind to many sites, only a small fraction undergoes cleavage, suggesting that the binding and cleavage processes are distinct. The findings highlight the importance of seed sequence and chromatin accessibility in determining Cas9 target specificity. The research provides insights into the mechanisms of Cas9 binding and cleavage, and suggests that off-target binding may be a significant factor in CRISPR-based applications. The study also demonstrates that the seed sequence influences sgRNA abundance and specificity, and that the use of multiple sgRNAs can help reduce off-target effects. Overall, the study underscores the complexity of Cas9 target recognition and the need for careful design of sgRNAs to minimize off-target effects in CRISPR-based applications.