The study demonstrates that the Cas9 protein from the Type II CRISPR immune system in bacteria can be programmed with single-guide RNAs (sgRNAs) to introduce site-specific double-strand DNA breaks (DSBs) in human cells. The researchers show that Cas9 assembles with sgRNAs in human cells and can induce DSBs at sites complementary to the guide RNA sequence. Experiments using cell extracts indicate that the expression and/or assembly of sgRNAs into Cas9 are limiting factors for DNA cleavage. Additionally, extending the 3' end of the sgRNA enhances DNA targeting activity in vivo. These findings suggest that RNA-programmed genome editing is a straightforward and versatile strategy for introducing site-specific genetic changes in human cells, potentially offering a simpler and more efficient alternative to existing methods like zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs).The study demonstrates that the Cas9 protein from the Type II CRISPR immune system in bacteria can be programmed with single-guide RNAs (sgRNAs) to introduce site-specific double-strand DNA breaks (DSBs) in human cells. The researchers show that Cas9 assembles with sgRNAs in human cells and can induce DSBs at sites complementary to the guide RNA sequence. Experiments using cell extracts indicate that the expression and/or assembly of sgRNAs into Cas9 are limiting factors for DNA cleavage. Additionally, extending the 3' end of the sgRNA enhances DNA targeting activity in vivo. These findings suggest that RNA-programmed genome editing is a straightforward and versatile strategy for introducing site-specific genetic changes in human cells, potentially offering a simpler and more efficient alternative to existing methods like zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs).