RNA-programmed genome editing in human cells enables precise genetic modifications by using RNA-guided Cas9 to induce double-strand DNA breaks. This study demonstrates that Cas9 can assemble with hybrid guide RNAs (sgRNAs) in human cells, leading to targeted DNA cleavage at sites complementary to the guide RNA sequence. The activity requires both Cas9 and the complementary binding of the guide RNA. Experiments show that RNA expression and/or assembly into Cas9 limit DNA cleavage, while extending the RNA sequence at the 3' end enhances DNA targeting in vivo. These findings indicate that RNA-programmed genome editing is a simple and effective strategy for introducing site-specific genetic changes in human cells. The system is more straightforward than existing methods like zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), and could lead to new applications in genome engineering. The study shows that Cas9 can be expressed and localized to the nucleus of human cells, and that it assembles with sgRNA in vivo to generate DNA breaks and stimulate non-homologous end joining (NHEJ) repair. The results demonstrate the feasibility of RNA-programmed genome editing in human cells, with the potential to revolutionize genome engineering in humans and other species with complex genomes. The study also highlights the importance of optimizing sgRNA design, expression levels, and subcellular localization to improve Cas9 function. The system offers distinct advantages due to the simplicity of sgRNA design. The study provides a framework for implementing Cas9 as a facile molecular tool for diverse genome editing applications. The system allows for programming Cas9 with multiple sgRNAs in the same cell, which could enhance targeting efficiency or enable simultaneous targeting of multiple loci. This could have broad applications in genome-wide experiments and large-scale research efforts. The programmable Cas9:sgRNA system offers an inexpensive and rapid mechanism for triggering site-specific genome modification, potentially transforming next-generation genome-scale studies.RNA-programmed genome editing in human cells enables precise genetic modifications by using RNA-guided Cas9 to induce double-strand DNA breaks. This study demonstrates that Cas9 can assemble with hybrid guide RNAs (sgRNAs) in human cells, leading to targeted DNA cleavage at sites complementary to the guide RNA sequence. The activity requires both Cas9 and the complementary binding of the guide RNA. Experiments show that RNA expression and/or assembly into Cas9 limit DNA cleavage, while extending the RNA sequence at the 3' end enhances DNA targeting in vivo. These findings indicate that RNA-programmed genome editing is a simple and effective strategy for introducing site-specific genetic changes in human cells. The system is more straightforward than existing methods like zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), and could lead to new applications in genome engineering. The study shows that Cas9 can be expressed and localized to the nucleus of human cells, and that it assembles with sgRNA in vivo to generate DNA breaks and stimulate non-homologous end joining (NHEJ) repair. The results demonstrate the feasibility of RNA-programmed genome editing in human cells, with the potential to revolutionize genome engineering in humans and other species with complex genomes. The study also highlights the importance of optimizing sgRNA design, expression levels, and subcellular localization to improve Cas9 function. The system offers distinct advantages due to the simplicity of sgRNA design. The study provides a framework for implementing Cas9 as a facile molecular tool for diverse genome editing applications. The system allows for programming Cas9 with multiple sgRNAs in the same cell, which could enhance targeting efficiency or enable simultaneous targeting of multiple loci. This could have broad applications in genome-wide experiments and large-scale research efforts. The programmable Cas9:sgRNA system offers an inexpensive and rapid mechanism for triggering site-specific genome modification, potentially transforming next-generation genome-scale studies.