The article describes the engineering of a type II bacterial CRISPR system to function in human cells using custom guide RNA (gRNA). The authors synthesized a codon-optimized Cas9 protein and expressed it in mammalian cells, along with gRNAs that target specific genomic sequences. They demonstrated that this system can efficiently induce homologous recombination (HR) and non-homologous end joining (NHEJ) events, achieving gene correction rates of up to 38% in different cell types. The study also computed a resource of approximately 190,000 unique gRNAs targeting about 40.5% of human exons, establishing a versatile and robust tool for RNA-guided genome engineering in humans. The results highlight the potential of CRISPR-Cas9 for facilitating gene targeting and multiplexed genome editing in mammalian cells.The article describes the engineering of a type II bacterial CRISPR system to function in human cells using custom guide RNA (gRNA). The authors synthesized a codon-optimized Cas9 protein and expressed it in mammalian cells, along with gRNAs that target specific genomic sequences. They demonstrated that this system can efficiently induce homologous recombination (HR) and non-homologous end joining (NHEJ) events, achieving gene correction rates of up to 38% in different cell types. The study also computed a resource of approximately 190,000 unique gRNAs targeting about 40.5% of human exons, establishing a versatile and robust tool for RNA-guided genome engineering in humans. The results highlight the potential of CRISPR-Cas9 for facilitating gene targeting and multiplexed genome editing in mammalian cells.