HEK293-Cas9 cell line stably expresses Cas9 nuclease, GFP and hygromycin resistance gene. The expression cassette of Cas9 and selection markers is flanked by LoxP sites and is integrated at the human AAVS1 site. The Cas9 nuclease in HEK293-Cas9 cell line has been functionally validated using T7 Endonuclease I assay. In combination with separately transfected sgRNAs, HEK293-Cas9 cell line can be used to efficiently generate targeted genomic modifications including gene knockout, gene knockin, gene mutagenesis, gene tagging etc. It is also an ideal cell line model for sgRNA screening and validation, either individually or in pools.