HEK293T-SpCas9-HF1 cell line stably expresses high-fidelity SpCas9-HF1 nuclease, GFP and hygromycin resistance gene. SpCas9-HF1 is integrated at the human AAVS1 site. The SpCas9-HF1 nuclease in HEK293T-SpCas9-HF1 cell line has been functionally validated using T7 Endonuclease I assay. In combination with separately transfected sgRNAs, HEK293T-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.