The High-Efficiency gRNA-Cas9-GFP plasmid (linear) Assembly Kit is used for easier and highly efficient cloning of gRNA fragments into Cas9 vectors. This vector has been constructed by integrating a suicide reporter gene into the gRNA insert site. Under the selection of this suicide report gene, almost 100% of clones are positive clones without screening. The High-Efficiency gRNA-Cas9-GFP plasmid is provided in both the circular and linear formats. The linear format allows omission of the digestion and purification process, which saves time. The GFP fluorescence reporter is linked by 2A, a self-cleavage peptide, and is expressed separately from Cas9, keeping the maximum activity of Cas9.