The High-Efficiency gRNA-Cas9-Puro plasmid 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-Puro plasmid is provided in both the circular and linear formats. The Puromycin resistant gene is linked by 2A, a self-cleavage peptide, and is expressed separately from Cas9, keeping the maximum activity of Cas9.