The Guide-it CRISPR/Cas9 systems are kits for the cloning and expression of single guide RNAs (sgRNAs) for mammalian genome editing using CRISPR/Cas9 technology. The vectors in these systems simultaneously express Cas9 nuclease, a target-specific sgRNA, and an exceptionally bright fluorescent protein for monitoring transfection efficiency and/or for further enriching/isolating transfected cells by flow cytometry (ZsGreen1 and tdTomato versions are available). Generating a plasmid that expresses a sequence-specific sgRNA with this system is simple: a pair of user-provided oligos corresponding to the target genomic sequence of interest are annealed to form a duplex, and the duplexed oligos are inserted into the pre-linearized vector using the included high-efficiency ligation mix. The kit also includes Stellar Competent Cells to ensure high-efficiency transformation.