Cloning-free CRISPR/Cas system. (a) Schematic diagram of the in vitro digestion assay with Cas9 protein. The PCR product is the Actb PCR product amplified from wild-type mouse genomic DNA with internal forward and internal reverse primers. (b) The dose-dependency of target digestion by chemically synthesized crRNA/tracrRNA with Cas9 protein (n = 3). RNA concentrations represent the concentration of each crRNA and tracrRNA. (c) The efficiencies of target digestion by Cas9 protein only, Cas9 protein and sgRNA, and Cas9 protein and chemically synthesized crRNA/tracrRNA (n = 4, respectively). Statistical significance was determined by one-way ANOVA and post hoc Tukey-Kramer test. **P < 0.01. M, molecular marker.