Highly efficient generation of knock-in mice carrying a gene cassette by the cloning-free CRISPR/Cas system. (a) Schematic diagram of pronuclear injection of Cas9 protein, chemically synthesized crRNA and tracrRNA and TetO-FLEX-EGFP-polyA targeting vector. (b) PCR screening of knock-in newborns derived from pronuclear protein injection. (c) Southern blotting of knock-in newborns derived from pronuclear protein injection. The knock-in mice, Protein_KI-#1, 3, 4, and 5, correspond to the newborn mice 1, 5, 8, and 11 in (b), respectively. IF, internal forward primer; IR, internal reverse primer; KI, knock-in; LF, left forward primer; LR, left reverse primer; RF, right forward primer; RR, right reverse primer; M, molecular marker; WT, wild type.