Generation of knock-in mice carrying a gene cassette by sgRNA combined with Cas9 protein injection. (a) Schematic diagram of pronuclear injection of Cas9 protein, sgRNA, and TetO-FLEX-EGFP-polyA targeting vector. (b,c) PCR screening of knock-in newborns derived from 30 (b) or 100 ng/μl (c) Cas9 protein injection. Note that the size of the IF+IR PCR product in lane 3 in (c) is lower than that of the knock-in PCR product. 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.