Generation of knock-in mice carrying gene cassette by sgRNA combined with Cas9 mRNA injection. (a) Targeting strategy for the generation of Actb-TetO-FLEX-EGFP-polyA knock-in mice. (b) Schematic diagram of pronuclear injection of Cas9 mRNA, Actb sgRNA and TetO-FLEX-EGFP targeting vector. (c) PCR screening of knock-in newborns derived from pronuclear RNA injection. (d) Sequences of boundaries between Actb and TetO-FLEX-EGFP-polyA cassette. 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; L-HA, left homology arm; R-HA, right homology arm; M, molecular marker; WT, wild type.