Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice

Abstract

Although the CRISPR/Cas system has enabled one-step generation of knockout mice, low success rates of cassette knock-in limit its application range. Here we show that cloning-free, direct nuclear delivery of Cas9 protein complex with chemically synthesized dual RNAs enables highly efficient target digestion, leading to generation of knock-in mice carrying a functional cassette with up to 50% efficiency, compared with just 10% by a commonly used method consisting of Cas9 mRNA and single guide RNA. Our cloning-free CRISPR/Cas system facilitates rapid one-step generation of cassette knock-in mice, accelerating functional genomic research by providing various in vivo genetic tools.

Figure 1

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.

Figure 2

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.

Figure 3

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.

Figure 4

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.

Figure 5

Functionality of the reporter cassette inserted at the Actb locus. (a) Schematic diagram of primary fibroblast cultures and transfection of three plasmids. (b) Confocal images of transfected fibroblasts derived from knock-in (KI) mice and their control littermates (wild type, WT).