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

Aida T, Chiyo K, Usami T, Ishikubo H, Imahashi R, Wada Y, Tanaka KF, Sakuma T, Yamamoto T, Tanaka K - Genome Biol. (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Neuroscience, Medical Research Institute (MRI), Tokyo Medical and Dental University (TMDU), Tokyo, 113-8510, Japan. aida.aud@mri.tmd.ac.jp.

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.

No MeSH data available.


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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4414275&req=5

Fig5: 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).

Mentions: Finally, we tested whether the TetO-FLEX-EGFP-polyA cassette inserted into the mouse Actb locus is effective. We transfected Cre-, tTA-, and DsRed-expressing plasmids into primary mouse fibroblasts derived from ear tips of three F0 knock-in and control littermates (Figure 5a). We found strong EGFP fluorescence only in fibroblasts derived from knock-in mice (Figure 5b). These results suggest that functional EGFP proteins are produced from the TetO-FLEX-EGFP-polyA cassette inserted into the endogenous Actb locus under the presence of Cre and tTA.Figure 5


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

Aida T, Chiyo K, Usami T, Ishikubo H, Imahashi R, Wada Y, Tanaka KF, Sakuma T, Yamamoto T, Tanaka K - Genome Biol. (2015)

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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4414275&req=5

Fig5: 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).
Mentions: Finally, we tested whether the TetO-FLEX-EGFP-polyA cassette inserted into the mouse Actb locus is effective. We transfected Cre-, tTA-, and DsRed-expressing plasmids into primary mouse fibroblasts derived from ear tips of three F0 knock-in and control littermates (Figure 5a). We found strong EGFP fluorescence only in fibroblasts derived from knock-in mice (Figure 5b). These results suggest that functional EGFP proteins are produced from the TetO-FLEX-EGFP-polyA cassette inserted into the endogenous Actb locus under the presence of Cre and tTA.Figure 5

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Neuroscience, Medical Research Institute (MRI), Tokyo Medical and Dental University (TMDU), Tokyo, 113-8510, Japan. aida.aud@mri.tmd.ac.jp.

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.

No MeSH data available.