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.


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

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

Fig4: 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.

Mentions: Next, we tested whether the knock-in efficiency was improved by the cloning-free CRISPR/Cas system. We first injected a mixture of 30 ng/μl Cas9 protein, 0.061 pmol/μl Actb crRNA, 0.061 pmol/μl tracrRNA, and 10 ng/μl targeting vector into the pronuclei of zygotes (Figure 4a). The molar concentrations of dual RNAs were equivalent to that of sgRNA in Figures 1 and 2. We obtained nine newborn mice, but none carried the TetO-FLEX-EGFP-polyA cassette at the Actb locus (Figure S8 and Table S3 in Additional file 1), consistent with the low digest activity at this concentration shown by IDA in vitro (Figure 3b), and the endogenous Actb loci were targeted by NHEJ in three out of eight mice (Figure S9 and Table S3 in Additional file 1). Next, we injected a mixture containing a higher dose of the dual RNAs (30 ng/μl Cas9 protein, 0.61 pmol/μl Actb crRNA, 0.61 pmol/μl tracrRNA, and 10 ng/μl targeting vector) into the pronulei of zygotes. Surprisingly, we found 5 correctly targeted knock-in mice among 11 newborn mice (Figure 4b). The efficiency of the targeted insertion of the transgene by Cas9 protein injection combined with a higher dose of chemically synthesized crRNA and tracrRNA was 45.5% (Table 1). We further confirmed genotypes by Southern blotting with genomic DNA of knock-in newborns (Figure 4c). We also found the endogenous Actb loci were targeted by NHEJ in four out of six non-knock-in mice (Table 1; Figure S10 and Table S3 in Additional file 1). These results suggest that the direct pronuclear delivery of the Cas9 protein-chemically synthesized dual RNAs complex majorly facilitates the generation of knock-in mice carrying a functional gene cassette.Figure 4


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)

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

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

Fig4: 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.
Mentions: Next, we tested whether the knock-in efficiency was improved by the cloning-free CRISPR/Cas system. We first injected a mixture of 30 ng/μl Cas9 protein, 0.061 pmol/μl Actb crRNA, 0.061 pmol/μl tracrRNA, and 10 ng/μl targeting vector into the pronuclei of zygotes (Figure 4a). The molar concentrations of dual RNAs were equivalent to that of sgRNA in Figures 1 and 2. We obtained nine newborn mice, but none carried the TetO-FLEX-EGFP-polyA cassette at the Actb locus (Figure S8 and Table S3 in Additional file 1), consistent with the low digest activity at this concentration shown by IDA in vitro (Figure 3b), and the endogenous Actb loci were targeted by NHEJ in three out of eight mice (Figure S9 and Table S3 in Additional file 1). Next, we injected a mixture containing a higher dose of the dual RNAs (30 ng/μl Cas9 protein, 0.61 pmol/μl Actb crRNA, 0.61 pmol/μl tracrRNA, and 10 ng/μl targeting vector) into the pronulei of zygotes. Surprisingly, we found 5 correctly targeted knock-in mice among 11 newborn mice (Figure 4b). The efficiency of the targeted insertion of the transgene by Cas9 protein injection combined with a higher dose of chemically synthesized crRNA and tracrRNA was 45.5% (Table 1). We further confirmed genotypes by Southern blotting with genomic DNA of knock-in newborns (Figure 4c). We also found the endogenous Actb loci were targeted by NHEJ in four out of six non-knock-in mice (Table 1; Figure S10 and Table S3 in Additional file 1). These results suggest that the direct pronuclear delivery of the Cas9 protein-chemically synthesized dual RNAs complex majorly facilitates the generation of knock-in mice carrying a functional gene cassette.Figure 4

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.