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. 2014 Jul 16;4:5705.
doi: 10.1038/srep05705.

Highly efficient targeted mutagenesis in one-cell mouse embryos mediated by the TALEN and CRISPR/Cas systems

Akihiro Yasue  1 , Silvia Naomi Mitsui  1 , Takahito Watanabe  2 , Tetsushi Sakuma  3 , Seiichi Oyadomari  4 , Takashi Yamamoto  3 , Sumihare Noji  2 , Taro Mito  2 , Eiji Tanaka  1
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Highly efficient targeted mutagenesis in one-cell mouse embryos mediated by the TALEN and CRISPR/Cas systems

Akihiro Yasue et al. Sci Rep. .
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Abstract

Since the establishment of embryonic stem (ES) cell lines, the combined use of gene targeting with homologous recombination has aided in elucidating the functions of various genes. However, the ES cell technique is inefficient and time-consuming. Recently, two new gene-targeting technologies have been developed: the transcription activator-like effector nuclease (TALEN) system, and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system. In addition to aiding researchers in solving conventional problems, these technologies can be used to induce site-specific mutations in various species for which ES cells have not been established. Here, by targeting the Fgf10 gene through RNA microinjection in one-cell mouse embryos with the TALEN and CRISPR/Cas systems, we produced the known limb-defect phenotypes of Fgf10-deficient embryos at the F0 generation. Compared to the TALEN system, the CRISPR/Cas system induced the limb-defect phenotypes with a strikingly higher efficiency. Our results demonstrate that although both gene-targeting technologies are useful, the CRISPR/Cas system more effectively elicits single-step biallelic mutations in mice.

Figures

Figure 1. Design and application of TALENs targeted to mouse Fgf10.
(a) Fgf10 genomic structure and target sequences of TALENs in the mouse Fgf10 locus. Red and blue sequences indicate the left and right binding sites of Fgf10-TALEN, respectively. (b) Example of the Surveyor endonuclease assay shows cleavage of the PCR amplicons from embryos injected with TALEN mRNAs. Asterisks are the lanes for embryos possessing undisrupted target sequences in the genome. Black arrowhead indicates fragments corresponding to predicted cleavage of the 521-bp amplicon. Red arrowheads indicate products generated from Surveyor nuclease assays. Arrow indicates the 500-bp size marker. (c) F0 embryo with limb defects after injection with TALEN mRNAs (E18.5). Complete limb deficiency was observed in the Fgf10-deficient embryo TALEN_LD-#1. (d) F0 embryo with limb defects after injection with TALEN mRNAs (E13.5). TALEN_LD-#2 showed an abnormal limb development only in the right forelimb (magnified in the right panels).
Figure 2. Sequence information of Fgf10 mutant alleles produced by TALEN.
(a) Sequences obtained from embryos with limb defects or from founder animals generated by microinjection of TALEN mRNAs. DNA sequences to which the TALEN monomers were designed are shown in blue text. Nucleotide mutations (deletions and insertions) are shown in red text. Microhomologous sequences adjacent to the breakpoint are underlined. For F0 embryos with limb defects, TA clones of the PCR products amplified from the genomic DNA were analysed. The total number of examined clones and the number of each genotype are listed at the right side. (b) Wild-type sequences of the Fgf10 protein are shown on the top. Amino acids corresponding to spacer region are highlighted in blue. Predicted consequences of the mutation on the amino acid sequences are highlighted in red. Stop codons are shown as x.
Figure 3. Fgf10 genomic structure and target sequences of CRISPR/Cas system, and examples of embryos produced by CRISPR_143.
(a) Fgf10 genomic structure and target sequences of CRISPR_143, 547 and 563 in the mouse Fgf10 locus. Protospacer adjacent motif (PAM) sequences are highlighted in red. TALEN-binding sites described in Fig. 1 are underlined. (b) Examples of embryos with limb defects (143-#3, #16 and #18) are shown (E16.5). Embryo 143-#3 shows mosaic phenotypes in both hindlimbs. The yellow arrowhead indicates an extra digit in the right forelimb. Embryo 143-#16 with only -14 allele shows the complete limb-deficiency phenotype typical of Fgf10-deficient embryos. Embryo 143-#18 exhibits a small prominence at the right forelimb area (white arrowhead).
Figure 4. Sequence information of Fgf10 mutant alleles produced by CRISPR_143 sgRNA and Cas9 mRNAs.
For F0 embryos, TA clones of the PCR products amplified from each embryo were analysed by DNA sequencing. Total number of clones examined and number of each genotype are listed at the right side. Protospacer and PAM sequences are highlighted in green and shown in blue text, respectively. Boxed sequences represent TALEN-binding sites described in Fig. 1. Microhomologous sequences adjacent to the breakpoint are underlined.
Figure 5. Sequence information of Fgf10 mutant alleles produced by CRISPR_547 and 563 sgRNA and Cas9 mRNAs.
For F0 embryos with limb defects, TA clones of the PCR products amplified from each embryo were analysed by DNA sequencing. The total number of clones examined and the number of each genotype are listed at the right side. Protospacer and PAM sequences are highlighted in green and shown in blue text, respectively. Microhomologous sequences adjacent to the breakpoint are underlined. Positions of β-strands are shown above the corresponding amino acid sequences (β10–β12).
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