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. 2015 Mar 5:5:8841.
doi: 10.1038/srep08841.

Precise in-frame integration of exogenous DNA mediated by CRISPR/Cas9 system in zebrafish

Yu Hisano  1 , Tetsushi Sakuma  2 , Shota Nakade  2 , Rie Ohga  3 , Satoshi Ota  3 , Hitoshi Okamoto  1 , Takashi Yamamoto  2 , Atsuo Kawahara  3
Affiliations

Precise in-frame integration of exogenous DNA mediated by CRISPR/Cas9 system in zebrafish

Yu Hisano et al. Sci Rep. .

Abstract

The CRISPR/Cas9 system provides a powerful tool for genome editing in various model organisms, including zebrafish. The establishment of targeted gene-disrupted zebrafish (knockouts) is readily achieved by CRISPR/Cas9-mediated genome modification. Recently, exogenous DNA integration into the zebrafish genome via homology-independent DNA repair was reported, but this integration contained various mutations at the junctions of genomic and integrated DNA. Thus, precise genome modification into targeted genomic loci remains to be achieved. Here, we describe efficient, precise CRISPR/Cas9-mediated integration using a donor vector harbouring short homologous sequences (10-40 bp) flanking the genomic target locus. We succeeded in integrating with high efficiency an exogenous mCherry or eGFP gene into targeted genes (tyrosinase and krtt1c19e) in frame. We found the precise in-frame integration of exogenous DNA without backbone vector sequences when Cas9 cleavage sites were introduced at both sides of the left homology arm, the eGFP sequence and the right homology arm. Furthermore, we confirmed that this precise genome modification was heritable. This simple method enables precise targeted gene knock-in in zebrafish.

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Figures

Figure 1
Figure 1. Precise integration of the donor vector into the tyr locus.
(a) A schematic representation of the tyrosinase (tyr) locus and the donor vector consisting of the eGFP-gRNA target sequence, homology arms, mCherry and polyA (pA) signal. The try-gRNA target sequences (tyr-gRNA site in red, PAM sequence in blue) and the upstream region of try-gRNA target sequences were integrated between the eGFP-gRNA target sequence and mCherry in the donor vector, while the downstream sequences of try-gRNA target sequences were integrated into the preceding site of eGFP-gRNA target sequences in the donor vector. When the donor vector, gRNAs and Cas9 mRNA were co-injected into 1–2-cell-stage embryos, the tyr gene and mCherry were connected in the same reading frame by precise integration of the donor vector into the genomic locus. (b) Precise integration of exogenous DNA into the targeted genomic locus. Genomic DNAs were prepared from 47 embryos injected with gRNAs, Cas9 mRNA and the donor vector containing homology arms of different lengths (0 to 40 bp). The integration events were assessed by genomic PCR using primers specific to the genomic locus and donor vector (Supplementary Fig. S1). Sequence analysis was performed in five randomly selected embryos among the integrated individuals to determine whether the donor vectors were integrated homology-dependently. (c) Sequence analysis at the 5′ junction of the genome integrated with the donor vector harbouring homology arms.
Figure 2
Figure 2. Precise integration of eGFP into the krtt1c19e locus.
(a) A schematic representation of the krtt1c19e locus and the donor vector consisting of eGFP-gRNA target sequences, homology arms, eGFP and polyA (pA) signal. The krtt1c19e-gRNA was designed to target the vicinity of the stop codon of the krtt1c19e gene. The upstream sequences of the krtt1c19e-gRNA target locus (krtt1c19e-gRNA sites in red, PAM sequence in blue) were inserted between the eGFP-gRNA target sequence and linker sequence on the donor vector, whereas the downstream sequences of the krtt1c19e-gRNA target locus were inserted between the polyA signal and eGFP-gRNA target sequence in the donor vector. When the donor vector, gRNAs and Cas9 mRNA were co-injected into 1–2-cell-stage embryos, the krtt1c19e gene and eGFP were connected in the same reading frame via the linker sequence by precise integration into the targeted genomic locus. (b) The injected embryo showed broad eGFP expression in the epidermis 2 days post-fertilisation (dpf). (c) The eGFP expression level was classified into three groups: broad, intermediate and narrow. Representatives of each expression level are shown in Supplementary Fig. S4. We observed no eGFP expression in embryos injected with the donor vector lacking homology arms. (d) Sequence analysis at the 5′ junction of the genome integrated with the donor vector harbouring homology arms.
Figure 3
Figure 3. Precise genome modification using CRISPR/Cas9 with our donor vector is heritable.
(a) An F1 embryo was obtained by mating wild-type fish with the F0 founder fish injected with eGFP-gRNA, krtt1c19e-gRNA, Cas9 mRNA and the donor vector, and it exhibited eGFP expression in the epidermis at 2 dpf. (b) The numbers of F0 fish screened and of founders bearing eGFP-positive progeny are indicated. (c) Genomic DNA was prepared from the embryo expressing eGFP, and sequence analysis confirmed the precise integration of eGFP into the krtt1c19e locus.
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