FIGURE SUMMARY
Title

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

Authors
Hisano, Y., Sakuma, T., Nakade, S., Ohga, R., Ota, S., Okamoto, H., Yamamoto, T., Kawahara, A.
Source
Full text @ Sci. Rep.

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.

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.

Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Sci. Rep.