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. 2019 Sep 18;10:875. doi: 10.3389/fgene.2019.00875

Figure 3.

Figure 3

Generation of the MC1R-knockout (KO) rabbits with a new coat color using the CRISPR/Cas9 system. (A) Photographs of the MC1R-KO rabbits at 6 months old generated by the CRISPR/Cas9 system; rabbit B15, black; rabbit W12, white; rabbit Y11, yellow; and rabbit Y31 and Y32, pale yellow. Y31 is a female rabbit, and Y32 is male. (B) The mutation was determined in founder rabbits by PCR. M, marker; 1, Y31; 2, Y32; 3, Y11; 4, W12; 5, W13; 6, W14; 7, B15; 8, B16; and 9, B17. The number in red represents the MC1R mutated pups. (C) T-cloning and Sanger sequencing in nine pups with modified MC1R alleles. The two alleles ED and e of parent Lianshan black rabbits (BR) are shown at the top of the targeting sequence. Spontaneous deletions are designated by black dashes, and the corresponding nondeleted bases are shown in blue. Sequences of single-guide RNAs (sgRNAs) are marked in green, the protospacer adjacent motif (PAM) sites are highlighted in red, insertions are highlighted in lowercase red letters, and deletions are designated by green dashes. Deletion: “−”.