Alteration of sheep coat color pattern by disruption of ASIP gene via CRISPR Cas9

Abstract

Coat color is an important characteristic and economic trait in domestic sheep. Aiming at alteration of Chinese merino sheep coat color by genome manipulation, we disrupted sheep agouti signaling protein gene by CRISPR/Cas9. A total of seven indels were identified in 5 of 6 born lambs. Each targeted lamb happened at least two kinds of modifications, and targeted lambs with multiple modifications displayed variety of coat color patterns. Three lambs with 4 bp deletion showed badgerface with black body coat color in two lambs, and brown coat color with light ventral pigmentation in another one. The black-white spotted color was observed in two lambs with 2 bp deletion. Further analysis unraveled that modifications happened in one or more than two copies of ASIP gene, and moreover, the additional spontaneous mutations of D₉ and/or D₅ preceding the targeting modification could also involve the formation of coat color patterns. Taken together, the entanglement of ASIP modifications by CRISPR/Cas9, spontaneous D₉/D₅ mutations, and ASIP gene duplications contributed to the variety of coat color patterns in targeted lambs.

Figure 1

Schematic of the structure and diagram of sgRNA targeting exon 2 of sheep ASIP gene. Three coding exons and intervening intron sequences are showed in black and white boxes respectively. D₉/N₉ and D₅/N₅ are showed in heather gray, closed boxes. The sgRNA-targeting sequence is underlined and the PAM sequence is indicated in green.

Figure 2

Coat color phenotypes of ASIP gene targeted lambs and primary detection of targeting modification. (A) Differential coat color patterns of ASIP gene targeted lambs. a, Photos of five lambs with ASIP gene targeted. b, Representative lambs of three distinctive coat color patterns. (B) Detection of sgRNA/Cas9 mediated modification by cleavage of ASIP gene with T7EI assay. a, PCR products of the targeting region of ASIP gene amplified by the DNA extracted from lamb tail tissues. b, Electrophoresis of T7EI cleavage assay of PCR products. M, 100 bp DNA ladder marker; Con, control of wild type lamb.

Figure 3

Distinctive coat color patterns and respective ASIP gene modifications of targeted lambs. (A) Targeted lambs with distinctive ASIP gene modifications. GM~ or WT~ numbers were the ear tags of each gene targeted (GM) or wild type (WT) lambs. “−” represents deletion, “+” represents insertion. (B) Photographs of sanger sequencing of each targeted lamb and non-targeted control. (C) Coat color patterns of each ASIP gene targeted lamb (GM081, GM106, GM110, GM109, GM105) and wild type or non-targeted control (WT108 and Control).

Figure 4

Diagram of the spontaneous deletions (D₉D₅ or N₉N₅) and sgRNA/Cas9 targeted modifications of five targeted lambs. “D₉” was designated as spontaneous mutation (versus N₉ as wild type), with 9 bp deletion downstream of ATG start codon. “D₅” was designated as spontaneous mutation (versus N₅ as wild type), with 5 bp deletion downstream of D₉. The sgRNA/Cas9 targeted modifications were illustrated in the right of spontaneous mutation diagram, which showed the forms of modifications, events of in frame or frame shift in translation, and predicted protein products of premature stop or ablation of amino acids. The sgRNA sequence was marked in red and PAM sequence in green.

Figure 5

Varieties of targeted modifications of five targeted lambs. The left listed the targeted and wild type lambs. Each type of modifications was illustrated in the sequence in right of each lamb, which was determined by T-A cloning sequencing. The sgRNA sequence was marked in red and PAM was in green. The targeted modifications and ratios of colonies with modifications in the total colonies were displayed in the right. At least 19 T-A cloning colonies derived from the PCR products of each targeted lamb were sequenced. “−” represented deletion, “+” or “∧” represented insertion. N/N indicated the number of sequences with respective modification to the total sequencing colonies.

Figure 6

Amino acid sequences and primary ASIP protein structure of seven animal species. The alignment of amino acid sequences of seven animal species was listed. Amino acids highlighted in blue and red indicated regions of high local constraint that correspond to the N-terminal domain or C-terminal domain of ASIP protein respectively. Accession numbers for ASIP orthologs (and their species and sources) are P42127 (human, UniProt), Q5UK76 (dog, UniProt), Q29414 (cow, UniProt), A7UGE3(sheep, Uniprot), Q6ZYM3 (pig, UniProt), Q03288 (mouse, UniProt), and Q99JA2 (rat, Uniprot).