Fig 1.
Evaluation of phenotypic changes in goats using CRISPR/Cas9 gene editing.
(a) Cas9-mediated FGF5-disrupted goats at D160. (The Photo was taken by X. Wang). (b) Staple length between FGF5-disrupted (GM) and WT goats at different growth stages. (c) The length of cashmere between FGF5-disrupted (GM) and WT goats at different growth stages. (d) Differences in cashmere yield between FGF5-disrupted (GM) and WT goats at D120. (e) Differences in the diameter of cashmere fibers between FGF5-disrupted (GM) and WT goats at D120. * p < 0.05, ** p < 0.01, Student's t-test.
Table 1.
Summary of the 19 alive gene-modified animals.
Fig 2.
Genotypes of Cas9-mediated FGF5 modified and WT goats.
(a) PCR products of the targeted region of FGF5 from three founder goats (#19, #41, #84) and two WT goats (#22, #66) at 120 days old. (b) Detection of sgRNA:Cas9-mediated on-target cleavage of FGF5 by using the T7E1 cleavage assay. All PCR products from (a) were subjected to the T7E1 cleavage assay. (c) Sequencing results of modified FGF5 loci detected in goat skins of founders (#19, #41, #84), 9/12 represents 9 out of 12 clones showing the given genotype.
Fig 3.
Morphological analyses of skin tissues from Cas9-mediated FGF5 disrupted and WT goats.
(a) H&E staining shows HF morphology in the skin of an aborted FGF5 gene-modified (MUT) goat and an aborted WT goat. Scale bar = 200 μm. (b) H&E staining shows HF morphology in the skin of FGF5 gene-modified (MUT) (#9) and WT goats at D120. (c) Immunohistochemistry of skin tissues from MUT and WT goats. Scale bar = 200 μm. (d) TEM analyses of HF from the skin of at 120-days old goats. Scale bars: left = 5 μm. (e) Quantitative RT-PCR analysis of FGF5 in the skin of Cas9-mediated (MUT) and WT goats. Data are expressed as the mean ± SD. (f) Western blot analysis using anti-FGF5 and anti-GAPDH (loading control) antibodies.
Fig 4.
Detection of germline transmission in the testis of FGF5-disrupted goats.
(a) PCR products of the targeted region of FGF5 in testis from founder goat #99; (b) Detection of sgRNA:Cas9-mediated on-target cleavage of FGF5 by T7E1 cleavage assay. PCR products from (a) were subjected to T7E1 cleavage assay. (c) Sequencing results of modified FGF5 loci detected in testis. (d) Immunostaining analysis of biopsied testis of the founder (#99) at 120-day-old, confirmed by germ cell specific marker VASA. Germ cells from gonads of founders were stained with an anti-VASA antibody (green) and Hoechst 33342 (blue). VASA positive cells are germ cells. VASA negative cells were set aside as the negative control. Scale bar = 200 μm.
Fig 5.
Germline transmission detection in the germ cells of FGF5-disrupted goats.
(a) PCR products of the targeted region of FGF5 in germ cells (GCs) and blood cells (BCs) from founder goats (#9 and #70) at 120 days old. (b) Detection of sgRNA:Cas9-mediated on-target cleavage of FGF5 by T7E1 cleavage assay. All PCR products from (a) were subjected to T7E1 cleavage assay. (c) Sequencing results of modified FGF5 loci detected in goat germ cells (GCs) and blood cells (BCs).