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. 2016 Oct 18;11(10):e0164640.
doi: 10.1371/journal.pone.0164640. eCollection 2016.

Disruption of FGF5 in Cashmere Goats Using CRISPR/Cas9 Results in More Secondary Hair Follicles and Longer Fibers

Xiaolong Wang  1 , Bei Cai  1 , Jiankui Zhou  2   3 , Haijing Zhu  4   5 , Yiyuan Niu  1 , Baohua Ma  6 , Honghao Yu  4   5 , Anmin Lei  6 , Hailong Yan  1   4   5 , Qiaoyan Shen  6 , Lei Shi  4   5 , Xiaoe Zhao  6 , Jinlian Hua  6 , Xingxu Huang  2   3 , Lei Qu  4   5 , Yulin Chen  1
Affiliations

Disruption of FGF5 in Cashmere Goats Using CRISPR/Cas9 Results in More Secondary Hair Follicles and Longer Fibers

Xiaolong Wang et al. PLoS One. .

Erratum in

Abstract

Precision genetic engineering accelerates the genetic improvement of livestock for agriculture and biomedicine. We have recently reported our success in producing gene-modified goats using the CRISPR/Cas9 system through microinjection of Cas9 mRNA and sgRNAs targeting the MSTN and FGF5 genes in goat embryos. By investigating the influence of gene modification on the phenotypes of Cas9-mediated goats, we herein demonstrate that the utility of this approach involving the disruption of FGF5 results in increased number of second hair follicles and enhanced fiber length in Cas9-mediated goats, suggesting more cashmere will be produced. The effects of genome modifications were characterized using H&E and immunohistochemistry staining, quantitative PCR, and western blotting techniques. These results indicated that the gene modifications induced by the disruption of FGF5 had occurred at the morphological and genetic levels. We further show that the knockout alleles were likely capable of germline transmission, which is essential for goat population expansion. These results provide sufficient evidences of the merit of using the CRISPR/Cas9 approach for the generation of gene-modified goats displaying the corresponding mutant phenotypes.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
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.
Fig 2
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
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
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
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).
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