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

Wang, Xiaolong; Cai, Bei; Zhou, Jianghua; Zhu, Haijing; Niu, Yiyuan; Ma, Baohua; Yu, Haiyang; Lei, Anmin; Yan, Hailong; Shen, Qiaoyan; Shi, Lei; Zhao, Xiaoe; Hua, Jinlian; Huang, Xingxu; Liu, Qu; Chen, Yulin

doi:10.1371/journal.pone.0164640

Cited by 76 publications

(58 citation statements)

References 22 publications

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“…Hematoxylin and eosin (H&E) staining indicated more secondary hair follicles in the skin of BE3 edited goats (Figs B and ). Considering our previous report , these results confirmed that inducing point mutations through BE3 yielded a similar phenotype to site‐directed nuclease gene disruption by DSB and repair.…”

Section: Resultssupporting

confidence: 86%

“…The encoded protein of FGF5 is secreted during the hair growth cycle to signal inhibition of hair growth by a mechanism that blocks dermal papilla cell activation [19]. FGF5 is regarded as the causative gene responsible for the angora phenotype (long hair) in mice [20], and we have previously shown that disruption of FGF5 via CRISPR/Cas9 resulted in longer hair fibers and a 30% increase in cashmere yield per animal [21,22]. In the present study, we demonstrated that the BE3 system can achieve high efficiency single base substitution in FGF5, when introduced by microinjection into single-cell embryos.…”

Section: Introductionmentioning

confidence: 99%

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Base pair editing in goat: nonsense codon introgression into FGF5 results in longer hair

Zhou

et al. 2019

The FEBS Journal

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The ability to alter single bases without homology directed repair (HDR) of double‐strand breaks provides a potential solution for editing livestock genomes for economic traits, which are often multigenic. Progress toward multiplex editing in large animals has been hampered by the costly inefficiencies of HDR via microinjection of in vitro manipulated embryos. Here, we designed sgRNAs to induce nonsense codons (C‐to‐T transitions) at four target sites in caprine FGF5, which is a crucial regulator of hair length in mammals. Initial transfections of the third generation Base Editor (BE3) plasmid and four different sgRNAs into caprine fibroblasts were ineffective in altering FGF5. In contrast, all five progenies produced from microinjected single‐cell embryos had alleles with a targeted nonsense mutation. The effectiveness of BE3 to make single base changes varied considerably based on sgRNA design. In addition, the rate of mosaicism differed between animals, target sites, and tissue type. The phenotypic effects on hair fiber were characterized by hematoxylin and eosin, immunofluorescence staining, and western blotting. Differences in morphology were detectable, even though mosaicism was probably affecting the levels of FGF5 expression. PCR amplicon and whole‐genome resequencing analyses for off‐target changes caused by BE3 were low at a genome‐wide scale. This study provided the first evidence of base editing in large mammals produced from microinjected single‐cell embryos. Our results support further optimization of BEs for introgressing complex human disease alleles into large animal models, to evaluate potential genetic improvement of complex health and production traits in a single generation.

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Section: Resultssupporting

confidence: 86%

Section: Introductionmentioning

confidence: 99%

Base pair editing in goat: nonsense codon introgression into FGF5 results in longer hair

Zhou

et al. 2019

The FEBS Journal

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“…; Wang et al . ). FGF5 was up‐regulated in the L group, indicating that FGF5 expression has a positive effect on wool growth.…”

Section: Resultsmentioning

confidence: 97%

Transcriptomic analysis reveals differentially expressed genes associated with wool length in rabbit

et al. 2018

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Rabbit fur characteristics are primarily genetically determined traits. We used Illumina high-throughput sequencing technology to assess gene expression in the skin tissues of rabbits derived from a cross between Wanxi Angora rabbits and Rex rabbits, which exhibit differential characteristics of short and long wool respectively, to investigate molecular mechanisms related to wool length determination. To identify key regulatory genes involved in rabbit wool length, genes that were differentially expressed between the long- and short-wool rabbits based on a P-value < 0.05 and log |fold change| > 1 were characterized. A total of 798 genes were up-regulated and 523 were down-regulated in the long-wool group compared to expression levels in the short-wool group, and these genes were annotated with GO terms and KEGG pathways, revealing wool-development-related biological functions. The Wnt, Hedgehog and TGF-β signaling pathways, which are related to cell proliferation, fibroblast proliferation and hair follicle regulation respectively, were identified. The expression levels of eight genes were validated by RT-qPCR. In addition, an interaction network was constructed to show the regulatory relationships among the differentially expressed genes. In this study, we found that FGF5, WNT5A, BMP4 and BMP7 showed significant differential expression between the two groups. These transcriptomic profiling results provide comprehensive gene expression information for improving understanding of the molecular mechanisms involved in the growth and development of rabbit wool.

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“…; Wang et al . ). The sequencing data from the F1 animal (#9_F1) further confirmed the high fidelity of germline transmission from edited animals (Fig.…”

Section: Discussionmentioning

confidence: 97%

CRISPR/Cas9‐mediated MSTN disruption and heritable mutagenesis in goats causes increased body mass

et al. 2018

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Genetic engineering in livestock has been greatly enhanced through the use of artificial programmed nucleases such as the recently emerged clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system. We recently reported our successful application of the CRISPR/Cas9 system to engineer the goat genome through micro-injection of Cas9 mRNA and sgRNAs targeting MSTN and FGF5 in goat embryos. The phenotypes induced by edited loss-of-function mutations of MSTN remain to be evaluated extensively. We demonstrate the utility of this approach by disrupting MSTN, resulting in enhanced body weight and larger muscle fiber size in Cas9-mediated gene-modified goats. The effects of genome modifications were further characterized by H&E staining, quantitative PCR, Western blotting and immunofluorescence staining. Morphological and genetic analyses indicated the occurrence of phenotypic and genotypic modifications. We further provide sufficient evidence, including breeding data, to demonstrate the transmission of the knockout alleles through the germline. By phenotypic and genotypic characterization, we demonstrated the merit of using the CRISPR/Cas9 approach for establishing genetically modified livestock with an enhanced production trait.

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

Cited by 76 publications

References 22 publications

Base pair editing in goat: nonsense codon introgression into FGF5 results in longer hair

Base pair editing in goat: nonsense codon introgression into FGF5 results in longer hair

Transcriptomic analysis reveals differentially expressed genes associated with wool length in rabbit

CRISPR/Cas9‐mediated MSTN disruption and heritable mutagenesis in goats causes increased body mass

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