No abstract

The cashmere goat breed is known to provide excellent quality cashmere. Here, we attempted to breed high-yielding cashmere goats by specifically inserting the Tβ4 gene into the goat CCR5 locus and provided an animal model for future research. We successfully obtained Tβ4 knock-in goat without any screening and fluorescent markers using CRISPR/Cas9 technology. A series of experiments were performed to examine physical conditions and characteristics of the Tβ4 knock-in goat. The goat exhibited an increase in cashmere yield by 74.5% without affecting the fineness and quality. Additionally, RNA-seq analysis indicated that Tβ4 may promote hair growth by affecting processes such as vasoconstriction, angiogenesis, and vascular permeability around secondary hair follicles. Together, our study can significantly improve the breeding of cashmere goat and thereby increase economic efficiency.

Recent advances in understanding the CRISPR/Cas9 system have provided a precise and versatile approach for genome editing in various species. However, no study has reported simultaneous knockout of endogenous genes and site-specific knockin of exogenous genes in large animal models. Using the CRISPR/Cas9 system, this study specifically inserted the fat-1 gene into the goat MSTN locus, thereby achieving simultaneous fat-1 insertion and MSTN mutation. We introduced the Cas9, MSTN knockout small guide RNA and fat-1 knockin vectors into goat fetal fibroblasts by electroporation, and obtained a total of 156 positive clonal cell lines. PCR and sequencing were performed for identification. Of the 156 clonal strains, 40 (25.6%) had simultaneous MSTN knockout and fat-1 insertion at the MSTN locus without drug selection, and 55 (35.25%) and 101 (67.3%) had MSTN mutations and fat-1 insertions, respectively. We generated a site-specific knockin Arbas cashmere goat model using a combination of CRISPR/Cas9 and somatic cell nuclear transfer for the first time. For biosafety, we mainly focused on unmarked and non-resistant gene screening, and point-specific gene editing. The results showed that simultaneous editing of the two genes (simultaneous knockout and knockin) was achieved in large animals, demonstrating that the CRISPR/Cas9 system has the potential to become an important and applicable gene engineering tool in safe animal breeding.

Increased cashmere yield and improved quality are some goals of cashmere goat breeding. Thymosin beta-4 (Tβ4) plays a key role in the growth and development of hair follicles. For the past ten years, we have evaluated the role of Tβ4 by establishing a flock of 15 cashmere goats that specifically overexpress the Tβ4 gene in the hair follicles. These Tβ4 overexpression (Tβ4-OE) cashmere goats had more secondary hair follicles than the WT goats and produced more cashmere. Meanwhile, combined analysis of the skin transcriptome and proteome in cashmere goats suggested that Tβ4 may affect hair growth by interacting with keratin type II cytoskeletal 4 epidermal (KRT4) to mediate the extracellular signal-regulated protein kinase (ERK) signaling pathway, thereby promoting the development of secondary hair follicles, and consequently, increasing cashmere yield. Thus, the specific overexpression of Tβ4 in the hair follicles of cashmere goats effectively increased the cashmere yield.

Objective Non-small-cell lung cancer (NSCLC) accounts for >85% of lung cancers, and its incidence is increasing. We explored expression differences between NSCLC and normal cells and predicted potential target sites for detection and diagnosis of NSCLC. Methods Three microarray datasets from the Gene Expression Omnibus database were analyzed using GEO2R. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis were conducted. Then, the String database, Cytoscape, and MCODE plug-in were used to construct a protein–protein interaction (PPI) network and screen hub genes. Overall and disease-free survival of hub genes were analyzed using Kaplan-Meier curves, and the relationship between expression patterns of target genes and tumor grades were analyzed and validated. Gene set enrichment analysis and receiver operating characteristic curves were used to verify enrichment pathways and diagnostic performance of hub genes. Results In total, 293 differentially expressed genes were identified and mainly enriched in cell cycle, ECM–receptor interaction, and malaria. In the PPI network, 36 hub genes were identified, of which 6 were found to play significant roles in carcinogenesis of NSCLC: CDC20, ECT2, KIF20A, MKI67, TPX2, and TYMS. Conclusion The identified target genes can be used as biomarkers for the detection and diagnosis of NSCLC.