Integrative analysis of transcriptomic and metabolomic profiles reveal the complex molecular regulatory network of meat quality in Enshi black pigs

CMAS and ST3GAL4 Play an Important Role in the Adsorption of Influenza Virus by Affecting the Synthesis of Sialic Acid Receptors

Influenza A viruses (IAVs) initiate infection by attaching Hemagglutinin (HA) on the viral envelope to sialic acid (SA) receptors on the cell surface. Importantly, HA of human IAVs has a higher affinity for α-2,6-linked SA receptors, and avian strains prefer α-2,3-linked SA receptors, whereas swine strains have a strong affinity for both SA receptors. Host gene CMAS and ST3GAL4 were found to be essential for IAV attachment and entry. Loss of CMAS and ST3GAL4 hindered the synthesis of sialic acid receptors, which in turn prevented the adsorption of IAV. Further, the knockout of CMAS had an effect on the adsorption of swine, avian and human IAVs. However, ST3GAL4 knockout prevented the adsorption of swine and avian IAV and the impact on avian IAV was more distinct, whereas it had no effect on the adsorption of human IAV. Collectively, our findings demonstrate that knocking out CMAS and ST3GAL4 negatively regulated IAV replication by inhibiting the synthesis of SA receptors, which also provides new insights into the production of gene-edited animals in the future.

The Advancements, Challenges and Future Implications of the CRISPR/Cas9 System in Swine Research

CRISPR screening of porcine sgRNA library identifies host factors associated with Japanese encephalitis virus replication

Abstract Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic flavivirus that causes encephalitis and reproductive disorders in mammalian species. However, the host factors critical for its entry, replication, and assembly are poorly understood. Here, we design a porcine genome-scale CRISPR/Cas9 knockout (PigGeCKO) library containing 85,674 single guide RNAs targeting 17,743 protein-coding genes, 11,053 long ncRNAs, and 551 microRNAs. Subsequently, we use the PigGeCKO library to identify key host factors facilitating JEV infection in porcine cells. Several previously unreported genes required for JEV infection are highly enriched post-JEV selection. We conduct follow-up studies to verify the dependency of JEV on these genes, and identify functional contributions for six of the many candidate JEV-related host genes, including EMC3 and CALR. Additionally, we identify that four genes associated with heparan sulfate proteoglycans (HSPGs) metabolism, specifically those responsible for HSPGs sulfurylation, facilitate JEV entry into porcine cells. Thus, beyond our development of the largest CRISPR-based functional genomic screening platform for pig research to date, this study identifies multiple potentially vulnerable targets for the development of medical and breeding technologies to treat and prevent diseases caused by JEV.

Genome editing with the CRISPR‐Cas system: an art, ethics and global regulatory perspective

Enhancing the antibacterial activities of sow milk via site-specific knock-in of a lactoferrin gene in pigs using CRISPR/Cas9 technology

AbstractColostrum quality is a vital factor in mortality and growth performance for piglets. Lactoferrin is an immuno-active milk protein that contributes to the formation of a protective layer above intestinal mucosa, possesses the antibacterial and antiviral activities that are favorable for piglet development. However, there is a notable reduction in lactoferrin in sow milk during lactation after the first few days, which causes many piglets to fail to ingest enough colostrum thereby leading to an increase in piglet mortality. In this study, we successfully constructed genome-edited Large-White pigs with marker-free site-specific knock-in of lactoferrin gene in the 3′-end of Casein alpha-s1 via CRISPR/Cas9 mediated homologous recombination. Thus, the lactoferrin protein can be expressed in the mammary gland in the control of Casein alpha-s1 promoter. As expected, the lactoferrin protein in genetically modified pigs sustained high expression in both colostrum and milk when compared with wild-type pigs. Moreover, the bacterial plate assay indicated that the milk from genetically modified pigs showed bacteriostatic effects when compared with control pigs. Taken together, our study demonstrated that the milk from genetically modified pigs had antibacterial activity which may reduce the costs of veterinary drug and improve the surviving rate of piglets, which is promising for pig breeding.

Identification of ACTB Gene as a Potential Safe Harbor Locus in Pig Genome

Application of CRISPR-Cas12a Enhanced Fluorescence Assay Coupled with Nucleic Acid Amplification for the Sensitive Detection of African Swine Fever Virus

Knockout of glutathione peroxidase 5 down-regulates the piRNAs in the caput epididymidis of aged mice

miR-208b modulating skeletal muscle development and energy homoeostasis through targeting distinct targets

Genome-Wide Patterns of Homozygosity and Relevant Characterizations on the Population Structure in Piétrain Pigs

Investigating the patterns of homozygosity, linkage disequilibrium, effective population size and inbreeding coefficients in livestock contributes to our understanding of the genetic diversity and evolutionary history. Here we used Illumina PorcineSNP50 Bead Chip to identify the runs of homozygosity (ROH) and estimate the linkage disequilibrium (LD) across the whole genome, and then predict the effective population size. In addition, we calculated the inbreeding coefficients based on ROH in 305 Piétrain pigs and compared its effect with the other two types of inbreeding coefficients obtained by different calculation methods. A total of 23,434 ROHs were detected, and the average length of ROH per individual was about 507.27 Mb. There was no regularity on how those runs of homozygosity distributed in genome. The comparisons of different categories suggested that the formation of long ROH was probably related with recent inbreeding events. Although the density of genes located in ROH core regions is lower than that in the other genomic regions, most of them are related with Piétrain commercial traits like meat qualities. Overall, the results provide insight into the way in which ROH is produced and the identified ROH core regions can be used to map the genes associated with commercial traits in domestic animals.

Selective sweep analysis reveals extensive parallel selection traits between large white and Duroc pigs

Evaluation of the effects of sequence length and microsatellite instability on single-guide RNA activity and specificity

Identification of Glyceraldehyde-3-Phosphate Dehydrogenase Gene as an Alternative Safe Harbor Locus in Pig Genome

The ectopic overexpression of foreign genes in animal genomes is an important strategy for gain-of-function study and establishment of transgenic animal models. Previous studies showed that two loci (Rosa26 and pH11) were identified as safe harbor locus in pig genomes, which means foreign genes can be integrated into this locus for stable expression. Moreover, integration of a transgene may interfere with the endogenous gene expression of the target locus after the foreign fragments are inserted. Here, we provide a new strategy for efficient transgene knock-in in the endogenous GAPDH gene via CRISPR/Cas9 mediated homologous recombination. This strategy has no influence on the expression of the endogenous GAPDH gene. Thus, the GAPDH locus is a new alternative safe harbor locus in the pig genome for foreign gene knock-ins. This strategy is promising for agricultural breeding and biomedical model applications.

Japanese Encephalitis Virus infection induces inflammation of swine testis through RIG-I—NF-ĸB signaling pathway

Production of homeobox A10 gene transgenic pigs by somatic cell nuclear transfer

Quantification of allelic differential expression using a simple Fluorescence primer PCR-RFLP-based method

Identifying Selection Signatures for Backfat Thickness in Yorkshire Pigs Highlights New Regions Affecting Fat Metabolism

Identifying the genetic basis of improvement in pigs contributes to our understanding of the role of artificial selection in shaping the genome. Here we employed the Cross Population Extended Haplotype Homozogysity (XPEHH) and the Wright’s fixation index (FST) methods to detect trait-specific selection signatures by making phenotypic gradient differential population pairs, and then attempted to map functional genes of six backfat thickness traits in Yorkshire pigs. The results indicate that a total of 283 and 466 single nucleotide polymorphisms (SNPs) were identified as trait-specific selection signatures using FST and XPEHH, respectively. Functional annotation suggested that the genes overlapping with the trait-specific selection signatures such as OSBPL8, ASAH2, SMCO2, GBE1, and ABL1 are responsible for the phenotypes including fat metabolism, lean body mass and fat deposition, and transport in mouse. Overall, the study developed the methods of gene mapping on the basis of identification of selection signatures. The candidate genes putatively associated with backfat thickness traits can provide important references and fundamental information for future pig-breeding programs.

Genomic Analysis To Identify Signatures of Artificial Selection and Loci Associated with Important Economic Traits in Duroc Pigs

Abstract Identifying genetic basis of domestication and improvement in livestock contributes to our understanding of the role of artificial selection in shaping the genome. Here we used whole-genome sequencing and the genotyping by sequencing approach to detect artificial selection signatures and identify the associated SNPs of two economic traits in Duroc pigs. A total of 38 candidate selection regions were detected by combining the fixation index and the Composite Likelihood Ratio methods. Further genome-wide association study revealed seven associated SNPs that were related with intramuscular fat content and feed conversion ratio traits, respectively. Enrichment analysis suggested that the artificial selection regions harbored genes, such as MSTN, SOD2, MC5R and CD83, which are responsible for economic traits including lean muscle mass, fertility and immunization. Overall, this study found a series of candidate genes putatively associated with the breeding improvement of Duroc pigs and the polygenic basis of adaptive evolution, which can provide important references and fundamental information for future breeding programs.

Identifying artificial selection signals in the chicken genome

sRNAPrimerDB: comprehensive primer design and search web service for small non-coding RNAs

DICER1 regulates antibacterial function of epididymis by modulating transcription of β-defensins

Abstract DICER1 is a key enzyme responsible for the maturation of microRNAs. Recent evidences suggested that DICER1 and microRNAs expressed in epididymis were involved in the control of male fertility. However, the exact mechanism remains to be elucidated. Here, we created a mouse line by targeted disruption of Dicer1 gene in the principal cells of distal caput epididymis. Our data indicated that a set of β-defensin genes were downregulated by DICER1 rather than by microRNAs. Moreover, DICER1 was significantly enriched in the promoter of β-defensin gene and controlled transcription. Besides, the antibacterial ability of the adult epididymis significantly declined upon Dicer1 deletion both in vitro and in vivo. And a higher incidence of reproductive defect was observed in middle-aged Dicer1−/− males. These results suggest that DICER1 plays an important role in transcription of β-defensin genes, which are associated with the natural antibacterial properties in a microRNA-independent manner, and further impacts the male fertility.

A Review of CRISPR-Based Genome Editing: Survival, Evolution and Challenges

CRISPR/Cas9‐mediated genome editing reveals the synergistic effects of β‐defensin family members on sperm maturation in rat epididymis

Expression of tamoxifen-inducible CRE recombinase in Lcn5-CreERT2 transgenic mouse caput epididymis

CRISPR-offinder: a CRISPR guide RNA design and off-target searching tool for user-defined protospacer adjacent motif

Alleviation of Toxicity Caused by Overactivation of P parα through P parα -Inducible miR-181a2

Epididymal Region-Specific miRNA Expression and DNA Methylation and Their Roles in Controlling Gene Expression in Rats

sgRNAcas9: A Software Package for Designing CRISPR sgRNA and Evaluating Potential Off-Target Cleavage Sites

Lcn5 Promoter Directs the Region-Specific Expression of Cre Recombinase in Caput Epididymidis of Transgenic Mice1

An Androgen Receptor-MicroRNA-29a Regulatory Circuitry in Mouse Epididymis

A Newly Identified MicroRNA, mmu-miR-7578, Functions as a Negative Regulator on Inflammatory Cytokines Tumor Necrosis Factor-α and Interleukin-6 via Targeting Egr1 in Vivo

MicroRNA-29a Inhibited Epididymal Epithelial Cell Proliferation by Targeting Nuclear Autoantigenic Sperm Protein (NASP)*

Localization, Expression Change in PRRSV Infection and Association Analysis of the Porcine TAP1 Gene

Discovery of Porcine microRNAs in Multiple Tissues by a Solexa Deep Sequencing Approach