Glucose restriction enhances oxidative fiber formation: A multi-omic signal network involving AMPK and CaMK2.

Skeletal muscle is a highly plastic organ that adapts to different metabolic states or functional demands. This study explored the impact of permanent glucose restriction (GR) on skeletal muscle composition and metabolism. Using Glut4m mice with defective glucose transporter 4, we conducted multi-omics analyses at different ages and after low-intensity treadmill training. The oxidative fibers were significantly increased in Glut4m muscles. Mechanistically, GR activated AMPK pathway, promoting mitochondrial function and beneficial myokine expression, and facilitated slow fiber formation via CaMK2 pathway. Phosphorylation-activated Perm1 may synergize AMPK and CaMK2 signaling. Besides, MAPK and CDK kinases were also implicated in skeletal muscle protein phosphorylation during GR response. This study provides a comprehensive signaling network demonstrating how GR influences muscle fiber types and metabolic patterns. These insights offer valuable data for understanding oxidative fiber formation mechanisms and identifying clinical targets for metabolic diseases.

CRISPR-Cas9-Mediated Cytosine Base Editing Screen for the Functional Assessment of CALR Intron Variants in Japanese Encephalitis Virus Replication.

The Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes viral encephalitis in humans, pigs and other mammals across Asia and the Western Pacific. Genetic screening tools such as CRISPR screening, DNA sequencing and RNA interference have greatly improved our understanding of JEV replication and its potential antiviral approaches. However, information on exon and intron mutations associated with JEV replication is still scanty. CRISPR-Cas9-mediated cytosine base editing can efficiently generate C: G-to-T: A conversion in the genome of living cells. One intriguing application of base editing is to screen pivotal variants for gene function that is yet to be achieved in pigs. Here, we illustrate that CRISPR-Cas9-mediated cytosine base editor, known as AncBE4max, can be used for the functional analysis of calreticulin (CALR) variants. We conducted a CRISPR-Cas9-mediated cytosine base editing screen using 457 single guide RNAs (sgRNAs) against all exons and introns of CALR to identify loss-of-function variants involved in JEV replication. We unexpectedly uncovered that two enriched sgRNAs targeted the same site in intron-2 of the CALR gene. We found that mutating four consecutive G bases in the intron-2 of the CALR gene to four A bases significantly inhibited JEV replication. Thus, we established a CRISPR-Cas9-mediated cytosine-base-editing point mutation screening technique in pigs. Our results suggest that CRISPR-mediated base editing is a powerful tool for identifying the antiviral functions of variants in the coding and noncoding regions of the CALR gene.

Sensitive and Specific Exonuclease III-Assisted Recombinase-Aided Amplification Colorimetric Assay for Rapid Detection of Nucleic Acids.

The development of a contamination-free and on-site nucleic acid detection platform with high sensitivity and specificity but low-cost for the detection of pathogenic nucleic acids is critical for infectious disease diagnosis and surveillance. In this study, we combined the recombinase-aided amplification (RAA) with the exonuclease III (Exo III)-assisted signal amplification into a platform for sensitive and specific detection of nucleic acids of African swine fever virus (ASFV). We found that this platform enabled a naked eye visual detection of ASFV at a detection limit as low as 2 copies/µL in 30 min. As expected, no cross-reactivity was observed with other porcine viruses. In addition, to avoid aerosol contamination, a one-tube RAA-Exo III colorimetric assay was also established for the accurate detection of ASFV in clinical samples. Taken together, we developed a rapid, instrument-free, and low-cost Exo III-assisted RAA colorimetric-assay-based nucleic acid detection platform.

Development of a Naked Eye CRISPR-Cas12a and -Cas13a Multiplex Point-of-Care Detection of Genetically Modified Swine.

The Rapid Visual CRISPR (RAVI-CRISPR) assay employs Cas12a and Cas13a enzymes for precise gene detection in a sample. However, RAVI-CRISPR is limited in single-tube multiplex detection applications due to the lack of specific single-strand (ss) DNA-fluorescently quenched (ssDNA-FQ) and RNA-fluorescently quenched (ssRNA-FQ) reporter cleavage mechanisms. We report the development of a sensitive and specific dual-gene Cas12a and Cas13a diagnostic system. To optimize the application for field testing, we designed a portable multiplex fluorescence imaging assay that could distinguish test results with the naked eye. Herein, dual gene amplified products from multiplex recombinase polymerase amplification (RPA) were simultaneously detected in a single tube using Cas12a and Cas13a enzymes. The resulting orthogonal DNA and RNA collateral cleavage specifically distinguishes individual and mixed ssDNA-FQ and ssRNA-FQ reporters using the green-red-yellow, fluorescent signal conversion reaction system, detectable with portable blue and ultraviolet (UV) light transilluminators. As a proof-of-concept, reliable multiplex RAVI-CRISPR detection of genome-edited pigs was demonstrated, exhibiting 100% sensitivity and specificity for the analysis of CD163 knockout, lactoferrin (LF) knock-in, and wild-type pig samples. This portable naked-eye multiplex RAVI-CRISPR detection platform can provide accurate point-of-care screening of genetically modified animals and infectious diseases in resource-limited settings.

Genome-scale CRISPR screen identifies TRIM2 and SLC35A1 associated with porcine epidemic diarrhoea virus infection.

Porcine epidemic diarrhoea (PED) caused by the porcine epidemic diarrhoea virus (PEDV) is the most devastating disease in the global pig industry due to its high mortality rate in piglets. The host factors critical for PEDV replication are poorly understood. Here, we designed a pooled African green monkey genome-scale CRISPR/Cas9 knockout (VeroCKO) library containing 75,608 single guide RNAs targeting 18,993 protein-coding genes. Subsequently, we use the VeroCKO library to identify key host factors facilitating PEDV infection in Vero E6 cells. Several previously unreported genes associated with PEDV infection are highly enriched post-PEDV selection. We discovered that knocking out the tripartite motif 2 (TRIM2) and the solute carrier family 35 member A1 (SLC35A1) inhibited PEDV replication. Virtual screening and molecular docking approaches showed that chem-80,048,685 (M2) s ignificantly inhibited PEDV attachment and late replication by impeding SLC35A1. Furthermore, we found that knocking out SLC35A1 in Vero E6 cells upregulated a disintegrin and metalloprotease protein-17 (ADAM17) by splicing porcine aminopeptidase N (pAPN) and angiotensin-converting enzyme 2 (ACE2) ectodomains to reduce PEDV-infection in a CMP-Sialic Acid (CMP-SA) cell entry-independent manner. These findings provide a new perspective for a better understanding of host-pathogen interactions and new therapeutic targets for PEDV infection.

Correction: Zhang et al. Genome-Scale CRISPR Knockout Screening Identifies BACH1 as a Key Regulator of Aflatoxin B1-Induced Oxidative Damage. Antioxidants 2022, 11, 1787.

In the original publication [...].

A Simple, Affordable, and Rapid Visual CRISPR-Based Field Test for Sex Determination of Earlier Porcine Embryos and Pork Products.

Sex selection technologies have immensely impacted swine production globally. Conventional earlier embryo sex identification methods require professional technicians and sophisticated laboratory instruments. Rapid on-site gender identification of porcine embryos and pork products remains challenging. In this study, we developed a CRISPR/Cas12a-based fluorescence visualization point-of-care sex determination test that is rapid, accurate and easy to implement on-site. The CRISPR/Cas12a assay coupled with either the polymerase chain reaction (PCR) or loop-mediated isothermal amplification (LAMP) employs precisely designed primers and single-guide RNAs targeting the sex-determining region Y (SRY) and the zinc finger protein X-linked (ZFX) genes. PCR and LAMP amplicons were cleaved with the subsequent generation of fluorescing products detectable with portable blue and ultraviolet light transilluminators. Approximately two copies per microliter of the ZFX and SRY genes were detected using the RApid VIsual CRISPR (RAVI-CRISPR) assay. This method is a sensitive, inexpensive, versatile, and point-of-care test. The technology has other potential applications like determining the sex of diverse livestock species, detecting livestock disease-causing pathogens and evaluating the quality of meat products.

Genome-Scale CRISPR Knockout Screening Identifies BACH1 as a Key Regulator of Aflatoxin B1-Induced Oxidative Damage.

Aflatoxin B1 (AFB1) is amongst the mycotoxins commonly affecting human and animal health, raising global food safety and control concerns. The mechanisms underlying AFB1 toxicity are poorly understood. Moreover, antidotes against AFB1 are lacking. Genome-wide CRISPR/Cas9 knockout screening in porcine kidney cells identified the transcription factor BTB and CNC homolog 1 (BACH1) as a gene required for AFB1 toxicity. The inhibition of BACH1 expression in porcine kidney cells and human hepatoma cells resulted in increased resistance to AFB1. BACH1 depletion attenuates AFB1-induced oxidative damage via the upregulation of antioxidant genes. Subsequently, virtual structural screening identified the small molecule 1-Piperazineethanol, α-[(1,3-benzodioxol-5-yloxy)methyl] -4-(2-methoxyphenyl) (M2) as an inhibitor of BACH1. M2 and its analogues inhibited AFB1-induced porcine and human cell death in vitro, while M2 administration significantly improved AFB1-induced symptoms of weight loss and liver injury in vivo. These findings demonstrate that BACH1 plays a central role in AFB1-induced oxidative damage by regulating antioxidant gene expression. We also present a potent candidate small-molecule inhibitor in developing novel treatments for AFB1 toxicity.

An Inexpensive CRISPR-Based Point-of-Care Test for the Identification of Meat Species and Meat Products.

The growing demand for and supply of meat and meat products has led to a proportional increase in cases of meat adulteration. Adulterated meat poses serious economic and health consequences globally. Current laboratory methods for meat species identification require specialized equipment with limited field applications. This study developed an inexpensive, point-of-care Loop-Mediated Isothermal Amplification (LAMP)-CRISPR/Cas12a colorimetric assay to detect meat species using a Texas Red-labelled single-strand (ssDNA) reporter. As low as 1.0 pg/µL of the porcine NADH4, the chicken NADH dehydrogenase subunit 2 (ND2) and the duck D-loop genes was detectable under white, blue and ultraviolet light. The test turnaround time from DNA extraction to visualization was approximately 40 min. The assay accurately detected pure and mixed-meat products in the laboratory (n = 15) and during a pilot point-of-care test (n = 8) in a food processing factory. The results are 100% reproducible using lateral flow detection strips and the real-time PCR detection instrument. This technology is fully deployable and usable in any standard room. Thus, our study demonstrates that this method is a straightforward, specific, sensitive, point-of-care test (POCT) adaptable to various outlets such as customs, quarantine units and meat import/export departments.

Identification of the CKM Gene as a Potential Muscle-Specific Safe Harbor Locus in Pig Genome.

Genetically modified pigs have shown considerable application potential in the fields of life science research and livestock breeding. Nevertheless, a barrier impedes the production of genetically modified pigs. There are too few safe harbor loci for the insertion of foreign genes into the pig genome. Only a few loci (pRosa26, pH11 and Pifs501) have been successfully identified to achieve the ectopic expression of foreign genes and produce gene-edited pigs. Here, we use CRISPR/Cas9-mediated homologous directed repair (HDR) to accurately knock the exogenous gene-of-interest fragments into an endogenous CKM gene in the porcine satellite cells. After porcine satellite cells are induced to differentiate, the CKM gene promoter simultaneously initiates the expression of the CKM gene and the exogenous gene. We infer preliminarily that the CKM gene can be identified as a potential muscle-specific safe harbor locus in pigs for the integration of exogenous gene-of-interest fragments.

Epigenomics analysis of miRNA cis-regulatory elements in pig muscle and fat tissues.

Although large-scale and accurate identification of cis-regulatory elements on pig protein-coding and long non-coding genes has been reported, similar study on pig miRNAs is still lacking. Here, we systematically characterized the cis-regulatory elements of pig miRNAs in muscle and fat by adopting miRNAomes, ChIP-seq, ATAC-seq, RNA-seq and Hi-C data. In total, the cis-regulatory elements of 257 (85.95%) expressed miRNAs including 226 known and 31 novel miRNAs were identified. Especially, the miRNAs associated with super-enhancers, active promoters, and "A" compartment were significantly higher than those associated by typical enhancers, prompters without H3K27ac, and "B" compartment, respectively. The tissue specific transcription factors were the primary determination of core miRNA expression pattern in muscle and fat. Moreover, the miRNA promoters are more evolutionarily conserved than miRNA enhancers, like other type genes. Our study adds additional important information to existing pig epigenetic data and provides essential resource for future in-depth investigation of pig epigenetics.

A transgene-free method for rapid and efficient generation of precisely edited pigs without monoclonal selection.

Gene-edited pigs for agricultural and biomedical applications are typically generated using somatic cell nuclear transfer (SCNT). However, SCNT requires the use of monoclonal cells as donors, and the time-consuming and laborious monoclonal selection process limits the production of large populations of gene-edited animals. Here, we developed a rapid and efficient method named RE-DSRNP (reporter RNA enriched dual-sgRNA/CRISPR-Cas9 ribonucleoproteins) for generating gene-edited donor cells. RE-DSRNP takes advantage of the precise and efficient editing features of dual-sgRNA and the high editing efficiency, low off-target effects, transgene-free nature, and low cytotoxic characteristics of reporter RNA enriched RNPs (CRISPR-Cas9 ribonucleoproteins), thus eliminating the need for the selection of monoclonal cells and thereby greatly reducing the generation time of donor cells from 3-4 weeks to 1 week, while also reducing the extent of apoptosis and chromosomal aneuploidy of donor cells. We applied RE-DSRNP to produce cloned pigs bearing a deletion edit of the wild-type p53-induced phosphatase 1 (WIP1) gene: among 32 weaned cloned pigs, 31 (97%) carried WIP1 edits, and 15 (47%) were homozygous for the designed fragment deletion, and no off-target event was detected. The WIP1 knockout (KO) pigs exhibited male reproductive disorders, illustrating the utility of RE-DSRNP for rapidly generating precisely edited animals for functional genomics and disease research. RE-DSRNP's strong editing performance in a large animal and its marked reduction in the required time for producing SCNT donor cells support its application prospects for rapidly generating populations of transgene-free cloned animals.

Long-term, multidomain analyses to identify the breed and allelic effects in MSTN-edited pigs to overcome lameness and sustainably improve nutritional meat production.

Beef and mutton production has been aided by breeding to integrate allelic diversity for myostatin (MSTN), but a lack of diversity in the MSTN germplasm has limited similar advances in pig farming. Moreover, insurmountable challenges with congenital lameness and a dearth of data about the impacts of feed conversion, reproduction, and meat quality in MSTN-edited pigs have also currently blocked progress. Here, in a largest-to-date evaluation of multiple MSTN-edited pig populations, we demonstrated a practical alternative edit-site-based solution that overcomes the major production obstacle of hindlimb weakness. We also provide long-term and multidomain datasets for multiple breeds that illustrate how MSTN-editing can sustainably increase the yields of breed-specific lean meat and the levels of desirable lipids without deleteriously affecting feed-conversion rates or litter size. Apart from establishing a new benchmark for the data scale and quality of genome-edited animal production, our study specifically illustrates how gene-editing site selection profoundly impacts the phenotypic outcomes in diverse genetic backgrounds.

Rapid Visual CRISPR Assay: A Naked-Eye Colorimetric Detection Method for Nucleic Acids Based on CRISPR/Cas12a and a Convolutional Neural Network.

Rapid diagnosis based on naked-eye colorimetric detection remains challenging, but it could build new capacities for molecular point-of-care testing (POCT). In this study, we evaluated the performance of 16 types of single-stranded DNA-fluorophore-quencher (ssDNA-FQ) reporters for use with clusters of regularly spaced short palindrome repeats (CRISPR)/Cas12a-based visual colorimetric assays. Among them, nine ssDNA-FQ reporters were found to be suitable for direct visual colorimetric detection, with especially very strong performance using ROX-labeled reporters. We optimized the reaction concentrations of these ssDNA-FQ reporters for a naked-eye read-out of assay results (no transducing component required for visualization). In particular, we developed a convolutional neural network algorithm to standardize and automate the analytical colorimetric assessment of images and integrated this into the MagicEye mobile phone software. A field-deployable assay platform named RApid VIsual CRISPR (RAVI-CRISPR) based on a ROX-labeled reporter with isothermal amplification and CRISPR/Cas12a targeting was established. We deployed RAVI-CRISPR in a single tube toward an instrument-less colorimetric POCT format that required only a portable rechargeable hand warmer for incubation. The RAVI-CRISPR was successfully used for the high-sensitivity detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and African swine fever virus (ASFV). Our study demonstrates this RAVI-CRISPR/MagicEye system to be suitable for distinguishing different pathogenic nucleic acid targets with high specificity and sensitivity as the simplest-to-date platform for rapid pen- or bed-side testing.

Delivery of CRISPR/Cas9 Plasmid DNA by Hyperbranched Polymeric Nanoparticles Enables Efficient Gene Editing.

Gene editing nucleases such as CRISPR/Cas9 have enabled efficient and precise gene editing in vitro and hold promise of eventually achieving in vivo gene editing based therapy. However, a major challenge for their use is the lack of a safe and effective virus-free system to deliver gene editing nuclease elements. Polymers are a promising class of delivery vehicle due to their higher safety compared to currently used viral vectors, but polymers suffer from lower transfection efficiency. Polymeric vectors have been used for small nucleotide delivery but have yet to be used successfully with plasmid DNA (pDNA), which is often several hundred times larger than small nucleotides, presenting an engineering challenge. To address this, we extended our previously reported hyperbranched polymer (HP) delivery system for pDNA delivery by synthesizing several variants of HPs: HP-800, HP-1.8K, HP-10K, HP-25K. We demonstrate that all HPs have low toxicity in various cultured cells, with HP-25K being the most efficient at packaging and delivering pDNA. Importantly, HP-25K mediated delivery of CRISPR/Cas9 pDNA resulted in higher gene-editing rates than all other HPs and Lipofectamine at several clinically significant loci in different cell types. Consistently, HP-25K also led to more robust base editing when delivering the CRISPR base editor "BE4-max" pDNA to cells compared with Lipofectamine. The present work demonstrates that HP nanoparticles represent a promising class of vehicle for the non-viral delivery of pDNA towards the clinical application of gene-editing therapy.

CD8+ T Cells Involved in Metabolic Inflammation in Visceral Adipose Tissue and Liver of Transgenic Pigs.

Anti-inflammatory therapies have the potential to become an effective treatment for obesity-related diseases. However, the huge gap of immune system between human and rodent leads to limitations of drug discovery. This work aims at constructing a transgenic pig model with higher risk of metabolic diseases and outlining the immune responses at the early stage of metaflammation by transcriptomic strategy. We used CRISPR/Cas9 techniques to targeted knock-in three humanized disease risk genes, GIPRdn , hIAPP and PNPLA3I148M . Transgenic effect increased the risk of metabolic disorders. Triple-transgenic pigs with short-term diet intervention showed early symptoms of type 2 diabetes, including glucose intolerance, pancreatic lipid infiltration, islet hypertrophy, hepatic lobular inflammation and adipose tissue inflammation. Molecular pathways related to CD8+ T cell function were significantly activated in the liver and visceral adipose samples from triple-transgenic pigs, including antigen processing and presentation, T-cell receptor signaling, co-stimulation, cytotoxicity, and cytokine and chemokine secretion. The similar pro-inflammatory signaling in liver and visceral adipose tissue indicated that there might be a potential immune crosstalk between the two tissues. Moreover, genes that functionally related to liver antioxidant activity, mitochondrial function and extracellular matrix showed distinct expression between the two groups, indicating metabolic stress in transgenic pigs' liver samples. We confirmed that triple-transgenic pigs had high coincidence with human metabolic diseases, especially in the scope of inflammatory signaling at early stage metaflammation. Taken together, this study provides a valuable large animal model for the clinical study of metaflammation and metabolic diseases.

The advancements, challenges, and future implications of the CRISPR/Cas9 system in swine research.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) genome editing technology has dramatically influenced swine research by enabling the production of high-quality disease-resistant pig breeds, thus improving yields. In addition, CRISPR/Cas9 has been used extensively in pigs as one of the tools in biomedical research. In this review, we present the advancements of the CRISPR/Cas9 system in swine research, such as animal breeding, vaccine development, xenotransplantation, and disease modeling. We also highlight the current challenges and some potential applications of the CRISPR/Cas9 technologies.

Phenotypes of CF rabbits generated by CRISPR/Cas9-mediated disruption of the CFTR gene.

Existing animal models of cystic fibrosis (CF) have provided key insights into CF pathogenesis but have been limited by short lifespans, absence of key phenotypes, and/or high maintenance costs. Here, we report the CRISPR/Cas9-mediated generation of CF rabbits, a model with a relatively long lifespan and affordable maintenance and care costs. CF rabbits supplemented solely with oral osmotic laxative had a median survival of approximately 40 days and died of gastrointestinal disease, but therapeutic regimens directed toward restoring gastrointestinal transit extended median survival to approximately 80 days. Surrogate markers of exocrine pancreas disorders were found in CF rabbits with declining health. CFTR expression patterns in WT rabbit airways mimicked humans, with widespread distribution in nasal respiratory and olfactory epithelia, as well as proximal and distal lower airways. CF rabbits exhibited human CF-like abnormalities in the bioelectric properties of the nasal and tracheal epithelia. No spontaneous respiratory disease was detected in young CF rabbits. However, abnormal phenotypes were observed in surviving 1-year-old CF rabbits as compared with WT littermates, and these were especially evident in the nasal respiratory and olfactory epithelium. The CF rabbit model may serve as a useful tool for understanding gut and lung CF pathogenesis and for the practical development of CF therapeutics.

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

Colostrum 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.