Location: Plant Genetics Research
2023 Annual Report
Objectives
Objective 1. Using gene editing techniques, systematically alter the DNA sequence predicted to cause beneficial changes in pig production and to test these changes in vitro and, where warranted, in vivo.
Sub-objective 1.A. Establish porcine cell lines that will be important for phenotyping various agriculture traits while identifying candidate gene loci to evaluate effects of variants on these cell lines.
Sub-objective 1.B. Characterize the range of phenomena of known variants identified by porcine QTL data, differentially expressed gene analysis, and known variants from other species in gene edited cells.
Sub-objective 1.C. Produce genetically modified pigs to validate in vivo effects of improved alleles.
Objective 2. Test the effects of sequence modifications for on- and off-target effects.
Sub-objective 2.A. Explore the application of Cas9 inhibitors or new editing systems to reduce off-site cleavage events.
Sub-objective 2.B. Modification of guide RNA to diminish unintended cleavage events.
Objective 3. Develop efficient methods for gene modification of poultry.
Sub-objective 3.A. Establish methods for producing genetically modified poultry.
Sub-objective 3.B. Identify candidate genes that are associated with beneficial changes in poultry production and test genetic modification(s) in vitro to characterize variant effects on cellular phenotypes.
Sub-objective 3.C. Produce genetically modified poultry to validate in vivo effects of improved alleles.
Approach
By using gene editing techniques, we will systematically alter DNA sequences predicted to cause beneficial changes in pig and poultry production and test these changes in vitro and in vivo where warranted. In addition, we will comprehensively test effects of sequence modifications for on- and off-target effects. Gene editing and other technologies will be used to systematically alter DNA sequence in ways that were predicted to improve traits to (1) determine whether the sequence variation is causal for the change in the trait, and (2) determine any other changes in traits that might simultaneously occur through pleiotropy effects. The research will focus on both pigs and poultry, and the goal will be to develop pigs and poultry with improved traits of interest, without having deleterious effects on animal production. Simultaneously, we will work toward understanding relationships between genes and various physiological functions within both pigs and poultry. Finally, we will refine editing methods for poultry production to increase efficiency of this process. Together, the information gleaned from this project will facilitate genetic gain, improve animal welfare, increase sustainability, and directly improve production efficiency of swine and poultry and will likely be valuable to humans and other livestock species.
Progress Report
In support of Objective 1, Sub-objective 1.A, multiple porcine primary cell cultures have been created for studying agriculturally important traits. In addition to fibroblasts and fetal fibroblasts, porcine intestinal and liver organoids have been established in our lab. Primary hepatocyte, tracheal epithelium, lung epithelium, and intestinal epithelium have also been established. New methods to immortalize epithelium cells are being investigated. Multiple immortalized cell lines have been purchased from American Type Culture Collection (ATCC): a muscle satellite cell line, porcine kidney (PK-15), a lung alveolar cell line (3D4/21), and an intestinal epithelium cell line (IPEC-J2 from ARS researchers in Ames, Iowa). Primary and secondary candidate gene target lists have been created and genes associated with disease resistance have been prioritized along with muscle quality, milk yield and composition, and heat stress. In support of Objective 1, Sub-objective 1.B, an immortalized porcine kidney cell line (PK-15, ATCC) has been propagated and transfected with the CRISPR/Cas9 system to produce genetically modified cells that will be used for an in vitro viral challenge (in collaboration with Dr. Alex Buckley in Ames, Iowa). Additionally, genetically modified intestinal organoids have been produced and will also be challenged with a virus of interest in collaboration with researchers at the University of Missouri. CRISPRs have been designed to target three other genes that may be important for disease resistance and are ready to be transfected into cells to test for infectivity by using multiple viruses. In support of Objective 1, Sub-objective 1.C, microinjection of the CRISPR/Cas9 into oocytes has been accomplished. The gene edited embryos were transferred to a surrogate which is currently pregnant. Once the surrogate has farrowed, the piglets will be genotyped and if they contain useful genome edits, they will be challenged with the virus of interest by ARS researchers in Ames, Iowa, once weaned.
In support of Objective 2, Sub-objective 2A, a whole genome sequencing (WGS) pipeline was established to identify potential off-targeting sites on a global scale in previously created genome edited pigs. WGS was performed on six immunoglobulin heavy chain knockout pigs as the guide ribonucleic acid (gRNA) was known to produce off-targeting events. Cas-OFFinder software was used to identify potential off-targeting sites in the genome, and the two genes with previously confirmed off-targeting events were detected in these pigs. After filtering and eliminating variation by aligning with the paternal genome, five additional potential off-target sites were identified and are currently being validated. In addition to establishing a global off-target identification pipeline, anti-CRISPR proteins (ACRs) have been synthesized and injected into embryos to significantly diminish levels of unwanted mutations in embryos. Promising results were obtained, and further experiments are being conducted to validate the results.
In support of Objective 3, Sub-objective 3.A, a breeding flock of white leghorn have been established, and primordial germ cells (PGCs) have begun to be cultured (in collaboration with Dr. Hongjo Lee, University of Missouri) for optimizing the PGC-mediated method of genome editing in chickens. For Sub-objective 3.B, primary and secondary candidate gene lists have been created, which primarily focus on disease resistance, meat quality, and nutrient utilization. Furthermore, primary fibroblast, preadipocyte, tracheal epithelium cultures have been established from white leghorn embryos, and myoblast and preadipocyte cultures have been established from broiler embryos. A candidate gene for a viral receptor was tested by altering a chicken fibroblast cell line. Five predicted knockout cell lines were further tested by viral infection. Experiments are underway to validate the findings. Similarly, gRNAs for another viral target have been validated, and transfections in DT-40 chicken B cells have been started. Lastly, retinoic acid-inducible gene I (RIG-I) is a cytosolic receptor that detects viral RNAs, and the gene is absent in the chicken and other Gallinaceous genomes. RIG-I was knocked out in duck fibroblasts (non-Gallinaceous) to determine its effects on the immune response to a viral mimic.
Accomplishments
1. Identification of a duck antiviral gene for possible use to improve chicken resistance to viruses. Ducks and other waterfowl serve as natural reservoirs for avian influenza viruses and typically display mild symptoms of infection. As part of the innate immune response, retinoic acid-inducible gene I (RIG-I) is a ribonucleic acid (RNA) helicase that senses short viral double-stranded RNAs, within the cytoplasm, to induce the production of type I interferons and is present in the duck genome but not in the chicken genome. ARS researchers in Columbia, Missouri, knocked out RIG-I in duck cells and challenged the cells with a strain of avian influenza. The cells showed a reduced antiviral response, indicating that RIG-I is essential for viral surveillance. Since it is not present in chickens, the duck RIG-1 gene could be a useful for improving immune responses in chickens by generating a transgenic chicken. These chickens could be incorporated into production flocks and would have an improve innate immune system that could respond to pathogens.
2. Porcine intestinal and liver organoid cultures for use in testing gene editing in pigs. Infectious diseases in pigs account for billions of dollars in economic loss, cause animal welfare concerns, and are a threat to our food supply. Methods to identify genes that play a role in virus and bacterial infectivity require animals to be produced and challenged with a pathogen, which is expensive and time consuming. ARS researchers in Columbia, Missouri, successfully established organoid cultures for intestine and liver. The establishment of organoid systems mimic the live animal tissue more closely than individual or combined cell cultures. The organoid cultures can model viral and bacterial infectivity in a culture dish without doing an animal challenge. Therefore, these organoids provide a less expensive screening tool to identify important genes involved in pathogen resistance or infectivity. This innovation will accelerate the development of pigs that contain beneficial gene edits for disease by allowing the inexpensive rapid screening of possible changes.
Review Publications
Chen, P.R., Uh, K., Monarch, K., Spate, L., Reese, E., Prather, R., Lee, K. 2023. Inactivation of growth differentiation factor 9 blocks folliculogenesis in pigs. Biology of Reproduction. 108(4):611-618. https://doi.org/10.1093/biolre/ioad005.
Whitworth, K.M., Green, J.A., Redel, B.K., Geisert, R.D., Lee, K., Telugu, B.P., Wells, K.D., Prather, R.S. 2022. Improvements in pig agriculture through gene editing. CABI Agriculture and Bioscience (CABI A&B). 3. Article 41. https://doi.org/10.1186/s43170-022-00111-9.
Gabriel, G., Devine, W., Redel, B.K., Whitworth, K., Samuel, M., Spate, L., Cecil, R., Prather, R., Wu, Y., Wells, K., Lo, C. 2022. Profiling development of abdominal organs in the pig. Scientific Reports. 12. Article number 16245. https://doi.org/10.1038/s41598-022-19960-5.
Geisert, R.D., Johns, D.N., Pfeiffer, C.A., Sullivan, R.M., Lucas, C.G., Simintiras, C.A., Redel, B.K., Wells, K.D., Spencer, T.E., Prather, R.S. 2022. Gene editing provides a tool to investigate genes involved in reproduction of pigs. Molecular Reproduction and Development. 1-10. https://doi.org/10.1002/mrd.23620.
