Project : USDA ARS

ARS Home » Midwest Area » Columbia, Missouri » Plant Genetics Research » Research » Research Project #443341

Research Project: Validate Causative Mutations in Agriculturally-Important Vertebrates

2025 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
This research benefits U.S. livestock and poultry producers as well as the broader industry by providing advanced genetics-based methods to improve economically relevant animal health and production traits. Key characteristics such as feed efficiency, growth, and disease resistance in pigs and poultry can be enhanced while minimizing off-target effects often associated with gene editing technology, ultimately contributing to a more sustainable food supply and helping the U.S. to meet growing food demands. In support of Objective 1, Sub-objective 1.A, immortalized cell cultures from pig lung, trachea, muscle, spleen, and kidney have been established. These cell cultures will greatly enhance the ability to study and modify genes related to important agriculture traits such as disease resistance, feed efficiency, and growth. Currently, muscle cell culture conditions are being optimized for the transition of young pig muscle cells into a more mature muscle cell type. Given the importance of pigs in agriculture and the physiological similarities between pigs and humans, the mature muscle cells can function as a robust model to study various biological processes related to muscle development, metabolism, pathology, and in gene editing studies using clustered regularly interspaced palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9) gene editing. In addition to the work described above, to support Objective 1, Sub-objective 1.A, the production of gene-edited animals requires the ability to produce and grow embryos outside the body for a certain period of time. Systems for producing both pig and cow embryos in the laboratory have been successfully established. Methods to enhance the system are currently being explored in an effort to increase the number of high-quality embryos produced, which will ultimately make the production of gene edited animals more efficient. In support of Objective 1, Sub-objective 1.B, eight different genes have been modified in either bovine or porcine cell cultures or special lab grown mini organs, called organoids. This allows researchers to assess whether specific genetic changes are linked to beneficial agricultural traits. Currently, two genes that play key roles in pathogen infectivity have been modified in cattle cells, resulting in reduced susceptibility to infection by their respective pathogens. Additionally, six genes have been modified in pig cells to study how these genetic changes affect susceptibility to influenza and coronaviruses. Two other genes have been modified in pig cell cultures to produce pigs with improved feed efficiency. In support of Objective 1, Sub-objective 1.C, gene-edited embryos have been transferred to recipient pigs to produce piglets with improved feed efficiency and growth. In support of Objective 2, Sub-objective 2.A, USDA researchers in Columbia, Missouri, used an anti-CRISPR protein last year as a method to decrease unwanted off-targeting events in pig embryos. This year, we demonstrated this same anti-CRISPR protein could control how many copies of a gene got edited in each embryo. To verify that this approach is safe, the gene edited embryos were transferred into a recipient to illustrate they can develop normally and are capable of surviving. In support of Objective 2, Sub-objective 2.B, modified single guide ribonucleic acids (sgRNAs) were designed to potentially decrease the number of unwanted changes that can sometimes occur when using the CRISPR-Cas9 gene editing system. However, after multiple attempts of gene editing with these modified guides, there was no clear reduction in off-target events observed. Therefore, an alternative strategy was explored to see if other versions of Cas9 could be used to decrease unwanted targeting events. This attempt successfully identified a Cas9 variant, that when injected into pig embryos, decreases known off-targeting compared to conventional gene editing Cas9. In support of Objective 3, Sub-objective 3.A, the CRISPR-Cas9 system was delivered into quail embryos to target a gene that has been implicated in susceptibility to infection by the influenza virus. Sixteen founder quail hatched, and 14 quail were confirmed to be able to pass this edit to their offspring. Moreover, chicken primordial germ cells (PGCs), a critical cell type for establishing gene edited chickens, have been successfully modified for other genes of interest. The edited PGCs have been injected into surrogate host embryos, and 15 hatched surrogates are currently being grown to sexual maturity to screen whether these gene edits can also be passed down to the next generation of birds. In support of Objective 3, Sub-objective 3.B, to improve the growth of chicken tracheal epithelial cells in culture, differentiation pathways were inhibited. Tracheal epithelial cells are one of the first cells to come in contact with avian influenza virus, making long-term culture of this cell type extremely important. Upon infection with avian influenza virus strains, the cells were susceptible and shed viruses into the medium, demonstrating their utility for studying host-pathogen interactions. Multiple sequencing datasets were integrated and mined to identify genes that are active in the respiratory tract during avian influenza virus infection. A network was constructed to visualize the connections between the genes, and over 4,000 genes from this network were selected to build a library for screening to narrow down the candidates that are involved in the infection process. In addition to the tracheal cell cultures, the development of new, immortalized cell cultures from chicken and quail species (from liver, small intestine, lungs, muscle, spleen, kidney etc.) are ongoing. A modified combination approach involving mechanical and biological methods is employed in this process. Special proteins, including telomerase and specific viral proteins help turn regular cell cultures into long-lasting, stable lines. These cell lines will subsequently be used to test susceptibility to a range of avian viruses and to explore genetic modifications to produce virus resistant cells. In support of Objective 3, Sub-objective 3.C, founder birds have been generated for disease resistance projects. The birds are either being grown to sexual maturity or the mature birds are being mated to screen whether the edits can be passed to the next generation to create birds for functional studies.

Accomplishments
1. Generation of influenza virus-resilient chicken and pig cells. Influenza viruses pose major threats to livestock and poultry production with economic impacts for both producers and consumers. Sialic acids are sugar molecules found on the cell surface that have been implicated in influenza virus infection. ARS researchers in Columbia, Missouri, modified a gene responsible for sialic acid production in pig and chicken cells and demonstrated decreased susceptibility to influenza virus infection for some strains. These results confirm a potential route to decreasing the impacts on influenza and improving livestock and poultry production efficiency to the benefit of both farmers and consumers.

2. Identification of key genes involved in avian influenza virus infection. Respiratory cells are considered the primary target for avian influenza virus infection, making them key to developing effective resistance strategies in poultry. ARS researchers in Columbia, Missouri, combined a gene expression dataset of the chicken lung with other datasets to uncover specific genes that are predicted to be involved in avian influenza virus infection. By mapping how these genes interact, the researchers could prioritize genes, thus identifying the most promising candidates for developing genetically resistant poultry which could save the poultry industry billions of dollars each year.

3. Establishment, characterization, and viral susceptibility of immortalized cell lines derived from pig tissue. Viruses pose a significant threat to the U.S. swine industry, leading to major losses in production and profits. To better understand how these viruses infect pigs, researchers need viable cell lines that these viruses can infect. Towards this goal, ARS researchers in Columbia, Missouri, successfully established cell lines derived from pig lungs, trachea, muscle, spleen, and kidney. Culture conditions were optimized to maximize cell growth and viability. These cell lines demonstrate stable growth and have transitioned into established cell lines, suggesting their suitability for long-term research. The pig lung-derived cell line supported the replication of several relevant pig viruses including porcine parvovirus, porcine circovirus type-2, porcine respiratory corona virus, Seneca Valley virus, and transmissible gastroenteritis virus. The lung cell line will be used for gene editing of host cell receptors essential for virus binding and entry into the cells to test if genetic manipulations confer infection resistance that can be further studied by in vivo trials. These cell lines will facilitate development of control measures (disease resistant pigs, vaccines, antiviral agents, etc.) against viral disease outbreaks to support the welfare of pigs thus benefiting the industry.

U.S. DEPARTMENT OF AGRICULTURE

Plant Genetics Research: Columbia, MO