Location: Plant Genetics Research
2024 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, a multitude of primary and immortalized cell lines have been generated for studying agriculturally important traits in both pigs and cows. In addition to porcine fibroblasts, fetal fibroblasts, intestinal and liver organoids, primary hepatocytes, tracheal epithelium, lung epithelium, and intestinal epithelium that were established last year, we have also established an immortalized porcine muscle satellite cell line, bovine leukemia (BL3) cell line, Madin-Darby bovine kidney (MDBK) cell line, as well as a porcine and bovine in vitro embryo production system to produce gene-edited pigs and cows. Primary candidate gene target lists are being used to create gene edited cell cultures for traits associated with disease resistance, muscle biology, and heat stress.
In support of Objective 1, Sub-objective 1.B, a gene edited porcine fibroblast cell culture has been generated to challenge with swine influenza virus in collaboration with researchers at the University of Missouri. Clustered regularly interspaced short palindromic repeats (CRISPRs) have been designed to target four other genes that may be important for disease resistance and are ready to be challenged with multiple viruses.
In support of Objective 1, Sub-objective 1.C, gene edited piglets were born and challenged with porcine epidemic diarrhea virus by researchers in Ames, Iowa (see accomplishment 1 for more).
In support of Objective 2, Sub-objective 2A, last year 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. Since the annual report last year, we have used this WGS pipeline to sequence and identify off-targets in two other gene-edited pig models: pigs that did not have functional recombination activating 2 (RAG2) and interleukin 2 receptor subunit gamma (IL2RG) genes and another pig model that does not have a function amyloid beta precursor protein (APP) gene. By using our pipeline, we did not identify any off-target events in any of the APP edited pigs but did identify mosaicism across tissues. The RAG2/IL2RG edited pigs did reveal off-targeting in two different genes at a low frequency. This pipeline allows us to identify true off-targeting events even with the high level of natural variation seen across different heterogeneous pig breeds and was recently published in The CRISPR Journal.
In addition to establishing a global off-target identification pipeline, anti-CRISPR proteins (ACRs) were synthesized and injected into fertilized oocytes. The optimal concentration and timing were established and used to examine the frequency of unintended off-targeting events in embryos. The introduction of an ACR resulted in a significantly reduced number of unintended events in embryos that were injected with a guide that had a history of producing off-target events while maintaining on-target editing.
In support of Objective 3, Sub-objective 3.A, the adenoviral system for gene editing in poultry has been established, and adenoviral particles containing the CRISPR/Cas9 system were injected into quail embryos to target genes related to ovary development. Currently, over 20 founder birds have been hatched and are being mated with wild type quail to determine rates of germline transmission of indel mutations. Concomitantly, culture and cryopreservation of primordial germ cells (PGCs) has been achieved, and PGCs have been edited for a target gene related to viral resistance.
In support of Objective 3, Sub-objective 3.B, further optimization of culture conditions for tracheal epithelial cultures are nearly complete, providing a new model for studying host-virus interactions. Preadipocytes that were established last year were cultured in low oxygen tensions and were shown to have reduced lipid accumulation during differentiation. Myoblasts are being collected at different points during differentiation to understand changes in transcriptional profiles. Regarding candidate identification, single cell ribonucleic acid (RNA)-seq and spatial transcriptomics are being performed for layer hen lung samples to detect different cell populations and identify transcripts in epithelial cells that potentially encode proteins that interact with respiratory viruses. Publicly available datasets of lung samples infected with highly pathogenic avian influenza are being mined to detect additional host factors that are potentially implicated in viral infection. One gene edited chicken fibroblast cell line is being challenged with avian influenza virus in collaboration with researchers at the University of Missouri. Two other gene edited fibroblast lines have been established and cryopreserved in preparation for influenza virus challenges.
In support of Objective 3, Sub-objective 3.C, founder birds have been generated to study the effects of editing two different genes on ovary development. This information will be used to understand genetic control of the ovary in avians and identify biomarkers for improving reproductive efficiency.
Accomplishments
1. Production of pigs that exhibit no symptoms to PEDV infection. The porcine epidemic diarrhea virus (PEDV) has major impacts on the global pig industry. This alphacoronavirus causes acute diarrhea, dehydration, and high rates of mortality in newborn piglets. Identifying the receptor or host factor that plays an important role in infectivity is critical to produce more resilient pigs. ARS researchers in Columbia, Missouri, targeted a candidate gene in pig embryos. The gene-edited embryos were then transferred to two recipients which both produced a pregnancy. The piglets born were sent to ARS researchers in Ames, Iowa, where they challenged the pigs with a PEDV isolate and monitored symptoms and amount of virus in the body. The two pigs that did not show symptoms upon infection were pigs that did not have a functional host factor protein but did show viral replication. Although not fully resistant, this is one step forward in identifying critical regulators of PEDV infection. Once pigs are found to be fully resistant to PEDV, pig producers could use these genetics to produce animals that do not get sick which would impact consumers and animal welfare concerns.
2. Generation of Cas9-expressing chicken cells for rapid gene editing. The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9) (CRISPR-Cas9) system has become one of the most popular methods for genetic engineering in all species. However, effective models and resources for gene editing in chickens are currently lacking. Often, cell lines are used for initial gene editing studies to reduce the number of animals for experiments, so having cell lines available for gene editing is extremely important. ARS researchers in Columbia, Missouri, generated chicken cell lines that stably express Cas9 to be used for rapid and efficient gene editing. These cells were shown to be successfully gene edited; therefore, they can be used by researchers (academia, government, and industry) to understand the functions of diverse genes in chickens to improve production traits.
3. Improvement of the ability to culture chicken tracheal cells. Respiratory viruses, such as avian influenza virus, are devastating for the poultry industry and create a significant economic burden. Models for studying the interactions between the animal and the virus are currently lacking. The trachea represents one of the first sites of infection by respiratory viruses, and the ability to culture tracheal cells allows for a rapid platform to study virus infection and reduce animal numbers for experiments. ARS researchers in Columbia, Missouri, modified culture conditions that improved growth and proliferation of chicken tracheal cells. Optimization of freezing methods was also performed to determine ways of preserving these cells for future use or transfer to other research groups. Improving the culture of chicken tracheal cells provides a platform for studying virus infection in chickens as a critical first step before moving to animal studies and increases the resources for poultry research.
4. Identification and optimization of anti-CRISPR (Acr) proteins to improve genome integrity of gene edited cells. Identification and optimization of anti-CRISPR (Acr) proteins to improve genome integrity of gene edited cells. The development of the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9) (CRISPR-Cas9) system has revolutionized our ability to produce genetically engineered livestock at a high efficiency, however, the safety of this technology has yet to be fully demonstrated. The Cas9 nuclease is used to introduce a specific double stranded break in the genome which then triggers the cell’s mechanism to repair this break. However, there may be unintended double stranded breaks elsewhere in the genome, creating concern. These events are referred to as “off-targeting” events. Gene-edited embryos were generated by injection of the CRISPR-Cas9 system that produces off- targeting events at a high efficiency and then injected with Acr proteins. By doing so, the frequency of off- targeting events was reduced. ARS researchers in Columbia, Missouri, identified a strategy to mitigate unwanted modifications elsewhere in the genome when using genome editing technology. The use of Acr proteins in pig embryos may provide genetic engineers a way to ensure the genome is not compromised in unwanted locations.
Review Publications
Spate, L.D., Yin, J., Sammel, L., Prather, R.S., Redel, B.K. 2023. Oocyte recovery after overnight ovary transport provides an alternative source of cumulus oocyte complexes that are competent to produce live piglets. Reproduction in Domestic Animals. 00:1-3. https://doi.org/10.1111/rda.14491.
Lucas, C.G., Sullivan, R.M., Pfeiffer, C.A., Chen, P.R., Kim, J., Sponchiado, M.S., Conley, A.J., Prather, R.S., Wells, K.D., Geisert, R.D. 2023. Porcine-specific expression of the three functional CYP19 paralogs in early conceptus, placenta, and gonads. Reproduction. 166(4):263-269. https://doi.org/10.1530/REP-23-0189.
Redel, B.K., Yoon, J., Reese, E., An, H., Uh, K., Chen, P.R., Prather, R.S., Lee, K. 2024. Novel off-targeting events identified after genome wide analysis of CRISPR-Cas edited pigs. The CRISPR Journal. 7(3): 141 - 149. https://doi.org/10.1089/crispr.2024.0012.