The pig has similar features to the human in aspects such as physiology, immunology, and organ size. Because of these similarities, genetically modified pigs have been generated for xenotransplantation. Also, when using the pig as a model for human diseases (e.g. cystic fibrosis transmembrane conductance regulator), the pig exhibited similar symptoms to those that human patients present. The main goal of this work was to examine the efficacy of direct injection of the CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/ CRISPR associated protein 9) in pigs and to overcome shortcomings that resulted after direct injection into the cytoplasm of developing zygotes. By using direct injection of CRISPR/Cas9 into developing zygotes, we successfully generated fetuses and piglets containing 9 different mutations. The total number of aborted fetuses was 20 and of live piglets was 55. Moreover, one issue that was encountered during the production of mutated pigs was that insertion or deletion (indel) mutations did not always introduce a premature stop codon because it did not interfere with the codon read. As a result of these triplet indel(s) mutations, a hypomorphic phenotype was presented; consequently, the mutated gene was partially functional. To prevent this hypomorphic phenotype, we introduced two sgRNAs to generate an intended deletion that would remove a DNA fragment on the genome by causing two double-strand breaks (DSB) during non-homologous end joining (NHEJ). The injection of two sgRNAs successfully generated the intended deletion on the targeted genes in embryos and live piglets. Results after using intended deletions, in IL2RG mutation pigs, did not show hypomorphic phenotypes even when a premature stop codon was not present. After using the intended deletion approach, function of the targeted genes was completely disrupted regardless of the presence or absence of a premature stop codon. Our next aim was to introduce (i.e. knock-in) a portion of exogenous (donor) DNA sequence into a specific locus by utilizing the homology direct repair (HDR) pathway. Because of the cytotoxicity of the linear form of the donor DNA, the concentration of the injected donor DNA was adjusted. After concentration optimization, four different donor DNA fragments targeting four different genes were injected into zygotes. Efficiency of knock-in was an average of 35%. Another donor DNA was used in this study which is IL2RG-IA donor DNA carried 3kb of exogenous cassette. It showed 15.6% of knock-in efficiency. IL2RG-IA Donor DNA injected embryos were transferred into surrogates, and a total of 7 pigs were born from one surrogate, but none of the 7 were positive for the knock-in. Future experiments need to be developed to optimize this approach. Overall, the direct injection of CRISPR/Cas9 is advantageous in cost, time, and efficiency for large animal production and for biomedical research. However, there are still unsolved challenges (off-targeting effects, low efficiency of knock-in, and monoallelic target mutation) that need to be elucidated for future application in humans and other species. / Doctor of Philosophy / The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) system is commonly used to make genetically modified pigs. The CRISPR/Cas9 system can break the DNA on a desired gene region. During the DNA repair process, random DNA base pairs can be inserted or deleted on the broken regions, thus generating a mutation on the desired gene. Scientists have adopted new methods to disrupt genes in many species. One of these new methods is the direct injection of CRISPR/Cas9 into a fertilized oocyte. In our first project, we used direct injection of the CRISPR/Cas9 system into the fertilized one-cell embryo. A total of 55 live pigs and aborted 20 fetuses with specifically disrupted genes were produced for biomedical research model. During these studies, one critical drawback of the direct injection method was encountered. Partial function of the gene was possible. To prevent this problem, two DNA broken regions were generated by the CRISPR/Cas9 system to remove the middle part the DNA by two DNA breaking. This method successfully removed the middle portion of the DNA targeted region in the pig embryos. Embryos injected with the CRISPR/Cas9 system to cut the two specific DNA regions were transplanted into surrogate pigs, and a total of 15 piglets were produced. All 15 pigs confirmed that a specific part of the gene had been removed by two DNA breakage. Also, no function of the desired gene was found in the 15 pigs.
The objective of the last experiment was to introduce a specific exogenous DNA sequences into specific region of DNA using the CRISPR/Cas9 system. For this study, four different exogenous DNA fragments were synthesized for four different genes. When injected, one exogenous DNA along with the CRISPR/Cas9 system, the average integration efficiency of the four exogenous DNA fragments was 35% in the embryo. Another exogenous DNA, which was longer than other four DNA fragments showed 15.6% integration efficiency. The embryos injected with the long exogenous DNA fragment, along with the CRISPR/Cas9 system, were transferred into surrogate pigs. The result was that a total of 7 piglets were born, but the exogenous DNA sequence was not found in none of the seven piglets.
In conclusion, the CRISPR/Cas9 system showed effective removal of the entire gene function of specific genes in the pig. However, for future application in the human and other species, some problems (un-wanted region mutation and low efficiency of exogenous DNA integration) continue to emerge and need to be addressed in future experiments.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/101763 |
| Date | 16 July 2019 |
| Creators | Ryu, Junghyun |
| Contributors | Animal and Poultry Sciences, Lee, Kiho, Rhoads, Michelle, Eyestone, Willard H., Ealy, Alan D. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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