Jennie Nicole Peterson

Title of Work: 

Limb Bud Development in Vertebrate Embryos Using CRISPR Cas12f

Abstract:

For my internship abroad, CRISPR Cas12f , as well as prime editing software, was used to modify avian primordial germ cells (PGCs) to regrow limbs in mammalian vertebrate species. Using the enzyme, Cas12, the SOX9 gene was targeted and knocked out, which plays a key role in embryonic development, particularly, the development of limbs. Genetically modified germ cells were created using PCR techniques, which rapidly amplified the segment of DNA that would bind to the avian embryo’s DNA after insertion of developing fertilized chicken eggs of Hypeco nera and White Leghorn. Through normal DNA repair mechanisms, such as Gibson Recombination, the amino acid that codes for the SOX9 gene would be removed, which would impair limb bud development. Using the modified Cas12f plasmid, however, if the creation was successful, then a developing limb bud could be observed. 

Elevator Pitch Video

Elevator Pitch Transcript: 

Hello, my name is Jennie Peterson and I am a dual major studying Biomedical Science and Comparative Cultural Studies with an emphasis in Asian Studies and a minor in Japanese. For my internship, I had the unique opportunity of researching for an entire academic year at Kyushu University in Fukuoka, Japan. Under Professor Yuji Atsuta, I studied Limb Bud Development in Vertebrate Embryos Using CRISPR Cas12f. For my internship abroad, CRISPR Cas12f , as well as prime editing software, was used to modify avian primordial germ cells (PGCs) to regrow limbs in mammalian vertebrate species. First a plasmid was created that contained the Cas12f enzyme along with a target DNA sequence that bound to the avian DNA, causing a frameshift mutation that knocked out the SOX9 gene, which plays a key role in embryonic development, particularly, the development of limbs. This was the guideline for how the avian DNA would respond. Using the Cas12f enzyme, the DNA was cut allowing for the modified DNA sequence from the plasmid to take its place. This meant that the SOX9 gene was targeted and knocked out. Genetically modified germ cells were created using PCR techniques, which rapidly amplified the segment of DNA that would bind to the avian embryo’s DNA after insertion of developing fertilized chicken eggs of Hypeco nera and White Leghorn. Through normal DNA repair mechanisms, such as Homologous Recombination, the amino acid that codes for the SOX9 gene would be removed, which would impair limb bud development. Using the modified Cas12f plasmid, however, if the creation was successful, then a developing limb bud could be observed. Although still in the development stage, because limb bud development has been successfully observed in avian embryos after DNA modification, this research has incredible potential within the healthcare industry. Understanding which genes play key roles in limb development could potentially cure genetic diseases that impair the development of limbs within the gestation phase of human organisms. Further beyond, this research may also provide solutions to those who have experienced amputations and provide a way to regrow previously lost limbs. Although I experimented using avian embryos, before I left, Professor Atsuta was beginning the second phase of his research which was applying the same DNA modifying software to snake DNA to produce the same outcome of growing a limb bud, however, this time, in a place where a limb bud is not expected to grow. Thank you.

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