CRISPR Cas9 targeted single nucleotide change at the (+15) loci of the alpha-lactalbumin gene
Long, Sierra Alicia
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https://hdl.handle.net/2142/116097
Description
Title
CRISPR Cas9 targeted single nucleotide change at the (+15) loci of the alpha-lactalbumin gene
Author(s)
Long, Sierra Alicia
Issue Date
2022-07-19
Director of Research (if dissertation) or Advisor (if thesis)
Wheeler, Matthew B
Committee Member(s)
Perez, Pablo P
Hurley, Walter L
Department of Study
Animal Sciences
Discipline
Animal Sciences
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
M.S.
Degree Level
Thesis
Keyword(s)
CRISPR
gene-editing
cattle
milk
alpha-lactalbumin
RNP
Cas9
Gyr
base-editor
HEK293
dairy
Abstract
The application for genome editing within agriculture is a sustainable option due to the recent FDA approval trends and various gene-editing tools available. One highly targeted aspect of genetic modification is bovine milk due to its versatility in genetic manipulation and user consumption. Bovine alpha-lactalbumin (α-LA) is a whey protein exclusively produced during lactation and is closely associated with the amount of lactose present in the mammary gland, therefore increasing milk yield. Previous studies identified a correlation between a single nucleotide change (+15) base-pairs upstream of the transcription start point with increased milk yield in Holstein cattle. The polymorphism was designated as α-LA (+15) A or α-LA (+15) B, where the A variant has an adenine, and the B variant has the wild-type guanine. The A variant has a higher predicted transmitting ability for milk, protein dollars, protein yield, fat dollars, and fat yield. Here, we look at the various ways to induce this mutation in bovine DNA to allow for a seamless introduction of the A variant. CRISPR/Cas9 system utilizing plasmid DNA or ribonucleoproteins (RNP) was paired with a guide RNA targeting the (+15) α-LA gene to induce a break in the DNA of fibroblast cells from Gyr cattle. The cells were also supplied with a 97bp or 126bp single-stranded-oligonucleotide repair template to allow homology-directed repair to induce the A allele at this locus. Using the CRISPR/Cas9 systems of plasmids and RNPs, we created double-stranded breaks resulting in insertions and deletions at the (+15) site in Gyr fibroblast cells. Additionally, we detected a 1-18% mutation efficiency in plasmid CRISPR/Cas9 systems at the mutation site, yet we could not detect homologs repair. To increase mutation efficiency at the (+15) site, base-editor CRISPR/Cas9 systems were applied under a transient transfection of bovine α-LA in HEK293 cells. Cytosine base-editor CRISPR/Cas9 systems are revolutionary for SNP mutations since no repair template is needed to repair the DNA because no DSB is induced. Due to the difficulty of choosing a guide RNA for base-editors, a transient transfection utilizing HEK293s as a medium of transfection was set up to select the most efficient gRNAs for inducing the G>A (C>T) mutation of a bovine reporter plasmid. Of the nine gRNAs tested, seven indicated a G>A mutation at the (+15) site with an average editing efficiency between 4.7% and 31%. Knowing that the guanine to adenine substitution is achievable in bovine cells and transient bovine DNA, there is a strong possibility of producing a tropically adapted cow with increased milk yield without crossbreeding.
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