Single nucleotide editing at the 11q23.3 adiposity risk locus by the prime editing technique
Master thesis
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https://hdl.handle.net/11250/3141752Utgivelsesdato
2024-06-03Metadata
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- Master theses [266]
Sammendrag
SummaryUnraveling the mysteries hidden within the small fraction of the genome that makes every human unique, is still ongoing. Most of these genetic variations require interactions with environmental factors to affect the risk of developing complex diseases, and uncovering their biological roles remains a big challenge. Genome-Wide Association Studies (GWAS) have been instrumental in identifying single nucleotide polymorphisms (SNPs) associated with diseases, although the causal variants often elude detection due to the complexity of the genome. Connecting the biology of SNPs to their statistically linked diseases can be achieved by gene editing technologies such as CRISPR/Cas9 and prime editing (PE).Accumulation of visceral adipose tissue (VAT) – fat stored around the internal organs – has been linked to insulin resistance, metabolic disease, and cardiovascular disease. While GWASs have identified numerous SNPs associated with overall adiposity (overweight and obesity as measured by BMI), few have focused on the distribution of fat itself. A recently identified locus, 11q23.3, has been found to be associated with VAT mass . Specifically, the A-allele of the SNP rs1799993 within this locus is associated with an increase in VAT. The current thesis is part of a larger project with the aim of understanding the biological mechanisms of this locus, with a special focus on the SNP rs1799993. Our lab has identified regions with enhancer activity within the locus, and the SNP rs1799993 was found to be located within a potential enhancer. Moreover, epigenetic data suggest this enhancer is active in adipose-derived mesenchymal stem cells. By utilizing prime editing in the mesenchymal stem cell line ASC52telo, the underlying mechanisms of rs1799993 can be uncovered. The overall aims of this thesis are therefore to first establish a proof-of-concept prime editing protocol in the easily transfected cell line HT1080, followed by developing a lentiviral transduction protocol for the PE components in the mesenchymal stem cell line ASC52telo with the prospect of using PE to generate ASC52telo clones homozygous for the risk and protective alleles of rs1799993.Sanger sequencing was applied to genotype the HT1080 cell line, and it was found to be heterozygous for the SNP rs1799993. This meant that the edit needed to be performed in both directions to achieve homozygous cell lines. First, different transient transfection protocols were tested and assessed using qPCR or fluorescence imaging. The PE components (PEmax-P2A-GFP, pegRNAs, and mismatch repair inhibitor) were transfected using the optimal protocol, and GFP positive cells were sorted as single cells by FACS for single-cell colony expansion. Single-cell expansion yielded 162 colonies, 87 of which were genotyped by Sanger sequencing. One colony (~1%) was positively edited from heterozygous A/C to the homozygous C protective allele, suggesting that the PE protocol worked, at least with the designed pegRNAs encoding the C-allele.
Based on the successful edit in the HT1080 cells, PE of the ASC52telo proceeded. Lentivirus carrying the PEmax sequence and blasticidin resistance were created in the HEK293T cell line, and subsequent quantification of viral particles and infectious units were determined by a rapid antigen detection test and estimated by a functional viral titer test, respectively. ASC52telo cells were transduced with a multiplicity of infection of 2 and 5, and successfully transduced cells were selected using blasticidin. The lentiviral transduction of ASC52telo and selection of transduced cells were successful, but no single-cell colonies were expanded despite comprehensive effort. Therefore, several heterogeneous cell lines with stable expression of PEmax were established instead. Genomic integration and mRNA expression of PEmax was demonstrated by qPCR but could be validated by western blotting.Finally, a pilot was conducted to introduce the remaining PE-components into the heterogenous cell lines expressing PEmax. The cells were sorted by FACS after nucleofection, but none of the cells survived. Therefore, more work should be done to further develop clonal expansion of this cell line, and to optimize nucleofection for improved viability. As a result, the SNP rs1799993 was not edited in the ASC52telo cell line. However, the groundwork has been laid for further the development of a PE protocol and clonal expansion, as well as utilizing the PEmax-expressing cells to edit rs1799993 and other genomic regions by differently designed pegRNAs. Once these protocols have been developed, any disease-associated SNP in these cells can in principle be edited, their downstream target genes be identified, and the affected biology be determined.