Alotiby, Amna
(2017)
Exploring gene editing using site-specific endonucleases as an approach to improve in vitro models of TNF Receptor-Associated Periodic Syndrome (TRAPS).
PhD thesis, University of Nottingham.
Abstract
Tumour necrosis factor receptor-associated periodic syndrome is an autoinflammatory disorder caused by mutations in the tumour necrosis factor receptor-type 1 (TNFR1) gene, leading to misfolding of the TNFR1 protein and a resultant chronic inflammatory condition. This study aimed to explore the whether a more physiologically relevant model of TRAPS could be made by gene editing the cell’s endogenous TNFR1 genes. We utilized two site-specific nucleases, TALEN and CRISPR-Cas9 as gene editing tools. These nucleases are able to introduce double-strand DNA breakage at a pre-determined DNA sequence, resulting in gene modification, by two mechanisms: non-homologous end joining (NHEJ) or homology directed repair (HDR).
TALEN pairs were designed and constructed for targeting six sites on exon 2 of TNFR1 in the SK-Hep-1 Cell line. However, despite evidence of correct TALEN construction, gene editing at the target sites in TNFR1 was undetectable by the methods employed (Surveyor mutagenesis assay, DNA sequencing, Flow cytometry). This suggested that our TALEN pairs might be of very low efficiency or be non-functional in vivo. Consequently, the relatively simpler CRISPR-Cas9 system was used as an alternative gene editing tool to target the TNFR1 gene.
Two plasmids expressing guide RNAs and Cas9 enzyme were designed to target exon 2 of TNFR1, and were functionally verified in vitro. Both were applied separately to induce gene editing in vivo, firstly by NHEJ, and subsequently by HDR. Analysis of DNA from pooled clones of transfected cells for NHEJ yielded little evidence of successful gene editing by Surveyor assay or pooled DNA sequencing. In silico analysis of exon 2 sequences using TIDE suggested a maximal efficiency of ≤3%. Cells co-transfected with the cas9-guideRNA plasmids and a HDR template, designed to insert the FLAG epitope sequence into TNFR1, was however more successful. PCR detection of FLAG sequence insertion into exon 2 of TNFR1 indicated successful editing had occurred. However, examination of transfected cells by flow cytometry staining for FLAG epitope expression indicated that gene editing was still very low efficiency, ≤ 1.37%, despite high efficiency of transfection.
In conclusion, the CRISPR-Cas9 system, in our hands, shows evidence which supports it’s use in gene editing of TNFR1. However, the very poor efficiency of editing detected, suggests much further reagent and process/detection optimization is needed, or more fundamental issues, including accessibility of the TNFR1 gene within the SK-Hep1 cell line need to be addressed, before it’s application to generate disease specific mutations
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