Synthesis of chemically modified guide RNAs and their applications

Thesis

The genome editing tool CRISPR has transformed our understanding of how genetics affects human health. The Cas9 endonuclease, guided by a guide RNA (gRNA) has been used for genome editing, live cell imaging as well as for diagnostic purposes. The CRISPR-Cas9 system has been repurposed for live-cell imaging by mutating the Cas9 protein to inactivate the DNA cleavage activity (dCas9) and by appending green fluorescent proteins (GFP). This enables sgRNA-programmed localisation of GFP to any genomic loci. However, this method results in significant background fluorescence. To overcome this, we designed a metastable fluorescent gRNA that is only activated once it reaches the nucleus, to label its target. Live cell imaging showed a significant reduction in cytoplasmic accumulation of the fluorophore. To study cells and the genome, key modifications must be made on the sgRNA such as fluorophores or ligands that can aid cell delivery. These site-specific modifications cannot be introduced by transcription-mediated RNA synthesis and are expensive and difficult to source by solid-phase synthesis approach. We developed a novel method of introducing chemical modifications into the sgRNA; by extending the 3'-end and by crosslinking a short oligonucleotide that bears the modification. All the modifications were well tolerated by Cas9 in vitro. However, further work is required to test the modified sgRNAs in cells. Lastly, the R-loop structure was exploited as a means to introduce modifications into the CRISPR-Cas9 system. To test this hypothesis, we designed a short fluorescent oligonucleotide to bind to the R-loop structure formed after the sgRNA-Cas9 complex binds to their target, the telomeres. This method proved to be a successful tool for live cell imaging and will be applied to other areas in the future.

Authors: Mungur, T

• DOI: 10.5287/ora-6rxyk0bkd
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: live cell imaging; CRISPR; modified gRNA