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Modification of single guide (sg) RNA in CRISPR-Cas9 gene editing Tool

Modification of single guide (sg) RNA in CRISPR-Cas9 gene editing Tool

Objective

CRISPR/Cas9 has been extensively studied for genome editing, but its therapeutic application has been hampered by off-target effects. Site-specific modification of single guide (sg) RNA in CRISPR system is a potential means to expand the utility of CRISPR-Cas genome editing. To address this, and reduce of synthetic burden of sgRNA we propose a ‘click’ ligation method to synthesize sgRNA where two chemically modified oligonucleotides are joined together using CuAAC chemistry. The resultant artificial linkage is biomimetic, so it should not affect Cas9 activity. This method offers significantly higher DNA-targeting specificity (i.e. less off-target effects) and most importantly provides a cost-effective means to access thousands of synthetic sgRNAs. Imaging in CRISPR-Cas9 system using fluorescence in situ hybridisation (FISH) probes have enabled significant advancements in understanding genomic structure and transcriptional control. We will explore novel approaches to image CRISPR-Cas9 system using fluorophore-quencher pairs. The activation of fluorescence can be performed upon nuclear localisation using RNase H, and upon sgRNA binding to the target DNA. These methods will provide highly fluorescent sgRNAs for live-cell imaging, potentially much brighter compared to other methods. To control CRISPR off-target effects we will also design a light induced DNA damage method where cyanovinylcarbazole nucleoside or psolaren will be incorporated in the DNA-targeting RNA. Therefore, modification of sgRNA for CRISPR system can be used to address the biological limitations of CRISPR and expand its functionality. This will open up many avenues for future development and lead to more application-focused studies on therapeutic gene editing.
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Coordinator

THE CHANCELLOR, MASTERS AND SCHOLARS OF THE UNIVERSITY OF OXFORD

Address

Wellington Square University Offices
Ox1 2jd Oxford

United Kingdom

Activity type

Higher or Secondary Education Establishments

EU Contribution

€ 212 933,76

Project information

Grant agreement ID: 836039

Status

Grant agreement signed

  • Start date

    1 November 2019

  • End date

    31 October 2021

Funded under:

H2020-EU.1.3.2.

  • Overall budget:

    € 212 933,76

  • EU contribution

    € 212 933,76

Coordinated by:

THE CHANCELLOR, MASTERS AND SCHOLARS OF THE UNIVERSITY OF OXFORD

United Kingdom