Optimizing Cas9-mediated HDR in HEK293
The most widely used genome editing tool is the Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated proteins (Cas) system. With its programmable guide RNA sequence for specific gene targeting, it allows for precise genome editing including substitutions, kn...
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| Format: | Final Year Project |
| Language: | English |
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Nanyang Technological University
2022
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| Online Access: | https://hdl.handle.net/10356/156957 |
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| Institution: | Nanyang Technological University |
| Language: | English |
| Summary: | The most widely used genome editing tool is the Clustered regularly interspaced
short palindromic repeats (CRISPR) and CRISPR associated proteins (Cas) system. With
its programmable guide RNA sequence for specific gene targeting, it allows for precise
genome editing including substitutions, knock-outs, and knock-ins. For the treatment of
genetic disorders, a precise edit is compulsory compared to inaccurate end-joining
modifications. Our focus will be on Homology Directed Repair (HDR) pathway as it is
more precise but is limited by its efficiency and dominance of non-homologous end
joining (NHEJ). It has been identified that the proximity of repair template is a rate
limiting factor of HDR. Since previous studies has shown that the “all-in-one” method
with ssODNs has limitations in replacing large fragments, we will use plasmid donors to
counter this limitation. This project aims to improve HDR efficiency, by increasing
proximity of ideal donor repair template to repair site via fusing donor plasmid to gRNA.
Our findings show that plasmid donors are able to achieve a consistent HDR efficiency. |
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