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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Main Author: Lee, Cheryl
Other Authors: Tan Meng How
Format: Final Year Project
Language:English
Published: 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
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spelling sg-ntu-dr.10356-1569572022-05-06T04:53:56Z Optimizing Cas9-mediated HDR in HEK293 Lee, Cheryl Tan Meng How School of Chemical and Biomedical Engineering mh.tan@ntu.edu.sg Engineering::Bioengineering 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. Bachelor of Engineering (Bioengineering) 2022-05-04T06:17:00Z 2022-05-04T06:17:00Z 2022 Final Year Project (FYP) Lee, C. (2022). Optimizing Cas9-mediated HDR in HEK293. Final Year Project (FYP), Nanyang Technological University, Singapore. https://hdl.handle.net/10356/156957 https://hdl.handle.net/10356/156957 en application/pdf Nanyang Technological University
institution Nanyang Technological University
building NTU Library
continent Asia
country Singapore
Singapore
content_provider NTU Library
collection DR-NTU
language English
topic Engineering::Bioengineering
spellingShingle Engineering::Bioengineering
Lee, Cheryl
Optimizing Cas9-mediated HDR in HEK293
description 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.
author2 Tan Meng How
author_facet Tan Meng How
Lee, Cheryl
format Final Year Project
author Lee, Cheryl
author_sort Lee, Cheryl
title Optimizing Cas9-mediated HDR in HEK293
title_short Optimizing Cas9-mediated HDR in HEK293
title_full Optimizing Cas9-mediated HDR in HEK293
title_fullStr Optimizing Cas9-mediated HDR in HEK293
title_full_unstemmed Optimizing Cas9-mediated HDR in HEK293
title_sort optimizing cas9-mediated hdr in hek293
publisher Nanyang Technological University
publishDate 2022
url https://hdl.handle.net/10356/156957
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