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postgraduate thesis: Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus aureus Cas9 (SaCas9) engineering with enhanced genome targeting accuracy
Title | Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus aureus Cas9 (SaCas9) engineering with enhanced genome targeting accuracy |
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Authors | |
Advisors | |
Issue Date | 2022 |
Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
Citation | Yuen, T. L. C. [袁紫璐]. (2022). Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus aureus Cas9 (SaCas9) engineering with enhanced genome targeting accuracy. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. |
Abstract | Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Cas9 has become the most popular genome editing technology in recent years. Its broad applications have covered various fields, including cancer study, immunology, virus defence, and drug research. Its high editing efficiency and simple implementation make it accessible for general users, overcoming the obstacles of previous genome editing tools. However, long-standing discussion on the specificity of CRISPR-Cas9 has limited its direct application in genome therapies, that CRISPR-Cas9 induced off-target cleavages have been found.
To improve the targeting accuracy of the CRISPR-Cas9 system, Cas9 protein engineering is frequently used to lessen target cleavages in non-specific sites. In this thesis work, I first characterised the genome editing performance of a newly engineered Cas9 variant, Opti-SpCas9, which was modified from the most commonly used Streptococcus pyogenes Cas9 (SpCas9). I further engineered on another protospacer adjacent motif (PAM) relaxed Cas9 ortholog, KKH-SaCas9, which is derived from Staphylococcus aureus and carries KKH mutations.
I found Opti-SpCas9 had reduced off-target activity in endogenous gene cleavages while retaining high on-target efficiency. I also demonstrated Opti-SpCas9 has the compatibility of extended 5′ guanine in single guide RNA (sgRNA), which was found to be not compatible in some specificity improved SpCas9 variants.
I generated two high-fidelity KKH-SaCas9 variants, KKH-SaCas9-SAV1 and KKH-SaCas9-SAV2, which showed remarkable accuracy. Amino acid substitution Y239H was discovered to be significant in decreasing non-specific editing by SAV2. The targeting accuracy of SAV1 and SAV2 were examined on single-base mismatch discrepancy, which confirmed that they were sensitive to mismatched targets at the single-nucleotide level and thus had better specificity.
These findings not only contribute to increasing the safety of using CRISPR-Cas9 systems, but also expand their potential target range, which can provide users more choices on sgRNAs design or access to target sites that could not be edited previously. The stringent characteristics of SAV1 and SAV2 are valuable in gene therapies as they may promote alternative targeting strategies for allele-specific targeting or for diseases with single nucleotide polymorphism. |
Degree | Doctor of Philosophy |
Subject | CRISPR-associated protein 9 Streptococcus pyogenes Staphylococcus aureus |
Dept/Program | Biomedical Sciences |
Persistent Identifier | http://hdl.handle.net/10722/327657 |
DC Field | Value | Language |
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dc.contributor.advisor | Wong, SL | - |
dc.contributor.advisor | Huang, J | - |
dc.contributor.author | Yuen, Tsz Lo Chaya | - |
dc.contributor.author | 袁紫璐 | - |
dc.date.accessioned | 2023-04-04T03:02:58Z | - |
dc.date.available | 2023-04-04T03:02:58Z | - |
dc.date.issued | 2022 | - |
dc.identifier.citation | Yuen, T. L. C. [袁紫璐]. (2022). Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus aureus Cas9 (SaCas9) engineering with enhanced genome targeting accuracy. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. | - |
dc.identifier.uri | http://hdl.handle.net/10722/327657 | - |
dc.description.abstract | Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Cas9 has become the most popular genome editing technology in recent years. Its broad applications have covered various fields, including cancer study, immunology, virus defence, and drug research. Its high editing efficiency and simple implementation make it accessible for general users, overcoming the obstacles of previous genome editing tools. However, long-standing discussion on the specificity of CRISPR-Cas9 has limited its direct application in genome therapies, that CRISPR-Cas9 induced off-target cleavages have been found. To improve the targeting accuracy of the CRISPR-Cas9 system, Cas9 protein engineering is frequently used to lessen target cleavages in non-specific sites. In this thesis work, I first characterised the genome editing performance of a newly engineered Cas9 variant, Opti-SpCas9, which was modified from the most commonly used Streptococcus pyogenes Cas9 (SpCas9). I further engineered on another protospacer adjacent motif (PAM) relaxed Cas9 ortholog, KKH-SaCas9, which is derived from Staphylococcus aureus and carries KKH mutations. I found Opti-SpCas9 had reduced off-target activity in endogenous gene cleavages while retaining high on-target efficiency. I also demonstrated Opti-SpCas9 has the compatibility of extended 5′ guanine in single guide RNA (sgRNA), which was found to be not compatible in some specificity improved SpCas9 variants. I generated two high-fidelity KKH-SaCas9 variants, KKH-SaCas9-SAV1 and KKH-SaCas9-SAV2, which showed remarkable accuracy. Amino acid substitution Y239H was discovered to be significant in decreasing non-specific editing by SAV2. The targeting accuracy of SAV1 and SAV2 were examined on single-base mismatch discrepancy, which confirmed that they were sensitive to mismatched targets at the single-nucleotide level and thus had better specificity. These findings not only contribute to increasing the safety of using CRISPR-Cas9 systems, but also expand their potential target range, which can provide users more choices on sgRNAs design or access to target sites that could not be edited previously. The stringent characteristics of SAV1 and SAV2 are valuable in gene therapies as they may promote alternative targeting strategies for allele-specific targeting or for diseases with single nucleotide polymorphism. | - |
dc.language | eng | - |
dc.publisher | The University of Hong Kong (Pokfulam, Hong Kong) | - |
dc.relation.ispartof | HKU Theses Online (HKUTO) | - |
dc.rights | The author retains all proprietary rights, (such as patent rights) and the right to use in future works. | - |
dc.rights | This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. | - |
dc.subject.lcsh | CRISPR-associated protein 9 | - |
dc.subject.lcsh | Streptococcus pyogenes | - |
dc.subject.lcsh | Staphylococcus aureus | - |
dc.title | Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus aureus Cas9 (SaCas9) engineering with enhanced genome targeting accuracy | - |
dc.type | PG_Thesis | - |
dc.description.thesisname | Doctor of Philosophy | - |
dc.description.thesislevel | Doctoral | - |
dc.description.thesisdiscipline | Biomedical Sciences | - |
dc.description.nature | published_or_final_version | - |
dc.date.hkucongregation | 2023 | - |
dc.identifier.mmsid | 991044657074303414 | - |