Fracea, Mihai-Sergiu (2024) High-Throughput Proteomics: Screening Methods for CRISPR/Cas9 DNA binding affinity. Bachelor's Thesis, Life Science and Technology.
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Abstract
The RNA-guided CRISPR/Cas9 nuclease is a revolutionary gene-editing tool which dramatically increased the extent to which genetic editing is possible, bringing unprecedented levels of precision and cost efficiency. CRISPR/Cas9 allows for gene editing and expression control and has applications in plant and animal biotechnology, climate and in advancing innovative medical therapies. To address the limitations related to Cas9 specificity and to improve the precision in gene editing, many techniques rely on developing large numbers of Cas9 or guide RNA (gRNA) mutant variants in vitro and testing their capabilities. However, probing a large number of variants can often be challenging due to limited throughput, low precision, cost or other underlying factors. Here, we identify the main high-throughput (HT) in vitro DNA-Cas9 binding assays and thoroughly evaluate and compare their capabilities, advantages and limitations. Generally, HITS-FLIP and versions derived from it were found to be versatile and more accessible methods that can perform high-throughput assays without overlooking precision, but we suggest each method to be taken under account depending on the experimental setup, as there is no fit-for-all solution.
| Item Type: | Thesis (Bachelor's Thesis) |
|---|---|
| Supervisor name: | Furst, M.J.L.J. |
| Degree programme: | Life Science and Technology |
| Thesis type: | Bachelor's Thesis |
| Language: | English |
| Date Deposited: | 30 Jul 2024 09:36 |
| Last Modified: | 30 Jul 2024 09:36 |
| URI: | https://fse.studenttheses.ub.rug.nl/id/eprint/33746 |
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