Parr, Lochlan
(2024)
Part A: Application of fluorescent markers onto CRISPR-associated proteins/ Part B: Substitution of cysteine residues in CRISPR associated Protein Cas1.
MRes thesis, University of Nottingham.
Abstract
Part A:
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems mediate prokaryotic adaptive immunity against mobile genetic elements (MGEs), where MGE sequences are captured and stored as spacers in CRISPR loci. CRISPR-associated (Cas) proteins Cas1 and Cas2 form a complex that integrates these spacers into CRISPR arrays, generating adaptive immunity through CRISPR interference. While the mechanisms of the Cas1-Cas2 complex are understood, less is known about its regulatory pathways, particularly because it has been shown to interact with proteins far outside its genetic locality, sometimes from different chromosomes. Fluorescent proteins (FPs) have successfully been used to tag proteins for over a century, enhancing our understanding of their intermolecular interactions. Site directed mutagenesis (SDM) can be used to substitute all Cas1 cysteine residues for alternative inert amino acids, and implementation of another cysteine at a more accessible site would allow the binding of fluorescent maleimide proteins, accommodating advanced microscopy techniques to investigate molecular interactions in vivo, thereby improving our understanding of Cas1 regulatory interactions. The methods to produce the target mutant Cas1 protein are discussed in this review, including techniques such as Sanger sequencing, pilot protein overexpression and spacer acquisition assays, which are used to verify the quality and viability of the product.
Part B:
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems mediate prokaryotic adaptive immunity against mobile genetic elements (MGEs), where MGE sequences are captured and stored as spacers in CRISPR loci. CRISPR associated (Cas) proteins Cas1 and Cas2 form a complex that integrates these spacers into CRISPR arrays, generating adaptive immunity through CRISPR interference. While the mechanisms of the Cas1 Cas2 complex are understood, less is known about regulatory pathways, particularly because it has been shown to interact with proteins far outside its genetic locality, sometimes from different chromosomes. Fluorescent proteins (FPs) have successfully been used to tag Cas1 to study its interaction with other proteins. mStayGold, a novel highly photostable FP, shows promise of enhancing the observation of Cas1 interactions in live cells. In contrast to challenges with photobleaching in conventional FPs, mStayGold offers tenfold superior photostability at no cost to brightness, which is helpful for advanced microscopy techniques. Wildtype Cas1 presents difficulties with mStayGold fusion due to the presence of multiple cysteine residues which can destabilize the tertiary structure of FPs. To address this, site directed mutagenesis was employed to substitute cysteine for serine residues in Cas1, aiming to create a mutant Cas1 with a single, strategically placed cysteine residue that optimally binds mStayGold. There were significant setbacks caused by low PCR yields and undesirable mutations, but the first successful mutant, Cas1 C51S does not exhibit impaired protein expression. There is no apparent reason the remaining cysteine residues cannot continue to be substituted, allowing advanced microscopy techniques to investigate molecular interactions in vivo, aiming to improve our understanding of Cas1 regulatory interactions.
| Item Type: |
Thesis (University of Nottingham only)
(MRes)
|
| Supervisors: |
Bolt, Edward
Harding, Stephen |
| Keywords: |
CRISPR associated proteins; mstayGold, photostable fluorescent protein marker |
| Subjects: |
Q Science > QP Physiology |
| Faculties/Schools: |
UK Campuses > Faculty of Science > School of Biosciences |
| Item ID: |
79508 |
| Depositing User: |
HARDING, Prof Stephen
|
| Date Deposited: |
13 Dec 2024 04:40 |
| Last Modified: |
13 Dec 2024 04:40 |
| URI: |
https://eprints.nottingham.ac.uk/id/eprint/79508 |
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