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Identifier

000419739

Title

Large scale expression and purification of a high fidelityCas9 nuclease

Alternative Title

Έκφραση και καθαρισμός σε μεγάλη κλίμακα μιας Cas9 νουκλεάσης υψηλής πιστότητας

Author

Κεφάλα, Ιωάννα-Μαρία Δ.

Thesis advisor

Κουτσιούλης, Δημήτριος

Reviewer

Κοκκινίδης, Μιχαήλ
Μπουριώτης, Βασίλης

Abstract

CRISPRCas9 technology has been the most enlightening tool for genome editing compared to until now existing systems (ZFNs, TALENs). It’s been originally recognized as a bacterial immunity system. Its function is based in the cooperation of a nuclease (Cas9) that introduces double stranded breaks and a gRNA molecule. The avoidance of protein engineering and the easily manageable gRNA molecule gave to scientists the ability of targeting any possible place in the genome and made this particular system the most efficient, simple and accurate way of editing. The last decade this breakthrough came out of the blue to “rock the boat” in scientific world and find a huge spectrum of applications. The extensive use of the system has attracted the interest for more scientific research in purpose of making the system even more efficient and overcome its weaknesses. Scientists manage to manipulate the RNA molecule for multiplex targeting and most importantly manufactured new variants of Cas9 nuclease with dynamic properties. Cas9 variants such as high fidelity nucleases have been the latest development of this technology with the most promising of all the newest model: HypaCas9. The current master thesis is focusing on expression and purification of HypaCas9. The aim of this project is to use HypaCas9 in a ribonucleoprotein complex for genome editing in the animal house facility of IMBB-FORTH. In this master thesis is presented the procedure followed for large scale expression and the development of an effective purification protocol. The final protein should be pure enough to pass all the quality control assays and the final in vitro testing that proves its functionality.

Language

English

Subject

CRISPR

High fidelity

HypaCas9

Issue date

2018-11-23

Collection

School/Department--School of Sciences and Engineering--Department of Biology--Post-graduate theses

Type of Work--Post-graduate theses

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