Srovnání metod pro cílené genetické manipulace mikrořas a jejich praktického využití

Marianna KABÁTOVÁ

Bachelor's thesis

Comparison of methods for targeted genetic manipulations of microalgae and their practical use

Abstract:

The discipline of genetic engineering has never been more captivating than it is nowadays. The potential of altering genes gives us the tools to change the nature of diseases and modify organisms to meet our requirements. Engineered nucleases enable us to make specific changes in the genome. It started with sequencing, for it was needed to explore the genome and fast enough continued with editing techniques such as Zinc fingers nuclease, TALENs (transcription activator-like effector nucleases), so well-known CRISPR/Cas systems, and the latest Fanzors. Each editing tool is more precise than the one before. Thanks to their precise targeting of genomic sites, the research can manipulate defined locations within the genome and observe cell changes, some convenient for biotechnological use. Microalgae represent a suitable option for biotechnology as renewable and natural sources. Their fast-growing rate and ability to synthesise biotechnological interesting biomolecules indicate their entry into industries such as biofuels, pharmaceuticals, fiber, and food. This thesis will explore the potential of using engineered nucleases specifically to manipulate the genome of microalgae for biotechnological purposes. The practical part of the thesis will concentrate on the use of the CRISPR/Cas9 system, for precise manipulation of E. gracilis. To confirm the success of this method, it will focus on a particular gene knock-out and quantify the success rate of the genetic modification. The objective was on the nuclear-encoded plastid PNPase targeted by gRNA. The CRISPR/Cas9 was delivered with the electroporation of RNPs. The culture samples after electroporation were cultivated, then the DNA was isolated, and cloned with PCR. The gene knock-out was transferred to E. coli cells via heat shock, and the DNA was isolated and sequenced. Macroscopically the efficiency of the experiment is around 50 %. Additional experiments and sequencing are needed to determine optimal conditions for the electroporator delivery method and higher gene modification efficiency.

Abstract:

The discipline of genetic engineering has never been more captivating than it is nowadays. The potential of altering genes gives us the tools to change the nature of diseases and modify organisms to meet our requirements. Engineered nucleases enable us to make specific changes in the genome. It started with sequencing, for it was needed to explore the genome and fast enough continued with editing techniques such as Zinc fingers nuclease, TALENs (transcription activator-like effector nucleases), so well-known CRISPR/Cas systems, and the latest Fanzors. Each editing tool is more precise than the one before. Thanks to their precise targeting of genomic sites, the research can manipulate defined locations within the genome and observe cell changes, some convenient for biotechnological use. Microalgae represent a suitable option for biotechnology as renewable and natural sources. Their fast-growing rate and ability to synthesise biotechnological interesting biomolecules indicate their entry into industries such as biofuels, pharmaceuticals, fiber, and food. This thesis will explore the potential of using engineered nucleases specifically to manipulate the genome of microalgae for biotechnological purposes. The practical part of the thesis will concentrate on the use of the CRISPR/Cas9 system, for precise manipulation of E. gracilis. To confirm the success of this method, it will focus on a particular gene knock-out and quantify the success rate of the genetic modification. The objective was on the nuclear-encoded plastid PNPase targeted by gRNA. The CRISPR/Cas9 was delivered with the electroporation of RNPs. The culture samples after electroporation were cultivated, then the DNA was isolated, and cloned with PCR. The gene knock-out was transferred to E. coli cells via heat shock, and the DNA was isolated and sequenced. Macroscopically the efficiency of the experiment is around 50 %. Additional experiments and sequencing are needed to determine optimal conditions for the electroporator delivery method and higher gene modification efficiency.

Keywords

Design nucleases, CRISPR/Cas9, microalgae, biotechnology, microalgal bioproducts, PNPase

Language used: English

Date on which the thesis was submitted / produced: 20. 5. 2024

Accessible from:: 31. 12. 2999

Thesis defence

• Date of defence: 3. 6. 2024
• Supervisor: Mgr. Jiří Pergner, Ph.D.

University of Ostrava

Faculty of Science

Bachelor programme / field:
Biology / Experimental Biology