Optimization of nucleic acid delivery via cationic polymers for genome engineering

Date

2019

Author

Öktem, Ayşegül

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One of the most challenging aspects of genome engineering is the delivery of genome editing components such as plasmids, oligonucleotides, RNA and protein. In this work, in-house synthetized cationic polymer poly (2-hydroxypropylene imine) (pHP) was tested in order to achieve substantial delivery efficiency while preserving high culture viability. Applicability of this cationic polymer mediated nucleic acid delivery method for both plant and mammalian cells were demonstrated. Several parameters of plasmid and oligonucleotide delivery were optimized. It was demonstrated that the working concentration of the synthesized pHP was 2 mg/mL for maize protoplasts and 10 mg/mL for HEK239 cell line. Most crucial parameter for plasmid delivery was DNA to polymer ratio. For oligonucleotide delivery, it was discovered that if the polyplex sizes kept smaller, the delivery efficiency increased. Additionally, it was shown that increased amounts of oligonucleotides and polymer have adversely affected culture viability. Finally, CRISPR/Cas9 and Oligonucleotide Targeted Nucleotide Exchange (OTNE) mediated genome editing by delivering the editing components via cationic polymer was investigated. Successful CRISPR/Cas9 mediated knockout of genomic GFP gene was demonstrated in plant cells. Use of this polymer as a mediator of nucleic acid delivery to plant cells may have further applications in genome engineering in hard to transform plant cells.

Subject Keywords

Genomes., Genome engineering, cationic polymer, plant transformation, CRISPR/Cas9, Oligonucleotide Targeted Nucleotide Exchange

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http://etd.lib.metu.edu.tr/upload/12623516/index.pdf
https://hdl.handle.net/11511/43801

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Graduate School of Natural and Applied Sciences, Thesis