Evaluation of various deletion strategies using PGI gene as a model
(2018) KMBM01 20171Applied Microbiology
Biotechnology
- Abstract
- As the gene engineering area is the basis of nowadays molecular biology, the efficient tool of gene modification is needed. Despite already existing tools, dependent on DNA-protein interactions, such as zink finger nucleases (ZNFs) or transcription activator-like effector nucleases (TALENs), they are limited by the specificity to the target sequence or the time need for the design of such molecule. Such limitations narrow down the potential application of these methods. Since the CRISPR-Cas9 method relies on the RNA-DNA interaction, it required only the proper gRNA sequence design, therefore both the efficiency and specificity are improved, compared to ZNFs and TALENs. On the other hand, the success of the experiment depends on the gRNA... (More)
- As the gene engineering area is the basis of nowadays molecular biology, the efficient tool of gene modification is needed. Despite already existing tools, dependent on DNA-protein interactions, such as zink finger nucleases (ZNFs) or transcription activator-like effector nucleases (TALENs), they are limited by the specificity to the target sequence or the time need for the design of such molecule. Such limitations narrow down the potential application of these methods. Since the CRISPR-Cas9 method relies on the RNA-DNA interaction, it required only the proper gRNA sequence design, therefore both the efficiency and specificity are improved, compared to ZNFs and TALENs. On the other hand, the success of the experiment depends on the gRNA target sequence’s uniqueness. To asses CRISPR-Cas9 as the editing tool, the phosphoglucose isomerase (PGI) in Saccharomyces cerevisiae has been chosen for the deletion, due to its vital role in the central carbon metabolism. Along with the novel method, widely established and naturally occurring, deletion via homologous recombination with the clonNAT resistance marker was conducted. Results gathered in this project, indicate that the proper gRNA sequence design is crucial for the successful CRISPR-Cas9 application and gene targeting. In terms of deletion via homologous recombination, the clonNAT marker did not replace PGI but was incorporated in the genome in the not specific position. Overall, results prove the vital role of the phosphoglucose isomerase in yeast’s metabolism. (Less)
- Popular Abstract
- For years, gene engineering has been a main interest of many researchers all over the world. The possibility of inserting, deleting or enhancing genes, and therefore developing organisms with desired features, has plays an important role in many industry areas. One of the newest tools within gene editing is CRISPR/Cas9 method. Due to the simple interaction of the chosen organism’s DNA and the user-designed guide RNA, this method works as the molecular scissors, cutting the chosen DNA sequence therefore making a room for the modifications.
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/8934860
- author
- Miroszewska, Dominika LU
- supervisor
- organization
- course
- KMBM01 20171
- year
- 2018
- type
- H2 - Master's Degree (Two Years)
- subject
- keywords
- ZNFs, TALENs, PGI, deletion, CRISPR-Cas9, gRNA, mutagenesis, OE PCR, applied microbiology
- language
- English
- id
- 8934860
- date added to LUP
- 2018-02-19 14:47:49
- date last changed
- 2018-02-19 14:47:49
@misc{8934860, abstract = {{As the gene engineering area is the basis of nowadays molecular biology, the efficient tool of gene modification is needed. Despite already existing tools, dependent on DNA-protein interactions, such as zink finger nucleases (ZNFs) or transcription activator-like effector nucleases (TALENs), they are limited by the specificity to the target sequence or the time need for the design of such molecule. Such limitations narrow down the potential application of these methods. Since the CRISPR-Cas9 method relies on the RNA-DNA interaction, it required only the proper gRNA sequence design, therefore both the efficiency and specificity are improved, compared to ZNFs and TALENs. On the other hand, the success of the experiment depends on the gRNA target sequence’s uniqueness. To asses CRISPR-Cas9 as the editing tool, the phosphoglucose isomerase (PGI) in Saccharomyces cerevisiae has been chosen for the deletion, due to its vital role in the central carbon metabolism. Along with the novel method, widely established and naturally occurring, deletion via homologous recombination with the clonNAT resistance marker was conducted. Results gathered in this project, indicate that the proper gRNA sequence design is crucial for the successful CRISPR-Cas9 application and gene targeting. In terms of deletion via homologous recombination, the clonNAT marker did not replace PGI but was incorporated in the genome in the not specific position. Overall, results prove the vital role of the phosphoglucose isomerase in yeast’s metabolism.}}, author = {{Miroszewska, Dominika}}, language = {{eng}}, note = {{Student Paper}}, title = {{Evaluation of various deletion strategies using PGI gene as a model}}, year = {{2018}}, }