Generation of new antibiotic selections for prokaryotic CRISPR gene editing
Disciplines
Molecular Genetics
Abstract (300 words maximum)
The investigation of gene function often involves gene knock-out or mutational analysis. These procedures can sometimes be insufficient or difficult to achieve. CRISPR technology is a promising new advance in this field, exploiting a naturally occurring prokaryotic defense system to effect changes in specific DNA sequences known as gene editing. This system can be used to generate direct insertion or deletions in targeted gene sequences. This is possible because the structural RNA molecule targeting the gene of interest (pTarget) and the endonuclease required for cutting the DNA and introducing the mutation (pCas) are carried on plasmids. These plasmids may be transformed into suitable hosts for selection and gene editing. Herein lies the obstacle and focus of our study. Some of the organisms we are interested in studying here at KSU, namely Pseudomonas (Griffin) and Aeromonas (McGarey) bacteria, are naturally resistant to the antibiotic markers on the plasmids carrying the CRISPR system. We present the generation of modified CRISPR plasmids with new resistance markers for gentamycin and tetracycline. We seek to knock-out the natural antibiotic resistance in the Pseudomonas and Aeromonas strains to test our new CRISPR plasmids before continuing onto our investigation of metabolic and regulatory pathways of these bacteria. Upon successful generation, these plasmids will be available to the scientific community for prokaryotic gene editing.
Academic department under which the project should be listed
CSM - Molecular and Cellular Biology
Primary Investigator (PI) Name
Melanie C. Griffin
Generation of new antibiotic selections for prokaryotic CRISPR gene editing
The investigation of gene function often involves gene knock-out or mutational analysis. These procedures can sometimes be insufficient or difficult to achieve. CRISPR technology is a promising new advance in this field, exploiting a naturally occurring prokaryotic defense system to effect changes in specific DNA sequences known as gene editing. This system can be used to generate direct insertion or deletions in targeted gene sequences. This is possible because the structural RNA molecule targeting the gene of interest (pTarget) and the endonuclease required for cutting the DNA and introducing the mutation (pCas) are carried on plasmids. These plasmids may be transformed into suitable hosts for selection and gene editing. Herein lies the obstacle and focus of our study. Some of the organisms we are interested in studying here at KSU, namely Pseudomonas (Griffin) and Aeromonas (McGarey) bacteria, are naturally resistant to the antibiotic markers on the plasmids carrying the CRISPR system. We present the generation of modified CRISPR plasmids with new resistance markers for gentamycin and tetracycline. We seek to knock-out the natural antibiotic resistance in the Pseudomonas and Aeromonas strains to test our new CRISPR plasmids before continuing onto our investigation of metabolic and regulatory pathways of these bacteria. Upon successful generation, these plasmids will be available to the scientific community for prokaryotic gene editing.