Presenter Information

Paula PandolfiFollow

Keywords

gene editing, proteins, endonuclease, DNA cleavage, directed evolution, efficiency, mutagenesis, plasmids

Select the category the research project fits.

Life Sciences

Is this submission part of ICaP/PW (Introductory Composition at Purdue/Professional Writing)?

No

Abstract

Genome editing optimizes traits of interest by introducing specific changes to the genome of organisms. A novel genome editing tool, ASGARD, was developed in our laboratory as a more flexible tool for editing genomes without a binding site requirement, in contrast to the currently popular CRISPR systems (clustered regularly interspaced short palindromic repeats). Despite ASGARD’s attractive characteristics, it suffers from low efficiency and off-target activity. We aim to mutate ASGARD and design a screening system that selects for mutants that display highly efficient DNA cleavage activity with minimal off-target effects. As current methods fail to fulfill our needs, we created a new positive screening method based on a homing endonuclease, I-SceI. I-SceI, encoded on a plasmid, could create a deadly double stranded break in the E. coli genome unless our ASGARD mutants efficiently cut the I-SceI plasmid, rescuing the cell from I-SceI activity and preventing a double stranded break in the genome. Survived cells will then contain DNA encoding highly efficient ASGARDs for the next round of directed evolution. Using the inducible I-SceI system, we have halted cell growth by over 50%, illustrating our system’s potential as a selection screen. We now aim to rescue cell growth targeting ASGARD to the I-SceI plasmid. Using a high efficiency selection system for directed evolution could lead to a highly active programmable endonuclease which will efficiently cut dsDNA without the limitation of a binding sequence, expanding the toolkits for genome engineering

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Highly Efficient Selection System For Directed Evolution of Novel Endonuclease

Genome editing optimizes traits of interest by introducing specific changes to the genome of organisms. A novel genome editing tool, ASGARD, was developed in our laboratory as a more flexible tool for editing genomes without a binding site requirement, in contrast to the currently popular CRISPR systems (clustered regularly interspaced short palindromic repeats). Despite ASGARD’s attractive characteristics, it suffers from low efficiency and off-target activity. We aim to mutate ASGARD and design a screening system that selects for mutants that display highly efficient DNA cleavage activity with minimal off-target effects. As current methods fail to fulfill our needs, we created a new positive screening method based on a homing endonuclease, I-SceI. I-SceI, encoded on a plasmid, could create a deadly double stranded break in the E. coli genome unless our ASGARD mutants efficiently cut the I-SceI plasmid, rescuing the cell from I-SceI activity and preventing a double stranded break in the genome. Survived cells will then contain DNA encoding highly efficient ASGARDs for the next round of directed evolution. Using the inducible I-SceI system, we have halted cell growth by over 50%, illustrating our system’s potential as a selection screen. We now aim to rescue cell growth targeting ASGARD to the I-SceI plasmid. Using a high efficiency selection system for directed evolution could lead to a highly active programmable endonuclease which will efficiently cut dsDNA without the limitation of a binding sequence, expanding the toolkits for genome engineering