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