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5’-exonuclease Increases Gene Editing Efficiency of Plants

Technology #20160020

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iagram depicting how the expressed 5′-exonuclease resects double-stranded break ends to promote repair by the homologous recombination (HR) pathwaySchematic of proteins resulting from expression of each configuration
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Researchers
Daniel Voytas, PhD
McKnight Presidential Endowed Professor; Director, Center for Precision Plant Genomics
External Link (cbs.umn.edu)
Managed By
BJ Haun
Technology Licensing Officer
Patent Protection

US Patent Pending 2017-0175140

Resects 5’-ends at SSN-induced double-stranded breaks

Introducing a T5 bacteriophage 5’-exonuclease simultaneously with traditional gene targeting reagents (i.e., site-specific nucleases such as CRISPR/Cas9 or a TALEN system) can increase the frequency/rate of homologous recombination in gene targeting. This novel eukaryotic (plant) cell gene editing method uses the 5’-exonuclease—applied as a protein or nucleic acid in the presence of a supplied or endogenous repair template—to cause homologous recombination between the chromosomal target broken by the nuclease and the repair template. The technology exploits the natural mechanism of homology search by exposed 3’-ends of broken double-stranded DNA that mediates homologous recombination. The 5’-exonuclease can resect the 5’-ends at the double-stranded break caused by the site-specific nuclease (SSN), potentially increasing the abundance and possibly the size of exposed 3’-ends. 5’-exonuclease is a small protein that can be expressed as a transcript fusion with the nuclease and is easily deliverable by current methods to introduce the other gene targeting reagents. In addition, it is compatible with transient editing strategies using DNA replicons to make the modification and then degrade without integrating unwanted foreign DNA.

Increased efficiency and yield of desired genetically engineered products

Low efficiency of gene targeting remains a challenge for genome engineering efforts, particularly in plants. This new method, which adds the 5’-exonuclease to a homologous recombination reaction, demonstrated a threefold increase in efficiency and yield of desired genetically engineered products (e.g., Nicotiana benthamiana and wheat cells), and when combined with geminivirus technology, achieved a 15 to 50 fold increase in gene targeting. By harnessing the natural biology of the cell, this technology does not require exposure to chemicals, small molecules or interfering RNA that could impact cellular processes unrelated to gene targeting. Furthermore, no negative effects are expected on the viability or regenerative capacity of cells exposed to this reagent.

Phase of Development

  • Proof of concept. Demonstrated in plants (tobacco and wheat cells) using CRISPR/Cas9.

Benefits

  • Threefold increase in efficiency of gene targeting and yield
  • 15-50 fold increase in gene targeting when combined with geminivirus template technology
  • No negative effects expected on viability or regenerative capacity of cells
  • Reduces labor in creating and identifying gene targeting events in eukaryotic cells
  • Easily deliverable by current methods

Features

  • T5 bacteriophage 5’-exonuclease
  • Eukaryotic (plant) cell gene editing
  • Increases the frequency/rate of homologous recombination in gene targeting
  • 5’-exonuclease resects 5’-ends at site-specific nuclease induced double-stranded breaks
  • Compatible with transient editing strategies using DNA replicons
  • Makes modification and degrades without integrating unwanted foreign DNA

Applications

  • Gene targeting in eukaryotic cells
  • Genome engineering (both plant and animal)
  • Increasing frequency/rate of homologous recombination


Interested in Licensing?
The University relies on industry partners to further develop and ultimately commercialize this technology. The license is for the sale, manufacture or use of products claimed by the patents. Please contact BJ Haun to share your business needs and licensing and technical interests in this technology.