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