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Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Published on: May 25, 2018

Trait stacking via targeted genome editing.

William M Ainley¹, Lakshmi Sastry-Dent, Mary E Welter

¹Dow AgroSciences LLC, Indianapolis, IN, USA.

Plant Biotechnology Journal

|August 20, 2013

Summary

This summary is machine-generated.

This study introduces a novel method for stacking multiple crop traits using targeted genome editing and

Keywords:

designed zinc finger nucleases gene targeting transgene stacking

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Area of Science:

Plant biotechnology
Crop genetics
Molecular biology

Background:

Modern agriculture requires crops with multiple traits for enhanced yield and resilience.
Current methods for introducing multiple traits into crops face significant breeding challenges due to random gene integration.
Efficient trait stacking is crucial for developing advanced crop varieties.

Purpose of the Study:

To develop and demonstrate a versatile platform for targeted transgene integration and trait stacking in crop plants.
To utilize engineered zinc finger nucleases (ZFNs) and modular 'trait landing pads' (TLPs) for precise, on-demand gene stacking.
To validate the technology by stacking herbicide resistance genes in maize.

Main Methods:

Integration of a herbicide resistance gene (pat) flanked by a TLP into the maize genome using WHISKERS™ transformation.
Targeted integration of a second herbicide resistance gene (aad1) into the TLP via microparticle bombardment of immature embryos.
Co-transformation with donor DNA containing the aad1 gene and a ZFN expression construct for precise targeting.

Main Results:

Successfully integrated the pat gene with a TLP into the maize genome.
Achieved precise integration of the aad1 transgene into the TLP in up to 5% of transgenic events, directly adjacent to the pat gene.
Demonstrated cosegregation of both herbicide resistance traits in subsequent generations, confirming transgene linkage.

Conclusions:

Nuclease-driven TLP technology provides a facile and rapid approach for on-demand trait stacking in crops.
This method overcomes downstream breeding challenges associated with random transgene integration.
The ZFN-mediated targeted integration platform is applicable across diverse crop species, offering a versatile solution for crop improvement.