Multiplex Plant Gene Targeting through Homologous Recombination (20160266, Dr. Daniel Voytas)

Technology No. 20160266
IP Status: Pending US Patent; Application #: 16/344,496

Precisely and simultaneously modifies multiple chromosomal sequences

This new method uses homologous recombination to achieve first-ever multiplexed gene targeting in plants. The novel approach uses a geminivirus replicon to overcome traditional gene targeting challenges for plant genome engineering. The technology efficiently achieves multiplexed gene targeting by integrating a selective marker into one of the loci to regenerate plants with modifications in other loci at a high frequency. Modified wheat plants in experiments were regenerated in less than six weeks and showed a promising multiplexed gene targeting frequency of 1.1% (e.g., 13.75% of the cells that underwent gene targeting contained both modifications). Advantages of multiplex homologous recombination include simultaneous gene edits to confer multiple traits, reduced time to introduce traits into crop plants, and introducing gene edits for academic purposes.

Geminivirus-based replicons and CRISPR/Cas9 deliver SSNs and DNA repair templates into plant cells

Unlike targeted mutagenesis, gene targeting and gene repair in plants is quite difficult. Targeted modification of plant genomes remains a challenge due to ineffective methods for delivering reagents for genome engineering to plant cells. Furthermore, the few reports that describe gene targeting in plants are limited to only one chromosomal target. This method uses geminivirus-based replicons and CRISPR/Cas9 as a delivery method for sequence-specific nucleases (SSNs) and DNA repair templates into plant cells. The technique achieves multiple targeted integration of nucleotide sequences into different loci of the plant genome, as well as into different genomes in polyploid species. This is the first time that homologous recombination at two different loci within the same cell has been demonstrated.

Phase of Development

  • Proof of concept. Demonstrated efficiency of replicon-based system. Showed that multiplexed integration can occur within the same cell.


  • Highly efficient gene targeting at multiple genomic sites in a single cell
  • Shortens the time to make multiple genetic changes to a plant species
  • Easier and cheaper to create crops with improved traits


  • Multiplex homologous recombination
  • Simultaneous gene edits confer multiple traits
  • Multiplexed targeted integration of more than one sequence into the same plant cell, or into different homoeoalleles of the same gene in polyploid species
  • Makes genetic changes in complicated polyploidy species (e.g., many crops)
  • High frequency (~1.1%) of gene targeting
  • Efficient targeted integration of promoter-less genes, complete genes or modified endogenous sequences


  • Ag biotech
  • Genome editing
  • Gene editing of crops

Daniel Voytas, PhD
McKnight Presidential Endowed Professor; Director, Center for Precision Plant Genomics
External Link (

High-efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR/Cas9
The Plant Journal, (2017) 89, 1251–1262

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