Nian Wang

Efficiently Generates Transgene-Free, Gene-Edited Plants in the T0 Generation by Co-Editing the ALS Gene and Gene(s) of Interest

This genome editing tool, stemming from a co-editing strategy, efficiently generates transgene-free, gene-edited plants in the T0 generation, improving plant genetics. “Transgene-free” is a prerequisite for the commercialization of genetically modified organisms. Transgenic crops are under robust and strict regulations in various countries and regions and have a negative public perception, impeding their commercialization. Agrobacterium-mediated transformation is a method for producing genome-editing plants, which are commonly transgenic. Transgenic approaches cause regulatory and public concerns, often generating new and off-target mutations in the next generation. For perennials and vegetatively propagated plants, removing transgenes is difficult and time-consuming. Many crops lose traits of the parental cultivars from backcrossing. It is indispensable to remove transgenes from CRISPR-edited transgenic plants. Additionally, transgenes in plants such as citrus and apple are not removable through seed segregation once they’ve integrated into the plant genome. Transgene-free genome editing is challenging but highly desirable for plant genetic improvement.

Researchers at the University of Florida have developed a genome editing tool for generating transgene-free, gene-edited plants in the T0 generation by co-editing plants’ ALS genes. The generation of the transgene-free gene-edited plants occurs via Agrobacterium-mediated transient expression of cytosine base editor (CBE)/gRNA-Cas12a/crRNA-GFP in planta. The ALS gene encodes acetolactate synthase, catalyzing the first step in the synthesis of the branched-chain amino acids. Once the ALS gene is inhibited in presence of herbicides, plants are killed due to starving of these amino acids .The CBE/gRNA-mediated ALS editing confers resistance to the herbicides, which can be employed as a selection marker. Pro188 of ALS in citrus was selected for base editing without fitness loss. Most importantly, Cas12a/crRNA can target any genes of interest. This co-editing strategy is a potent tool with broad applications for plant genetic improvement.

Application

Co-edits the ALS gene and gene(s) of interest, generating transgene-free, genome-edited plant varieties in the T0 generation

Advantages

• Generates transgene-free lines without T-DNA integration, eliminating gene function disruptions at the insertion site
• Requires no backcrossing or selfing, eliminating the loss of traits from parental cultivars
• Generates transgene-free gene-edited plants in the T0 generation, bypassing the removal of transgenes in future generations and expediting genetic improvement
• Co-edits the ALS gene (Pro188 or its equivalents) in plants, generating herbicide-resistant plants with no negative effects on their phenotypes or fitness
• This strategy is easily adoptable, enabling its use for gene-editing perennials, vegetatively propagated plants, and citrus
• A highly efficient tool, generating transgene-free plant varieties with no off-target mutations

Technology

This genome editing tool generates transgene-free, gene-edited plants in the T0 generation through Agrobacterium-mediated transient expression of cytosine base editor (CBE)/gRNA-Cas12a/crRNA-GFP in planta. It is highly desirable for plant genetic improvement and expedition, particularly in perennials and vegetatively propagated plants. Transgene-free genome-edited plants originate by employing T-DNA carrying CBE/gRNA-Cas12a/crRNA-GFP to co-edit the ALS gene. By mutating the ALS genes using CBE, diverse plant species develop herbicide resistance, providing a practical selection marker as a gain-of-function against sulfonylurea herbicides. Precise editing by CBE targets the proline residue, disrupting the recognition and binding of the herbicides without affecting ALS function. Cas12a/crRNA edits gene(s) of interest, while GFP enables the selection of transgene-free transformants.

The co-editing strategy hinges on the Agrobacterium-mediated delivery of CRISPR components into recipient plant tissues. It is easily adoptable because Agrobacterium-mediated transformation is one of the most widely used and convenient methods. It has efficiently generated transgene-free, genome-edited genes in the T0 generation of tomato and tobacco plants (annuals), citrus plants (a perennial tree crop), and potato plants (vegetatively propagated tetraploid crop). The biallelic/homozygous transgene-free mutation rates for target genes among herbicide-resistant transformants range from 8% to 50%. The higher efficiency and herbicide resistance make this strategy a vital tool for plant genetic improvement and genetic studies of plants.