Marker-free gene editing platform for cereal crops using inducible cre-loxp auto-excision

Overview

This platform addresses a critical bottleneck in cereal crop gene editing: the absence of a cost-effective method to produce transgene-free edited plants that can be rapidly moved from tissue culture to field trials. Although SDN-1 gene editing outcomes have been deregulated in Australia and many other countries, regulators require confirmation that CRISPR components have been segregated out before field testing can proceed. By incorporating an inducible Cre-LoxP auto-excision system into the editing construct, the platform removes selective markers and CRISPR/Cas9 components during late callus stages, yielding regenerants that are free of integrated transgenes. This allows edited cereal crops to enter field testing in the T1 generation, accelerating the translation of gene editing research into agronomic validation.

Technical specifications

• Auto-excision mechanism: Heat-inducible promoter drives expression of Cre recombinase, which recognises LoxP sites flanking the CRISPR/Cas9 components and selective marker, excising them from the genome during late callus stages.
• Delivery method: Agrobacterium-mediated transformation with newly designed plasmids containing all editing components within LoxP sites.
• Target crops: Sorghum and barley, both with established, robust in-house transformation pipelines using transformable genotypes.
• Editing target: The PDS gene serves as a visual reporter to assess editing efficiency.
• Validation approach: PCR-based screening of regenerated plantlets to confirm the absence of transgenes before field release.
• Alternative promoters: Backup inducible promoters are available should the heat-inducible system compromise callus regenerability due to heat stress.

Technology readiness level

The platform is at an early development stage. Plasmids incorporating the heat-inducible Cre-LoxP system are being designed and constructed, with planned Agrobacterium-mediated transformation of embryogenic calli in sorghum and barley. The estimated timeline for initial proof-of-concept completion is approximately one year, covering construct design, plasmid purchase, tissue culture work, and PCR-based transgene screening. Successful demonstration of transgene-free regenerants in these two cereals would establish a foundation for broader application across additional monocot crops.