Co-editing strategy for isolating rare chromosomal inversion events in plants

Overview

This solution addresses a key bottleneck in plant genome engineering: the isolation of rare chromosomal inversion events induced by RNA-guided nucleases. Because such inversions occur at very low frequency, researchers traditionally need to screen large populations of edited individuals to find the desired event. This approach uses a co-editing strategy that leverages herbicide-resistance variants of the ALS or EPSP genes as dominant markers to identify edited plants, without requiring T-DNA integration. A conditionally toxic counter-selection gene eliminates T-DNA-containing events, leaving only clean, non-transgenic inversion events for screening.

Technical specifications

• Dominant herbicide markers: Precision edits in ALS (chlorsulfuron resistance) or EPSP (glyphosate resistance) serve as co-editing markers, enabling visual identification of edited individuals after Agrobacterium-mediated reagent delivery
• No T-DNA integration required: The herbicide-resistance edits function as selection markers without stable transgene insertion, yielding non-transgenic edited plants
• Counter-selection system: A conditionally toxic gene included in the T-DNA kills transformed cells in the presence of an anti-metabolite, applied during regeneration on herbicide-resistant calli to eliminate T-DNA insertion events
• Orthogonal CRISPR systems: Compatible with multiple nuclease platforms, including SpCas9 for base or prime editing, and SaCas9 or Cas12 for double-strand break induction at inversion breakpoints
• Multi-species validated: Negative selection with the conditionally toxic gene has been tested in four different species with near 100% efficiency
• Pooled PCR screening: DNA pooling strategies enable efficient inversion detection across large edited populations

Technology readiness level

The individual components have been validated in prior work: herbicide-resistance ALS and EPSP variants have been successfully used as co-editing markers in multiple plant species, and the conditionally toxic counter-selection gene has demonstrated near-complete elimination of T-DNA-containing events across four species. The full co-editing-to-inversion pipeline has not yet been executed end-to-end. Near-term validation steps include analyzing ALS and EPSP genes in target species to design editing approaches, assembling and testing T-DNA vectors in transient assays with amplicon sequencing confirmation, identifying efficient guide RNAs for inversion breakpoints, engineering orthogonal CRISPR T-DNA constructs, and producing editing events through Agrobacterium-mediated transformation followed by regeneration, counter-selection, and PCR-based inversion screening on DNA pools.