Crispr/cas9 targeted genome inversion platform for vegetable crop improvement

Overview

BetterSeeds is developing a CRISPR/Cas9-based platform for creating targeted genome inversions in vegetable crops. Chromosomal inversions are powerful breeding tools that can lock in favorable gene combinations, suppress recombination in specific regions, and accelerate the development of elite varieties. By inducing double-strand breaks at two flanking sites around a target region, the platform enables precise chromosomal rearrangements without introducing foreign DNA. This approach offers seed companies a non-transgenic route to novel trait variation and improved crop performance.

The platform addresses two key challenges in plant genome engineering: inversion efficiency and heritability. By working in a Ku70 knockout background, the system promotes error-prone repair pathways that increase inversion frequency. By driving Cas9 expression with gamete-specific promoters, the platform reduces chimerism and ensures that induced inversions are stably transmitted to subsequent generations.

Technical specifications

Core editing strategy:

• Dual guide RNAs (gRNAs) designed upstream and downstream of a selected genomic region, typically around 2 Mbp, to flank the inversion target
• Cas9 induces simultaneous double-strand breaks at both sites, stimulating chromosomal rearrangement through endogenous repair mechanisms

Efficiency enhancement via Ku70 knockout:

• Knockout of the Ku70 homolog in the target crop shifts DNA repair toward microhomology-mediated non-homologous end joining
• This error-prone pathway increases inversion event frequency compared to wild-type backgrounds
• Trade-off: reduced ligation precision, though acceptable for many breeding applications

Heritability enhancement via gamete-specific expression:

• Gamete-specific promoters (including egg cell-specific promoters) cloned upstream of Cas9 to restrict expression to reproductive cells
• Reduces somatic mosaicism and chimeric outcomes
• Ensures stable, heritable inversions transmitted across generations

Validation approach:

• Dual background experiments comparing inversion efficiency and ligation precision in Ku70 knockout versus wild-type plants
• Phenotypic comparison of edited plants against unedited wild-type controls to assess trait impacts

Technology readiness level

The proposed inversion platform is at an early-to-mid stage of development. The scientific foundation is established through published studies in Arabidopsis and related species demonstrating the feasibility of CRISPR/Cas9-mediated chromosome engineering and gamete-specific editing. BetterSeeds' broader gene-editing platform is operational and applied across multiple specialty crops, but the specific combination of Ku70 knockout backgrounds with gamete-specific promoter-driven Cas9 for targeted inversions in vegetable crops has not yet been validated in-house. Next steps include gRNA design for target regions, identification of crop-specific Ku70 homologs, cloning of gamete-specific promoters, and controlled experiments to benchmark inversion efficiency and phenotypic outcomes.