Engineered crispr-cas nucleases for precise chromosomal inversions in plants

Overview

This technology leverages engineered CRISPR-Cas9 nucleases fused to viral 5'-exonucleases to enable precise chromosomal inversions in plants. By introducing double-strand breaks at each end of a target chromosomal fragment and supplying short donor templates with homology arms matching the desired inverted outcome, the platform dramatically increases the frequency of homology-directed repair (HDR). This addresses a major bottleneck in plant genome engineering, where HDR is naturally rare, and opens new possibilities for synthetic chromosome restructuring, gene regulatory studies, and crop improvement.

Technical specifications

Key features:

• Engineered Cas9 fused to viral 5'-exonucleases that resect DNA ends to generate free 3' overhangs, promoting strand invasion into supplied homologous templates
• Up to 30-fold increase in HDR frequency compared to non-engineered Cas9 in both transient and stable transformation systems
• Intron-containing Cas9 architecture that boosts expression and editing efficiency
• Dual sgRNA strategy targeting junctions flanking the chromosomal fragment to be inverted
• Donor template design with 250-300 bp homology arms on each side of the inversion to ensure precise stitching of the inverted fragment
• Agrobacterium-mediated transformation delivery incorporating selection markers, Cas9-Exo fusions, sgRNAs, and donor fragments

Technology readiness level

The core HDR-enhancing Cas9-exonuclease fusion technology has been validated in transient and stable plant transformation experiments, with results available in a preprint. Preliminary work on intron-enhanced Cas9 expression has been published. The chromosomal inversion application is at an advanced research and proof-of-concept stage, with planned validation including sgRNA efficiency screening via amplicon sequencing, PCR-based identification of inversion events among approximately 100-200 transformants, and segregation of T-DNA constructs in subsequent generations. The technology is ready for collaborative validation and pilot applications in partner-relevant plant species.