Efficient induction of a heritable chromosomal inversion in Solanaceae using CRISPR/Cas

Overview

This research program focuses on developing an efficient method to induce heritable chromosomal inversions in Solanaceae species using the CRISPR/Cas system. Chromosomal rearrangements such as inversions are valuable for breeding and trait improvement, but current CRISPR-based methods produce them only rarely. By combining DNA-free protoplast transformation, optimized Cas protein selection, and a hierarchically structured bulk-to-individual screening workflow, the program aims to dramatically increase the throughput and frequency of identifying inversion events in tomato and related species.

Technical specifications

Core approach:

• DNA-free genome editing using pre-assembled CRISPR-Cas9 ribonucleoprotein (RNP) complexes delivered into protoplasts
• Protoplast transfection and regeneration methods optimized for Solanaceae, achieving targeted mutagenesis efficiencies of approximately 60%
• Selection of the most efficient Cas proteins and target sites through protoplast-based cleavage efficiency analysis
• Induction of two simultaneous double-strand breaks to generate inversions in the megabase pair (Mbp) range
• PCR-based bulk screening protocol adapted from Arabidopsis to efficiently process more than 1,500 transformants
• Junction-specific primers and droplet digital PCR (ddPCR) to determine genome-engineering efficiency
• Whole genome sequencing to confirm inversion accuracy
• Alternative Agrobacterium-mediated transformation pathway available if protoplast regeneration is not successful

Key capabilities:

• High-throughput generation and screening of edited transformants
• DNA-free approach suitable for breeding and commercialization requirements
• Expertise spanning Solanaceae genome editing, transformation, and regeneration

Technology readiness level

The underlying CRISPR-based inversion protocols have been demonstrated in Arabidopsis and Maize, where screening of approximately 1,500 plants yielded inversion events at frequencies of 0.1 to 0.5%. In Solanaceae, DNA-free protoplast-based editing has already achieved high mutagenesis efficiency, but inducible chromosomal rearrangements have not yet been reported. The current program is at the early-to-mid R&D stage, focused on adapting and validating these protocols for Solanaceae species, optimizing protoplast regeneration, and establishing the high-throughput screening pipeline needed to reliably recover inversion events.