(470f) Targeted DNA Insertion in Plants By CRISPR-Associated Transposons

Synthetic biology and biomolecular engineering advances have developed powerful approaches to control gene expression and editing spatiotemporally to ensure engineered organisms do not suffer from fitness cost and that the product titer can be increased. However, the inability to control the genomic insertion site of biosynthetic pathways in plants given tool limitations have been crippling plant engineering and biomanufacturing efforts. Researchers have used CRISPR-Cas9 homology-directed repair (HDR) for targeted gene insertion in plants to express biosynthetic pathways¹. Yet, this still has a single-digit efficiency hindering progress.

Here, we developed a high efficiency targeted gene insertion resource in plants to better understand, control, and improve plant bioengineering. We establish the CRISPR-associated transposon (CAST) system in plants for programmable and high efficiency DNA integration, merging CRISPR RNA-guided targeting with high insertion efficiency of transposases. CAST was discovered in bacteria and has recently been adapted to human cells^2,3. As of yet, its feasibility and efficiency in plants is unknown.

We reconstituted the Type I-F CASTs from V. cholerae and Pseudoalteromonas in model plants Arabidopsis thaliana and Nicotiana benthamiana. We achieved episomal and chromosomal integration of a ~1 kb fluorescent reporter DNA in single plant cells in solution and in intact plant leaf cells, respectively, with efficiencies around 15%, substantially higher than of HDR. Ongoing efforts focus on increasing the cargo size, insertion efficiency, performing heritable transformations using CASTs, and translating to crops.

An efficient and targeted gene insertion tool facilitates fundamental biological understanding of plant genomes and identifies genomic safe harbor sites for inserting biosynthetic pathways with optimal outputs in plant biomanufacturing.

References:

1. Dong & Ronald. Targeted DNA insertion in plants. PNAS (2021).
2. Vo et al. CRISPR RNA-guided integrases for high-efficiency, multiplexed bacterial genome engineering. Nat. Biotechnol. (2021).
3. Lampe et al. Targeted DNA integration in human cells without double-strand breaks using CRISPR-associated transposases. Nat. Biotechnol. (2023).