Research Projects

Conventional plant genetic engineering methods, including Agrobacterium-mediated transformation and biolistic delivery, rely on random transgene integration, which is a major bottleneck that often leads to positional silencing, erratic transgene expression patterns, and the need for extensive screening to isolate optimal events. Although strategies such as in planta gene targeting have attempted to address these limitations by leveraging homology-directed repair (HDR), their low insertion efficiency remains a significant challenge (Molla et al., 2022). Recent advances in hybrid systems, which combine CRISPR/Cas with recombinases or transposases, offer a breakthrough by enabling precise and high-efficiency targeted integration (Sun et al., 2024; Liu et al., 2024). For staple crops like rice, where unintended disruptions from random insertion can impair agronomic performance, developing robust methods for site-specific integration of large DNA fragments (approx. 5 kb) into genomic safe harbours (GSHs) is crucial. To this end, we propose to develop a robust CRISPR-based tool for integrating large DNA fragments in genomic safe harbour (GSH) loci by comparing and combining the components of two cutting-edge platforms: the recombinase-based PrimeRoot system (Sun et al., 2024) and the transposon-based TATSI (Transposase-Assisted Targeted Site Integration) approach (Liu et al., 2024). By optimizing a robust system for GSH-directed insertion in rice (utilizing the GSH for Oryza sativa var. Kitaake established by Sun et al. in 2024), we aim to establish standardized protocols for reliable, position-effect-free transgene expression for synthetic biology. As a translational demonstration, we will integrate the 5.2 kb Golden Rice carotenoid biosynthesis cassette into a validated GSH, generating vitamin A-biofortified lines with stable, high-level carotenoid production. Our experimental pipeline begins with rigorous validation of diverse design of vectors using transient expression in rice protoplasts. We will use a well established Protoplast transfection system developed in the host lab (Panda et al. 2024) for rapidly validating different editing reagents and strategies through deep sequencing. For stable transformation, we will employ a two-step Agrobacterium-mediated protocol: first introducing CRISPR/Cas constructs under hygromycin selection, followed by delivery of donor plasmids carrying the reporter or the Golden Rice payload. To validate large DNA integrations at scale, we will employ Nanopore adaptive sampling, selectively enriching and sequencing genomic safe harbour (GSH) loci in positive transformants to resolve complete insertion structures with single-molecule precision. For the Golden Rice-3 prototype plants, β-carotene accumulation will be quantified using HPLC across independent transgenic lines. Whole-genome sequencing of selected lines will also be performed to detect potential off-target integrations.

CRISPR/Cas, Genomic Safe Harbours (GSHs), Large DNA Insertion, Golden Rice, Biofortification, Site-Specific Integration