Advancing genetic engineering toolbox in plants
Plant genetic engineering emerges as a promising avenue for imparting new traits, including enhanced resilience to environmental conditions, heightened crop yields, and improved quality. Nevertheless, engineering of plants faces multiple hurdles, ranging from the need for better understanding of plant genomes and expression profiles to the development of advanced DNA editing tools.
We aim to establish the CRISPR-associated transposases (CAST) in plants for programmable and high efficiency DNA integration, merging CRISPR RNA-guided targeting with high insertion efficiency of transposases. Such biotechnology for plants will enable basic discoveries in plant genomics, such as the identification of essential genes and screening of ideal locus for exogenous gene insertion and expression. It will also allow improved capabilities, such as building developmental or metabolic pathways to provide biotic and abiotic stress tolerance, battle new plant epidemics and adverse effects of climate change, and enable scalable and affordable biosynthesis of valuable products in plants.
Targeted DNA Insertion in Plants by CRISPR-Transposases
The CAST system is separated into three parts for expression. Helper plasmid (pHelper) contains all essential CAST proteins and guide RNA in monocistronic cassettes. We designed monocistronic cassettes for helper expression. Donor plasmid (pDonor) contains the donor DNA, which is the cargo sequence flanked by transposon left and right ends (LE and RE). The whole transposon part is incorporated in a geminivirus-derived replication vector, which is designed to increase the donor DNA copy number to increase the integration efficiency.
Target plasmid (pTarget) contains the intended target site for integration, which is only used in the episomal approach. In the chromosomal approach, target sites are selected from native genomic sequences.
The proof of concept of CAST functioning in plant system is separated into two paradigms. Episomal integration means the integration taking place in a free plasmid, and the only common and feasible method to introduce free plasmids is PEG-mediated protoplast transfection. Chromosomal integration means the integration taking place in genomic DNA. Depending on the tissue being leaf or flower, the transformation can be transient or stable. Going forward, we aim to optimize integration efficiency, cargo size, and eventually engineer a stable transgenic line. Our efforts hold promise for advancing precision plant genome engineering with potential impact in plant biological research and agricultural biotechnology.
Develop assays for detecting heterologous protein expression
The expression constructs were designed with a CAST component fused to both HiBiT and NLS tags. Among many methods explored, we eventually developed an optimized HiBiT lytic assay for nuclear localized protein.
Individual constructs were infiltrated into the N. benthamiana leaves using the Agroinfiltration approach and samples were collected two days after the infiltration. LgBiT reagent was added to protein lysate solution. LgBiT will complex with HiBiT tag, generating luminance as an indicator of the presence of the HiBiT-tagged protein. Our successful detection of protein expression establishes a robust foundation for future endeavors aimed at optimizing heterologous protein expression.
Related publications
Yunqing Wang and Gozde S. Demirer. Synthetic biology for plant genetic engineering and molecular farming.
Trends in Biotechnology (2023).
