
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.
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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.
Retroelement-mediated genome insertion into the 28s rDNA safe harbor loci in plants
Another group of transposons, non-long terminal repeat (LTR) retrotransposons, utilize an RNA intermediate to transpose into genomic safe harbor loci, specifically within ribosomal RNA sites, which are present in multiple copies across most eukaryotic genomes, including those of plants. During target primed reverse transcription (Figure 1), the open reading frame encoded by the retrotransposons (retroelements) utilizes its own RNA as a template to synthesize complementary DNA within a very specific site of the 28s rDNA. Non-LTR retroelements hold the advantage of having smaller open reading frames ~3kb and they are able to integrate similar size payloads ~4kb with high on-target specificity. They also achieve these genomic insertion events without reliance on double-stranded DNA breaks or heavy reliance on host machinery.

We have adapted the eukaryotic non-LTR retroelements from Bombyx mori, and Taeniopygia guttata for targeted insertions into the multicopy 28S ribosomal DNA safe harbor site via RNA-mediated target-primed reverse transcription (TPRT) in plants, which is the first-time application of retroelements for plant genetic engineering. A retrotransposition reporter carrying the YFP reporter gene containing an intron (Figure 2), was used to genetically encode the RNA template to be utilized for genome insertion. We successfully integrated a fluorescent reporter casette in Nicotiana benthamiana leaves using retroelements confirmed via imaging and amplicon sequencing.
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Next, we aim to associate retrotransposons to CRISPR effectors, such as Cas9, to achieve RNA guided-targeted insertions in other genomic locations. Current work is aimed at enhancing the efficiency of retroelement-mediated targeted insertions, hence providing a more effective tool for targeted plant genome insertions. Recent studies demonstrate that RNA molecules can be modified for mobility to meristem tissue. We aim to engineer retroelement RNA templates for meristematic mobility, enabling stable genomic integration without introducing transgenic sequences—addressing the challenge of transformation tools that can penetrate meristematic tissues.
Related publications
Yunqing Wang and Gozde S. Demirer. Synthetic biology for plant genetic engineering and molecular farming.
Trends in Biotechnology (2023).
