Targeted site integration in plants using a transposon system [insertion-seq]

Genome editing tools with high precision are key to develop improved crops but current technologies to place new DNA into specific locations in plant genomes are low frequency and error-prone. Transposable elements (TEs) evolved to insert their DNA seamlessly into genomes, albeit in a quasi-random pattern. We developed a genome engineering tool that controls the TE insertion site and subsequently the delivery of any cargo attached to this TE. Using our tool, we demonstrated sequence-specific targeted delivery (guided by the CRISPR gRNA) of enhancers, an open reading frame and gene expression cassette into the genome of the model plant Arabidopsis, and we translated this technology to the crop soybean. We have engineered a 'junk' TE into a useful and accessible toolkit that enables the sequence-specific targeting of custom DNA into plant genomes. Overall design: Insertion-Seq was performed to detect genome-wide insertion sites of the transposable element mPing in various transgenic lines of both Arabidopsis and soybean. Custom libraries were constructed using an adapted HtStuf protocol (PMID: 33228280) with modifications as mentioned below.