Gerald Klaas

Abstract The current technologies to place new DNA into specific locations in plant genomes are low frequency and error-prone, and this inefficiency hampers genome-editing approaches to develop improved crops 1,2 . Often considered to be genome ‘parasites’, transposable elements (TEs) evolved to insert their DNA seamlessly into genomes 3–5 . Eukaryotic TEs select their site of insertion based on preferences for chromatin contexts, which differ for each TE type 6–9 . Here we developed a genome engineering tool that controls the TE insertion site and cargo delivered, taking advantage of the natural ability of the TE to precisely excise and insert into the genome. Inspired by CRISPR-associated transposases that target transposition in a programmable manner in bacteria 10–12 , we fused the rice Pong transposase protein to the Cas9 or Cas12a programmable nucleases. We demonstrated sequence-specific targeted insertion (guided by the CRISPR gRNA) of enhancer elements, an open reading frame and a gene expression cassette into the genome of the model plant Arabidopsis . We then translated this system into soybean—a major global crop in need of targeted insertion technology. We have engineered a TE ‘parasite’ into a usable and accessible toolkit that enables the sequence-specific targeting of custom DNA into plant genomes.

The success of many engineered crop traits depends on the stable expression of transgenes, but their effectiveness is frequently at risk due to transgene silencing. The reason why certain transgenes are targeted by silencing pathways while others remain highly expressed and durable has remained a major question for decades due to the lack of technologies to study the initiation of transgene silencing. We developed 2 technologies to identify the trigger of transgene silencing in Arabidopsis (Arabidopsis thaliana) and in lettuce (Latuca sativa): one using the RUBY transgene to visualize the precise developmental time point of transgene silencing and the second to identify all transcripts produced from a transgene. By combining these 2 methods with Machine Learning, we identified an aberrant transgene-derived RNA that accumulates to high levels and closely correlates with the onset of transgene silencing. Our data suggest that a ribosome stalled at an unusual 3-consecutive-histidine peptide sequence on the RUBY transcript triggers No-Go RNA Decay and cleavage of the RUBY mRNA. The production of this cleaved aberrant RNA precedes RNA interference during the triggering of transgene silencing; it is innate to the transgene coding sequence, independent of the promoter used or whether it is transformed into a model plant or crop.

<title>Abstract</title> The current technologies to place new DNA into specific locations in plant genomes are low frequency and error-prone, and this inefficiency hampers genome editing approaches to develop improved crops. Often considered genome ‘parasites’, transposable elements (TEs) evolved to insert their DNA seamlessly into genomes. TEs select their site of insertion based on preferences for chromatin contexts, which differ for each TE type. We developed a genome engineering tool that controls the TE insertion site and cargo delivered, taking advantage of the TE’s natural ability to precisely insert into the genome. Inspired by CRISPR-associated transposases (CASTs) that target transposition in a programmable manner in bacteria, we generated a synthetic CAST by fusing the rice <italic>Pong </italic>TE transposase protein to the Cas9 or CPF1 programmable nucleases. We demonstrated sequence-specific targeted delivery (guided by the CRISPR gRNA) of enhancer elements, an open reading frame and gene expression cassette into the genome of the model plant Arabidopsis. We additionally translated this system into soybean, a major global crop in need of targeted insertion technology. We have engineered a TE ‘parasite’ into a usable and accessible toolkit that enables the sequence-specific targeting of custom DNA into plant genomes.

Improvement and research of plants depends on the long-term expression of transgenes. However, the durability of transgene expression is routinely hampered by silencing pathways that start as the post-transcriptional process of mRNA degradation by RNA interference (RNAi). To avoid transgene silencing, we aimed to inhibit the sorting of transgene mRNAs into RNAi. We manipulated a well-studied protein/RNA-binding module from Arabidopsis into a transgene transcript, where the transcript is now bound by an engineered RNA-binding protein that preferentially sorts the RNA into translation. We used the Cas9 transcript as a proof-of-principle and demonstrated higher Cas9 protein production and gene editing rates. In addition, transgenes with the engineered protein/RNA-binding module had improved long-term durability of transgene expression, as after several inbred generations these plants had higher Cas9 protein accumulation and lower levels of DNA methylation, a hallmark of transgene silencing. Our engineered system represents a successful manipulation of post-transcriptional RNA sorting for improved transgene performance, and could be applied to any transgene transcript.