Transposase-assisted target-site integration for efficient plant genome engineering.

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 crops1,2. Often considered to be genome 'parasites', transposable elements (TEs) evolved to insert their DNA seamlessly into genomes3-5. Eukaryotic TEs select their site of insertion based on preferences for chromatin contexts, which differ for each TE type6-9. 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 bacteria10-12, 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.

Broken up but still living together: how ARGONAUTE's retention of cleaved fragments explains its role during chromatin modification.

Throughout the eukaryotic kingdoms, small RNAs direct chromatin modification. ARGONAUTE proteins sit at the nexus of this process, linking the small RNA information to the programming of chromatin. ARGONAUTE proteins physically incorporate the small RNAs as guides to target specific regions of the genome. In this issue of Genes & Development, Wang and colleagues (pp. 103-118) add substantial new detail to the processes of ARGONAUTE RNA loading, preference, cleavage, and retention, which together accomplish RNA-directed chromatin modification. They show that after catalytic cleavage by the plant ARGONAUTE protein AGO4, the cleaved fragment remains bound. This happens during two distinct RNA cleavage reactions performed by AGO4: first for a passenger RNA strand of the siRNA duplex, and second for a nascent transcript at the target DNA locus. Cleaved fragment retention of the nascent transcript explains how the protein complex accumulates to high levels at the target locus, amplifying chromatin modification.