Genome editing of upstream open reading frames enables translational control in plants

Translational regulation by upstream open reading frames (uORFs) is becoming established as a general mechanism for controlling the amount of protein that is synthesized from downstream primary ORFs (pORFs)(1-5). We found that genome editing of endogenous uORFs in plants enabled the modulation of translation of mRNAs from four pORFs that are involved in either development or antioxidant biosynthesis. A single-guide RNA that targeted the region harboring a uORF initiation codon can produce multiple mutations. Following uORF editing, we observed varying amounts of mRNA translation in four pORFs. Notably, editing the uORF of LsGGP2, which encodes a key enzyme in vitamin C biosynthesis in lettuce, not only increased oxidation stress tolerance, but also increased ascorbate content by similar to 150%. These data indicate that editing plant uORFs provides a generalizable, efficient method for manipulating translation of mRNA that could be applied to dissect biological mechanisms and improve crops.