An efficient <scp>mRNA</scp> delivery system for genome editing in plants

Transgene-free genome editing is important for crop improvement as it reduces unanticipated genomic changes. While mRNA delivery systems offer a powerful method for achieving transgene-free genome editing, they remain inefficient and challenging in plants. Here we describe an efficient mRNA delivery system for plants with substantially improved editing efficiency. By optimizing the 5' untranslated regions (5'UTRs) and poly(A) tails of in vitro-transcribed (IVT) mRNAs and coating the mRNA with protamine during particle bombardment, we have developed an optimized mRNA delivery system termed v2_TMV/DEN2. This system enhanced the efficiencies of knock-out, A-to-G and C-to-T base editing by an average 4.7-, 3.4- and 2.5-fold at various endogenous sites compared with plasmid-based transient delivery system via particle bombardment in rice suspension cells and wheat immature embryos 48 h post-transformation. Furthermore, we obtained edited plants with efficiencies of 5.0-180.8% and 26.1-26.2% using v2_TMV/DEN2 in rice and wheat, respectively, compared with 0.0-43.2% and 4.7-10.4% using plasmids. Our study provides a convenient and efficient mRNA delivery system for transgene-free genome editing in plants.