LAUSR

RNA editing is currently attracting attention as a method for editing genetic information without injury to the genome. The most common approach to edit RNA sequences involves the induction of an A-to-I change by adenosine deaminase acting on RNA (ADAR). However, this method only allows point editing. Here, we report a highly flexible RNA editing method called "RNA overwriting" that employs the influenza A virus RNA-dependent RNA polymerase (RdRp) comprising PA, PB1, and PB2 subunits. RdRp binds to the 5'-cap structure of the host mRNA and cleaves at the AG site, followed by transcription of the viral RNA; this process is called cap-snatching. We engineered a targeting snatch system wherein the target RNA is cleaved and extended at any site addressed by guide RNA (gRNA). We constructed five recombinant RdRps containing a PB2 mutant and demonstrated the editing capability of RdRp mutants by using short RNAs in vitro. PB2-480-containing RdRp exhibited good performance in both cleavage and extension assays; we succeeded in RNA overwriting using PB2-480-containing RdRp. In principle, this method allows RNA editing of any type including mutation, addition, and deletion, by changing the sequence of the template RNA to the sequence of interest; hence, the use of viral RdRp could open new avenues in RNA editing and be a powerful tool in life science.