Engineering an RNA-protein binding module for higher transgene protein production and improved long-term durability [BSAS]

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 transcript cleavage 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, rather than RNAi. We used the Cas9 transcript as a proof-of-principle and demonstrated higher Cas9 protein production and editing rates. In addition, transgenes with the engineered protein / RNA-binding module had improved long-term durability of transgene expression, as after four 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. Methylation profiling by high throughput sequencing of 4 GmRPS5a:Cas9 with various 3'UTR transgenic Arabidopsis lines, with 2-6 biological replicates for each line.