CRISPR/Cas9-mediated knockout of the RDR6 gene in Nicotiana benthamiana for efficient transient expression of recombinant proteins

Main ConclusionRDR6 gene knockout Nicotiana benthamiana plant was successfully produced using CRISPR/Cas9 technology.AbstractThe production of recombinant proteins in plants has many advantages, such as safety and reduced costs. However, there are several problems with this technology, especially low levels of protein production. The dysfunction of the RNA silencing mechanism in plant cells would be effective to improve recombinant protein production because the RNA silencing mechanism efficiently degrades transgene-derived mRNAs. Therefore, to overcome this problem, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology was used to develop RNA silencing-related gene knockout transgenic Nicotiana benthamiana. We successfully produced RNA-dependent RNA polymerase 6 (RDR6), one of the most important components of the RNA silencing mechanism-knockout N. benthamiana (Delta RDR6 plants). The Delta RDR6 plants had abnormal flowers and were sterile, as with the Arabidopsis RDR6 mutants. However, a transient gene expression assay showed that the Delta RDR6 plants accumulated larger amounts of green fluorescent protein (GFP) and GFP mRNA than the wild-type (WT) plants. Small RNA sequencing analysis revealed that levels of small interfering RNA against the GFP gene were greatly reduced in the Delta RDR6 plants, as compared to that of the WT plants. These findings demonstrate that the Delta RDR6 plants can express larger amounts of recombinant proteins than WT plants and, therefore, would be useful for recombinant protein production and understanding the contributions of RDR6 to genetic and physiological events in plants.