Use of the pre-assembled ribonucleoprotein (RNP) complex for gene editing of wheat and poplar

Genetic modification has been a useful research tool to study overexpression and down-regulation of genes. Gene editing offers a much more targeted approach to make more subtle changes to genes already present within the plant. However, integration of Cas9 and guide RNA sequences into the genome can be disadvantageous causing offsite cleavage and/or continued cleavage of target sites in subsequent generations. Use of purified protein could overcome this issue and would also avoid reliance on the cell’s expression machinery thus making effective promoters less essential. The use of purified proteins would also address regulatory issues as no recombinant DNA of any kind would have been involved in the process so resultant plants need not be classified as GMOs. Without the use of selectable markers, screening is likely to be challenging especially if efficiencies of editing with the RNP complex are not anticipated to be very high. However, with constantly improving sequencing methods, speed of data output and reducing costs, screening may become progressively more straightforward. Attempts are therefore being made to utilise a pre-assembled ribonucleoprotein complex for gene editing in wheat and poplar. Initial experiments using purified gus protein have demonstrated that it is possible to deliver protein to cells using biolistics. Formation of an active ribonucleoprotein complex has been achieved in vitro using purified Cas9 enzyme and synthetic single guide RNAs. Both wheat and poplar are now being targeted with a pre-assembled RNP complex which is being delivered via gold particles and the BioRad PDS-1000/He particle gun.