Genome Editing in Plants with CRISPR/Cas9

Zinc finger nucleases (ZFNs), were introduced by Sigma-Aldrich less than 8 years ago, but in that time the technology of targeted genome editing has advanced rapidly. Most recently, the discovery of the CRISPR/Cas9 pathway has accelerated interest in this field, opening up new possibilities for research and development. Although the CRISPR pathway was identified in bacteria as part of a putative adaptive immune system, it was quickly adapted to the purpose of modifying eukaryotic genomes.

While tools like ZFNs laid the groundwork for genome editing today, there are limitations to this founding technology and others like it: the protein:DNA interaction of ZFNs makes designing them complex, the assembly of the ZFN expression construct is time-consuming, and the options for ZFN targeting are limited in many A-T rich plant genomes.

The CRISPR pathway, as it has been adopted and modified for eukaryotic genome editing, overcomes many of these hurdles: it relies on an RNA:DNA interaction to find its genomic target, the recognition sequence required for this binding event is an easily altered 18-20 base pairs, and the only requirement for the nuclease binding is the presence of an NGG next to the target site.

The first reports of the use of CRISPR/Cas9 in plants studying transient expression assays using Agrobacterium came out in 2013. The CRISPR/Cas9 technology has been successfully applied in model plants (Nicotiana benthamiana, Arabidopsis thaliana) and crops (rice, wheat) and the list is growing.