Delivery of CRISPR/Cas Components into Higher Plant Cells for Genome Editing

CRISPR/Cas genome editing of plants is realized in three basic variants, including knockout mutations as indels, insertion of alien DNA fragments, and base editing via deamination of nitrogenous bases. The most important stages of the CRISPR/Cas-based genome editing are the choice of a target site, design of guide RNAs, creation of genetically engineered constructions, and delivery of CRISPR/Cas components into a plant cell. Rapid developments in the field of plant genome editing with the use of CRISPR/Cas systems requires more detailed consideration of the last stage, so this review is dedicated to the description of the main ways to deliver CRISPR/Cas components into cells of higher plants. In the first studies on the genome editing of different plant species, these components were delivered to the target site mainly by Agrobacterium tumefaciens. This approach supposes integration of T-DNA into a genome and a stable expression of CRISPR/Cas components or their transient expression in the case of agroinfiltration. Another widespread approach included the use of plant viruses as delivery platforms; in this case, viruses were used mainly for production of an increased amount of guide RNAs that significantly improved the efficiency of genome editing. Another approach provides for the use of another bacterium, A. rhizogenes, as a platform for delivery of CRISPR/Cas components. This bacterium induces hairy root formation that may be an indirect confirmation of successful genome editing and assist in the selection of genetically modified forms. Other common ways to obtain genetically edited plants are the biolistic delivery of genetically engineered constructions into explants and various protoplast transformation technologies. The review also considers some issues transgenic and GM status of CRISPR/Cas-edited plants to transgenic and GM plants. There are a number of cases in which new organisms created by a CRISPR/Cas genome editing without any introduction of alien DNA were not considered as transgenic ones; it is quite possible that such plants will not fall under Russian legislation prohibiting GMO cultivation.