CRISPR/CAS9 SYSTEMS FOR PLANT GENOME EDITING

Abstract

The increasing burden of the world population on agriculture requires the development of more robust crops. Dissecting the basic biology that underlies plant susceptibility to pathogens and drives crop loss will inform the design of better crops. One powerful tool for studying plants at the molecular level is the RNA-programmed genome editing system comprised of a clustered regularly-interspaced short palindromic repeats (CRISPR) encoded guide RNA (gRNA) and the nuclease Cas9. CRISPR/Cas9 evolved as form of acquired immunity in bacteria and archaea but can be re-engineered to edit and regulate eukaryotic genomes. The Cas9 nuclease encoded by Streptococcus pyogenes (SpCas9) is most commonly used for genome editing but is accompanied by strict requirements for nuclease activity. Here, we constructed and applied Cas9 systems for genome editing in plants with Cas9 derived from Streptococcus thermophiles (StCas9) and Neisseria meningitidis (NmCas9) to expand on methods for gneome editing with CRISPR/Cas9 in tobacco (Nicotiana benthamiana),. These results demonstrate an orthogonal CRISPR/Cas9 system that could be further utilized for genome editing and simultaneous gene regulation in plants, also providing a framework for synthetic transcriptional programming. Translating this toolkit into crop species will provide increased control over plant genomes, catalyzing new basic and applied discoveries to advance plant biology.