Editing Crop Genomes to Advance Agriculture

Targeted DNA double-strand breaks (DSB) can substantially increase the frequency of genome editing through homology directed repair. In the past decade, several site-directed nucleases have been utilized toward this end, but the recently discovered RNA-guided Cas9 endonuclease has truly revolutionized the field due to its simplicity, activity, and versatility. In plants, the ability to generate targeted DSB has three major applications: gene mutagenesis, gene editing, and site-specific gene insertion. Here we report a successful application of the Cas9-guide RNA system as an efficient tool for all three genome editing approaches in maize. Biolistic transformation of Cas9, guideRNAs, without or with repair templates, faithfully directed sequence alteration at five different genomic regions: upstream of the liguleless-1 gene, two male fertility genes, MS26 and MS45, and two acetolactate synthase genes, ALS1 and ALS2. Mutations were identified at all target sites often recovering plants with bi-allelic multiplex mutations. DSBs generated Cas9-guide RNA also stimulated gene insertion at a site near liguleless-1 while gene editing of ALS2 resulted in plants resistant to chlorsulfuron. In addition, transient delivery of guide RNA (as RNA molecules) and repair template directly into immature embryo cells containing pre-integrated Cas9 also resulted in ALS2 editing. These examples represent advances in new breeding technologies and demonstrate the potential of Cas9-guide RNA to accelerate plant biology to help meet demands on global agriculture.