Ribonucleoprotein (RNP)-Mediated Targeted Mutagenesis in Barley (Hordeum vulgare L.)

The crop species barley is a genetic model for the small grain temperate cereals. Thanks to the availability of whole genome sequence and the development of customizable endonucleases, site-directed genome modification has recently revolutionized genetic engineering. Several platforms have been established in plants, with the most flexible one offered by the clustered regularly interspaced short palindromic repeats (CRISPR) technology. In this protocol, commercially available synthetic guide RNAs (gRNAs), Cas enzymes, or custom-generated reagents are used for targeted mutagenesis in barley. The protocol has been successfully used with immature embryo explants to generate site-specific mutations in regenerants. As the double-strand break-inducing reagents are customizable and can be efficiently delivered, pre-assembled ribonucleoprotein (RNP) complexes allow efficient generation of genome-modified plants.