CRISPR-Cas9 Technology in Plant Genomics Research: A Review and Tutorial for Implementation with Considerations for Orchid Genetic Studies

The study and identification of gene mutations resulting in alterations to plant phenotype form the basis of plant genetic screens. Assigning function to particular loci or genes based on mutated alleles is critical for breeding programs in order to establish key genetic markers responsible for desirable traits. Forward genetics studies involving traditional mutagenesis by physical or chemical mutagens have largely been limited to plants with rapid generations, as identifying causal mutations requires repeated crossing to eliminate as many background mutations as possible. Orchids, with generations as long as 3–10 years, have been mainly excluded from genetic screens. Therefore, much remains unknown about their genomes and genes controlling key traits of floricultural, medicinal, or agronomic value. Targeted mutagenesis technologies such as CRISPR-Cas9 provide precise control over genetic manipulations in as few as one generation but are heavily dependent on past functional genomics research (in the same plant or a close relative) and accurate genetic records. Further efforts toward the assembly and annotation of orchid and other plant genomes will allow for greater implementation of targeted mutagenesis technology for rapid, selective breeding as well as reverse genetics studies.