RNAi-mediated silencing of target native gene(s) and CRISPR-mediated, targeted knockout of a gene represent two powerful tools to study gene function and to engineer useful traits in plants. Gossypol, present in most parts of the cotton plant, serves as a defense chemical against certain pests and pathogens. Gossypol and related terpenoids are synthesized and stored in lysigenous glands present in most aboveground parts of the plant. However, gossypol’s presence also limits the suitability of the seeds for human and animal nutrition. Selective, seed-specific RNAi-mediated silencing of the members of a gene family encoding δ-cadinene synthase, which catalyzes a key step in gossypol biosynthesis, reduced gossypol levels by 97% exclusively in the seeds. One such ultra-low gossypol cottonseed (ULGCS) line, TAM66274, has been deregulated in the U.S. and its seeds are considered safe for consumption as food and as feed for animals. Although CRISPR/Cas9-mediated knockout of the CGF3 gene, a key regulator of gland development, in a cotton plant eliminated the glands and their contents including gossypol in the seeds and other aboveground parts of the plant, it could not achieve the specificity of selective RNAi silencing. Thus, although it is possible to achieve seed-specific silencing of the target gene(s) using RNAi, it is not possible to eliminate their expression. In contrast, using the CRISPR system it is possible to knock out a target gene and its function entirely, tissue-specificity is difficult to obtain. Our investigations revealed both the positive attributes and limitations of RNAi and CRISPR/Cas9 technologies.
