Gene and Genome Editing with CRISPR/Cas Systems for Fruit and Vegetable Improvement

Ever since the advent of agriculture, breeding new varieties has relied upon crosses between individuals from a single species, and since the early twentieth century with relatives or via mutagenesis. Two major problems have been found time and again. First, combining genomes to improve a character often times causes decreases in other traits as a result of genetic linkage. The second is that natural variation does not always comprise all the possibilities a genome may have in terms of allelic combinations suitable for further improving a set of characters. In the last twenty years a number of technologies have been developed allowing the perturbation of a single gene. Development of genome editing technologies includes zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and Clustered Regularly Interspaced Palindromic Repeats (CRISPR). Here we review current methodologies regarding to the use of gRNA targeted gene and genome editing strategies by various CRISPR/Cas9 systems in agriculture. The molecular mechanism of DNA modification by CRISPR/Cas relies on guide RNA molecules comprising 20–25 DNA bases homologous to the target locus. This has opened the possibility of tackling single loci or multiple paralogs in a gene family. Importantly, complex genomes with polyploid structures such as wheat or camelina have been successfully engineered with single guides. This opens a new window of opportunities to engineer gene families, pathways and complex genomes that was unfeasible before the advent of CRISPR/Cas.