Page 1 Page 2 Page 3 Page 4 Page 5 Page 6 Page 7 Page 8 Page 9 Page 10 Page 11 Page 12 Page 13 Page 14 Page 15 Page 16 Page 17 Page 18 Page 19 Page 20 Page 21 Page 22 Page 23 Page 24 Page 25 Page 26 Page 27 Page 28 Page 29 Page 30 Page 31 Page 32 Page 33 Page 34 Page 35 Page 36 Page 37 Page 38 Page 39 Page 40 Page 41 Page 42 Page 43 Page 44 Page 45 Page 46 Page 47 Page 48 Page 49 Page 50 Page 51 Page 5248 I EUROPEAN SEED I EUROPEAN-SEED.COM GIANT VIEWS BY: JAN SCHAART AND RENÉ SMULDERS enome editing is a promising new technique for plant breeding. With designer nucleases called meganucleases, zinc finger nucleases, and TALENs, mutations can be precisely directed to any gene of interest. CRISPR- Cas9 is the latest innovation, as it can easily be ‘programmed’ by a separate guide RNA and is currently the chief genome editing tool used. The nucleases only create a DNA break at the desired location in the genome, which is subsequently repaired by the cell’s DNA repair mechanism. The mode of repair used by the cell determines the outcome of the reparation. DNA repair following non-homologous end joining (NHEJ) (which simply joins the ends of the broken DNA together) is relatively error-prone and may result in small errors (small deletions or insertions) at the location of the repaired DNA break. Homology-directed repair (HDR) uses a repair template (for example the homologous chromosome, or one that is supplied during the experiment) for accurate, error-free repair. In plants NHEJ is the predominant repair mechanism. LOSS-OF-GENE-FUNCTION MUTATIONS Deletions induced by CRISPR-Cas followed by NHEJ-repair may cause a reading frame shift. This often leads to premature G Jan Schaart GENOME EDITING AS NEW PLANT BREEDING TECHNIQUE: OPPORTUNITIES AND CHALLENGES stop codons in the coding sequence of a gene, causing loss of gene function. Almost all publications about genome editing in plants so far describe the induction and selection of plants with such loss-of- function mutations. These studies have resulted in a substantial series of plants with novel traits. For example, mutation of disease susceptibility genes (S-genes) has resulted in powdery mildew resistance in bread wheat, in a broad potyvirus resistance in cucumber, while rice was made resistant to certain pathotypes of bacterial leaf blight. Deletion of genes involved in starch biosynthesis produced high amylopectin (“waxy”) maize and potato varieties. High oleic acid oilseed crops soybean and Camelina were made by knockouts in genes involved in further steps in the fatty acid biosynthesis. IMPROVED GENE FUNCTION So, switching off gene functions may create improved varieties, but even more is possible by improving the function of already existing genes. Using CRISPR- Cas and a repair template containing a superior allele of the targeted gene could result in plants with improved gene functions. The reason why few publications report on allele replacements is the extremely low frequency of HDR (DNA repair using a template) in plants. Tricks such as co-inserting selection markers (e.g. herbicide resistance genes) together with the new allele enable positive selection for HDR events, but this gives end products containing additional undesirable genes. Undoubtedly, various future improvements of the methods used will enable allele replacement without leaving behind ‘foreign DNA’. GENOME EDITING IS NOT EASY An important reason why researchers succeeded in the application of HDR for replacement of promoter sequences in maize was the availability of extremely optimized methods for maize plant transformation. In addition, the simple genetics (diploid) and the availability of a high-quality genomic DNA sequence also contributed to the success of gene editing experiments in maize. It is clear that (optimal) transformation methods, simple genetics and genome information are lacking for many small crops, including many horticultural species. OTHER ISSUES TO CONSIDER To achieve targeted mutations by CRISPR- Cas, the nuclease system has to be delivered into the plant cell. Straightforward methods stably introduce the gene constructs for CRISPR-Cas and its accompanying guide RNA(s) into the plant’s genome, and after the intended mutations have been induced, the CRISPR-Cas genes are removed, for exam- ple by crossing and selecting null-segregants that inherit the induced mutation, but not the construct. Crossing is not suitable for heterozygous crops, diploid or polyploid, in which varieties are vegetatively propagated or that have a long generation time. As a solution, methods for transient expression of the CRISPR-Cas9 machinery have been developed and several publications show that plants with the intended mutations can be recovered without presence of the nuclease constructs. CONCLUSION A lt hou g h genome ed it i n g u si n g programmable nucleases is recognized as a revolution in plant breeding, there are still several technical hurdles to take. The quick succession of nucleases and improvements of methods indicates that over time genome editing may evolve into a generally applied technology. Editor’s Note: Jan Schaart in Scientific Researcher and René Smulders is Business Unit Manager at Wageningen UR Plant Breeding in The Netherlands René Smulders