Novel Genetic Variation Generated By CRISPR-Cas System for Improving Drought Tolerance in Maize

Lack of sufficient water is a major limiting factor to crop production worldwide and developing drought tolerant germplasm is needed to improve crop productivity. Natural genetic variation for traits that impact drought tolerance has been used in maize breeding programs to increase grain yield. Transgenes also showed efficacy in enhancing plant tolerance to drought stress. Targeted improvement of genes responsible for drought tolerance is desirable, but until recently, generating allelic variation with physically or chemically induced mutagenesis was a random process, which made it difficult to produce intended DNA sequence changes at a target locus. In the past few years, efficient genome editing technologies have emerged, enabling rapid and precise manipulation of DNA sequences. Maize ARGOS8 is a negative regulator of ethylene responses. When overexpressed, ARGOS8 confers drought tolerance in maize. Although natural variation exists in the ARGOS8 gene, the native diversity is limited and unsuitable for targeted drought breeding. To generate novel variants, we essentially duplicated and relocated genomic DNA fragments in maize genome to modify the ARGOS8 locus by using a CRISPR-Cas-enabled breeding technology. Natural rearrangement of DNA fragments via segmental duplication and translocation occurs spontaneously at a low frequency in plants and has been exploited by breeders over the decades for maize improvement. Using the CRISPR-Cas9 system, the native maize GOS2 promoter, which confers a moderate level of constitutive expression, was inserted into the 5’-untranslated region of the native ARGOS8 gene or was used to replace the native promoter of ARGOS8. Precise genomic DNA modification at the ARGOS8 locus was verified by PCR and sequencing. The ARGOS8 variants had elevated levels of ARGOS8 transcripts relative to the native allele and these transcripts were detectable in all the tissues tested. A field study showed that compared to the WT, the ARGOS8 variants increased grain yield by 5 bushels per acre under flowering stress conditions and had no yield loss under well-watered conditions. These results demonstrate the utility of the CRISPR-Cas9 system in generating novel allelic variation for breeding drought tolerant crops.