UC Berkeley

Climate change imposes a severe threat to agricultural systems, food security, and human nutrition. Meanwhile, efforts in crop and livestock gene editing have been undertaken to improve performance across a range of traits. Gene editing applications for climate change specifically have converged on four major traits: nutritional quality improvement, yield enhancement, disease tolerance, and abiotic stress tolerance, with the fewest current applications directed towards abiotic stress tolerance. While only few applications of gene editing have been translated to agricultural production thus far, numerous studies in research settings have demonstrated the potential for potent gene editing based solutions to address climate change in the near future. Gene editing of rice (Oryza sativa) specifically holds promise for generating climate resilient foodscapes. Rice is of paramount importance for global nutrition, supplying at least 20% of global calories. However, water scarcity and increased drought severity are anticipated to reduce rice yields globally. Rice stomatal developmental genetics were explored as a mechanism to improve drought resilience while maintaining yield under climate stress. CRISPR/Cas9-mediated knockouts of EPFL10 and STOMAGEN yielded lines with c. 80% and 25% of wild-type stomatal density, respectively. epfl10 lines with moderate reductions in stomatal densities are able to conserve water to similar extents as stomagen lines, but do not suffer from the concomitant reductions in stomatal conductance, carbon assimilation, or thermoregulation observed in stomagen knockouts. Moderate reductions in stomatal densities achieved by editing EPFL10 may be a climate-adaptive approach in rice that can safeguard yield. Editing the paralog of STOMAGEN in other species may provide a means to tune stomatal density in agriculturally important crops beyond rice.

Negative pleiotropic effects of gene editing may be mitigated by editing a single copy of a duplicated gene underlying a trait of interest. However, this approach is limited by a narrow set of duplicated genes whose null phenotype is not deleterious to overall plant fitness. Promoter editing is emerging as an increasingly relevant tool to generate subtle trait variation while mitigating against harmful pleiotropy. We applied a multiplexed, guide design approach informed by bioinformatic analyses to generate genotypic variation in the promoter region of OsSTOMAGEN. Engineered genotypic variation corresponded to continuous variation stomatal density and size. This near-isogenic panel of stomatal variants was leveraged in physiological assays to establish discrete relationships of stomatal density with a range of gas exchange parameters. Developmental plasticity in response to vegetative drought was inhibited in some promoter alleles and in stomagen. Derived stomatal variants can be matched with similarly broad environmental conditions to optimize. Collectively our data suggest a role of promoter editing as a tool for establishing trait variation including phenotypic gain-of-function that can be leveraged for establishing relationships of anatomy and physiology and for crop improvement along diverse environmental clines.

In securing food systems against the severe implications of climate change, gene editing approaches towards the adaptation of rice to abiotic stress has shown promise. An additional approach makes use of gene editing for improving crop quality in crops with existing tolerance. To this end, we sought to improve the safety of the drought-stress-tolerant cassava crop, for human consumption using CRISPR/Cas9. Cassava accumulates cyanogenic glucosides which are human-toxic-metabolites that must be removed to avoid severe human health consequences. Cyanogenic glucosides may play an important physiological role in cassava plants, so eliminating their synthesis entirely may also limit overall productivity. Our work sought to engineer tissue specific accumulation of cyanogenic glucosides by editing, MeCGTR1, a putative systemic transporter using CRISPR/Cas9. cgtr1 lines exhibited depletions of cyanogenic glucosides in upper leaves while maintaining wild-type levels in tuberous roots. Together with a phloem girdling assay, our data indicated a root-to-shoot mode of cyanogenic transport which stands in contrast to previously documented modes of detected movement. Our work provides the first in-vivo validation of a cyanogenic glucoside transporter in cassava providing evidence for the de novo biosynthesis of cyanogenic glucosides in roots.