Browsing by Subject "Tool Development"

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    Developing Methods To Overcome The Bottlenecks In Plant Gene Editing
    (2020-10) Nasti, Ryan
    Altering plant species to make crops for human use is a centuries old practice. Through traditional breeding, most of the extant crop varieties used today have been optimized to increase yield and growth potential. Despite advancements over time, current breeding is limited in its throughput. In order to address increasingly problematic issues such as climate change, a multitude of technologies have been developed to improve the speed and ease of this process. Technologies such as CRISPR/Cas based gene editing and innovative plant transformation methods promise to greatly democratize the ability to generate new cultivars of crops. Plant gene editing is typically performed by delivering gene editing reagents (e.g. Cas9 and sgRNAs) to explants in culture. Edited cells are induced to differentiate into whole plants by exposure to various hormone regimes. This process serves as a major bottleneck in current plant biological studies since creating edited plants through tissue culture has many drawbacks: the process is often inefficient, requires considerable time, works with limited species and genotypes, and causes unintended changes to the genome and epigenome. Developing methods to circumvent these issues by allowing for simpler delivery and less intensive regeneration across a wide number of species would obviate this bottleneck. Looking first to optimize editing reagent delivery, a simple and scalable reagent delivery method was required. To this end, improvements were made to existing Agrobacterium tumefaciens based co-culture methods, yielding a streamlined fast treated co-culture (Fast-TrACC) reagent delivery method for rapid reagent testing. The Fast-TrACC procedure combines treatment of Agrobacterium to increase T-DNA transfer and a luciferase reporter to allow for high-throughput screening after co-culture. Application of this method to deliver reporters, gene editing components and test expression elements in a number of dicot species has successfully demonstrated the broad usefulness of this delivery method. Next looking at the major bottleneck of regeneration, ways to overcome the tedious and time consuming nature of current regeneration practices were necessary. Advances in ectopic expression of plant developmental regulators have demonstrated the feasibility of inducing somatic cells to form meristems. A high-throughput method was developed to optimize combinations of developmental regulators for induction of meristems on dicot seedlings. After analysis of several different developmental regulator combinations, the combinations of ZmWUS2 & AtSTM as well as ZmWUS2 & ipt were found to be most effective in generating de novo shoots. When these developmental regulators are co-delivered with gene editing reagents, induced meristems produce fertile plants that transmit gene edits to progeny. The de novo induction of gene edited meristems sidesteps the need for tissue culture, promising to overcome a current bottleneck in plant gene editing. These methods were designed and established to function effectively in the model plant, Nicotiana benthamiana, but application in a crop species, such as tomato, is necessary to realize the technology’s full potential. Classical editing with tissue culture in tomatoes has been used to alter traits such as locule number and branching. Despite this ability to generate desired mutations, the key bottlenecks still exists in regenerating whole plants after editing has occurred. This limitation comes from the combination of tedious explant manipulation and subsequent regeneration. Based upon the inferences made from N. benthamiana, ectopic overexpression of ZmWUS2 & ipt was shown to promote re-differentiation of somatic tissues into meristems. By combining DRs with gene editing reagents, we have demonstrated edited tomato lines can be quickly established, thus circumventing the need for tissue culture. Using these tools, new avenues of engineered across plant species become more accessible, thus allowing for improvements in accelerated breeding, de novo domestication and reengineering metabolism in a simpler fashion than what was previously been possible.