This project seeks to combine two powerful, new approaches to accelerate breeding of crops adapted to environmental stresses, providing new tools to deal with agricultural challenges such as minimizing loss from drought or heat. One of the technical approaches used in the project, known by its acronym CRISPR -- creates genetic variation much the same way it occurs randomly in nature but with much greater speed, ease and target precision. The second technique, known as single-cell RNA-sequencing, allows researchers to measure activity of, potentially, every gene in a single cell. Together, the two methods allow a "miniaturization" of the breeding approach -- testing many crop variants to find ones with useful agricultural traits. Different, single cell variants can be generated by the millions using CRISPR, subjected to a stress in which only the fittest cells survive, and those cells analyzed for the precise changes that allowed them to survive using single-cell RNA-sequencing. This method, used in conjunction with classical breeding approaches, can serve as a resource for plant breeders and geneticists working to understand the genetic regulators of traits that are important for stable crop production. In the education goals of the project, the investigators will extend a successful program that trains talented New York City high student interested in a career in science.
More specifically, this project aims to develop pooled CRISPR/single-cell RNA-seq screens in maize protoplasts to introduce a novel approach to plant breeding and genetic analysis. Using CRISPR and associated endonucleases like Cas9, large-scale genetic screens will be carried out in which a diverse set of guide-RNAs is transfected into a pool of millions of cells. The cells are then subjected to a selection; survivors identify beneficial perturbations while underrepresented genotypes in the pool identify genes necessary to survive the stress. Coupled with single-cell RNA-seq, this approach provides a sensitive readout of genetic pathways, and has been used successfully in animal cells. The method has not yet been applied to plants and could not only represent an increase in the scale of crop screening but also reveal genetic targets for plant breeding that have previously been obscured by genetic redundancy. With the ability to screen millions of cells, libraries can also be transfected at titers that lead to multiple guides per cell, effectively allowing higher order mutants to be screened quickly and easily. Many important stress tolerance traits are known to be mediated at the cell level, and marker assisted selection allows screening for targeted pathways that regulate tissue level traits. The project will assess the efficiency of CRISPR in maize cells in pilot experiments that target the auxin signaling pathway using auxin response markers as a selection tool.
This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
