High-Throughput CRISPR/Cas9 Mutagenesis Streamlines Trait Gene Identification in Maize

Abstract

Maize (Zea mays) is one of the most important crops in the world. However, few agronomically important maize genes have been cloned and used for trait improvement, due to its complex genome and genetic architecture. Here, we integrated multiplexed CRISPR/Cas9-based high-throughput targeted mutagenesis with genetic mapping and genomic approaches to successfully target 743 candidate genes corresponding to traits relevant for agronomy and nutrition. After low-cost barcode-based deep sequencing, 412 edited sequences covering 118 genes were precisely identified from individuals showing clear phenotypic changes. The profiles of the associated gene-editing events were similar to those identified in human cell lines and consequently are predictable using an existing algorithm originally designed for human studies. We observed unexpected but frequent homology-directed repair through endogenous templates that was likely caused by spatial contact between distinct chromosomes. Based on the characterization and interpretation of gene function from several examples, we demonstrate that the integration of forward and reverse genetics via a targeted mutagenesis library promises rapid validation of important agronomic genes for crops with complex genomes. Beyond specific findings, this study also guides further optimization of high-throughput CRISPR experiments in plants.

Long non-coding RNAs in plants: emerging modulators of gene activity in development and stress responses

“…Large-scale functional screens of lncRNAs by CRISPR/Cas9-based mutagenesis have been established in human and flies, although only a small percentage of lncRNAs showed context-specific phenotypic changes (Liu et al 2017 ). A similar system has yet to be developed for plant lncRNAs although large-scale mutagenesis populations have been created in several plant species by transformation of sgRNA libraries targeting protein-coding genes (Jacobs et al 2017 ; Lu et al 2017 ; Meng et al 2017 ; Zhang et al 2019 ; Liu et al 2020 ; Bai et al 2020 ). Finally, we need to investigate how we can effectively utilize the knowledge on beneficial lncRNAs in breeding programs to develop novel plant germplasm and elite crop varieties.…”

Optimization of multiplexed CRISPR/Cas9 system for highly efficient genome editing in Setaria viridis

“…In recent years, the CRISPR/Cas9 system has become the reagent of choice to achieve efficient genome editing in many plant and animal species due to its simplicity, robust activity, versatility, and multiplexing capability (Yin, Gao and Qiu, 2017). Using this system, several gene knockout resources have been created in rice and maize (Meng et al, 2017; Liu et al ., 2020). To generate gene knock-out resources in a plant species, high mutagenesis frequency and multiplexing capability, i.e.…”

Use of virus‐induced gene silencing to characterize genes involved in modulating hypersensitive cell death in maize

“…It should be noted that new technologies promise to improve this situation (Jones et al, 2019), but they are not yet in the public domain. The recent availability of CRISPR/Cas9 technology in maize is revolutionizing the field by allowing us to make targeted genome edits (Svitashev et al, 2016;Liu et al, 2020) but is still limited by a relatively inefficient transformation system. Populations of sequence-indexed mutant maize lines also exist, the most extensive and available of which is the UniformMu population, which consists of a population of 14,024 stocks in a uniform background of the W22 maize line, each of which carries a number of unique, stabilized transpositions of the Mu transposable element.…”