Genome editing of grass species via CRISPR/Cas9

Abstract

Sugarcane (Saccharum spp.) is a C4 grass widely cultivated in tropical and subtropical regions and used as a feedstock for sugar and biofuel production. Brazil is the world leader in sugarcane production, with an estimated harvest of 600,000 tons in 2021, which results in the sugar-energy sector representing 2% of Brazil's GDP. However, climate change and its environmental impacts limit the extent of cultivable areas. In this context, simultaneous biotic (agricultural pests and phytopathogens) and abiotic stresses (physical and chemical factors) directly affect the survival of agronomically important crops. Therefore, the farming industry demands new sustainable crop varieties to overcome the damage caused by these environmental and biological factors. In particular, among the abiotic stresses caused by climate change, drought is considered the main limiting factor for sugarcane yield. Under water-limiting conditions, plants trigger adaptive responses involving morphological, physiological, biochemical, and molecular changes, underpinned by the differential expression of several drought tolerance-associated genes. In recent years, "omics" technologies (e.g., genomics, transcriptomics, proteomics) contributed substantially to a better understanding of the molecular basis of plant-environment interactions. Moreover, the plant genetic transformation has provided experimental validation of genomic data, as well as the development of commercial transgenic plants. Recently, the genome editing approach has gained support with the advent of CRISPR/Cas technology due to its feasibility of application in diverse contexts, such as agriculture, and has become the focus of many companies and research laboratories worldwide. In contrast to the mechanism of action of conventional transgenesis, CRISPR/Cas allows precise manipulation of the genome by an RNA-guided nuclease enzyme (Cas9, for example). Additionally, this technology enables the development of transgene-free plants with insertions/deletions of a few nucleotides, avoiding time-consuming deregulation processes for Genetically Modified Organisms (GMOs), thereby speeding up the launch of commercial varieties to the market. Thus, the present project aims to establish the CRISPR/Cas technology at the Centro de Cana (Instituto Agronômico de Campinas, IAC) to develop sustainable plants with agronomic traits. Initially, we will perform knockout of the fructokinase-like 2 gene (fls2), which results in an easily detectable albino phenotype. For this, a CRISPR-Cas9 plasmid vector will be delivered into rice embryogenic calli via Agrobacterium-mediated transformation. Gene knockout will be validated by enzymatic mismatch cleavage assay, followed by DNA sequencing. Sugarcane genes functionally validated by our team (via global expression analyses and gene overexpression) will be used as targets for gene editing via CRISPR-Cas in rice plants. Finally, physiological and biometric trials will be carried out to highlight possible changes in the development of genetically edited plants. Prospectively, the current study has the potential to set up a genome editing platform at the Centro de Cana, and may pave the way for the precise engineering of yield-associated genes in sugarcane and other grasses. (AU)