Developing a platform for fast and efficient evaluation of CRISPR/Cas-based genome editing tools in plant cells

• Grant number: 22/09418-7
• Support Opportunities: Research Grants - Visiting Researcher Grant - International
• Start date: September 01, 2022
• End date: August 31, 2024
• Field of knowledge: Biological Sciences - Genetics - Plant Genetics
• Principal Investigator: Ricardo Augusto Dante
• Grantee: Ricardo Augusto Dante
• Visiting researcher: Sofya Gerasimova
• Visiting researcher institution: Siberian Branch of the Russian Academy of Sciences (SB RAS), Russia
• Host Institution: Centro de Biologia Molecular e Engenharia Genética (CBMEG). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
• Associated research grant: 16/23218-0 - The Genomics for Climate Change Research Center, AP.PCPE

Abstract

Genome editing is a breakthrough technology allowing directed modification of crop characteristics. First-generation genome editing methods based on the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated proteins (CRISPR/Cas) show high efficiency in targeted mutagenesis of known genes. The further development of genome editing techniques involves different modifications of Cas proteins and gRNA molecules. The set of gRNA/Cas derivatives allows performing tiny correction of cell molecular systems including transcriptional, post transcriptional and post translational regulation tuning or desired allele conversion. The emerging methods of precise genome editing are still not well established for crop plants and, accordingly, current the genome modification methods performed at the Genomics for Climate Change Research Center (GCCRC) are restricted to conventional targeted mutagenesis with RNA guided Cas9 nuclease from Streptococcus pyogenes. This method is used for gene knockout and in most cases cannot be implemented for more precise editing. Establishment of base editing and prime editing for maize will give options for inducing knock-downs instead of knock-outs of target genes, or for customized regulation of target gene expression. For example, the modification of regulatory elements using base and prime editing can be used for overexpression or induced expression of agronomically valuable genes imparting higher biomass production and stress tolerance. Such a transgene-free approach will accelerate integration of new technologies into practical breeding. Genome editing capabilities at the GCCRC would also benefit from using Cas proteins other than Cas9. Cas12a nucleases are more optimal than Cas9 if an AT-rich sequence needs to be modified, and it is also more efficient at gene knockouts and amenable to multiplexing approaches. Establishing new protocols for genome editing requires multiple steps of optimization. The most efficient systems for such optimization in crop plants are based on protoplast or other cell models. Construction of various DNA vectors for genome editing and their transient expression in plant cells gives full information about all possible types of genome modifications, mutation frequency, or other specific properties of the tested tool prior to the laborious and resource-consuming stable transformation. Establishing such a transient expression based test systems for maize is a main goal of Dr. Sofya Gerasimova period in Campinas, Brazil, as a Fapesp Visiting Researcher. Dr. Sofya Gerasimova has broad expertise in crop genetics and plant genetic engineering and experience in protoplast genome editing utilizing both Cas9 and Cas12a nucleases. The one-year research plan for Dr. Gerasimova includes established protocols for genome editing tool validation in plant cells, new generic vectors for precise genome editing and ready-to-use constructs for customized modification of candidate genes previously identified at GCCRC. During her visit Dr. Gerasimova will also contribute with lectures and talks at various campi at São Paulo state aiming at fostering future collaborations with its plant molecular biology community. (AU)