Modification of the lignin biosynthesis pathway through the editing of the HCT gene by CRISPR/Cas9

• Grant number: 17/19181-6
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: November 01, 2017
• End date: September 30, 2018
• Field of knowledge: Agronomical Sciences - Agronomy
• Principal Investigator: Paulo Mazzafera
• Grantee: Ewerton Felipe da Silva Ribeiro
• Host Institution: Instituto de Biologia (IB). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil

Abstract

Genome editing mediated by the technology of clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein 9 systems (CRISPR/Cas9) is a powerful tool that allows us to generate plants containing a desired mutation in a "transgenic free" form with high reproducibility. In this project, we propose to continue the project 2016/15834-2 which proposes to edit the genome of tobacco (N. tabacum), using CRISPR/Cas9 technology, in order to understand the carbon flux between the metabolism of lignin and chlorogenic acid (CGA). The key gene for lignin production, hydroxycinnamoyl transferase (HCT), will be used for mutagenesis. In addition to cellulose, lignin represents the main component of the cell wall in plants and a great challenge in the processing of biomass. The genomic editing technique using CRISPR/Cas9 allows the inactivation haplotypes and isoforms individually in a precise way, which will give us a more complete answer on the role of HCT in the lignin and CGA biosynthesis pathway. We believe we could overcome the dwarfism previously described in HCT down-regulated plants. Our strategy is to make the transformation and the genotyping of plants with mutations in only one HCT haplotype. In the project 2016/15834-2 sgRNAs were constructed and CRISPR/Cas9 vectors were validated for different genes of the lignin pathway through Agro-transient expression in tobacco leaves. We propose to perform a stable transformation of one HCT haplotype and genotype the plants obtained to select the ones containing the desired CRISPR mutations. (AU)