CRISPR/Cas9 genome editing to modify lignin biosynthesis in Nicotiana tabacum

Abstract

Genome editing using clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein 9 system (CRISPR/Cas9) has been proven as a powerful tool in genome editing and promises to revolutionize the use of biotechnology for crop breeding. Engineered from prokaryotes immune system, genome editing by CRISPR enable the progress of more accurate science considering its high reproducibility. CRISPR/Cas9 allows the development of biallelic homozygous in T0, and open a new horizon in crop breeding for the possibility to develop "transgenic-free" genome-edited mutants. Here, we propose genome editing of tobacco (N. tabacum) using CRISPR/Cas9 technology in order to understand the carbon flow in lignin metabolism through chlorogenic acid (CGA). Two genes, HCT and CCoAOMT will be used for target mutagenesis. Besides cellulose, lignin represents the main compound in plant cell wall composition and a major challenge in biomass processing, such as production of second generation bioethanol. On the other hand, CGA is part of a group of phenolic antioxidants highly important for human dietary and plant defense response. These two biosynthesis routes are inter-connected by caffeoyl CoA and probably CGA acts as a carbon skeleton donor to lignin metabolism. In the attached proposal (FAPESP 2014/1781-5) we developed double and single mutants for HCT, HQT and CSE using traditional transformation methods to prove this hypothesis. To develop knockout mutants for CCoAOMT and HCT would complete our work considering their connection with CGA pathway have not been completely understood. We believe we can overcome the dwarfism described previously in HCT silenced plants adding Paclobutrazol (PAC) to block SA accumulation. We also propose to exploit the lignin pathway flexibility adding caffeate as a substrate during plant development. Theoretically, the addition of caffeate can lead to production of ferulate by COMT and subsequently to feruyol CoA by 4CL, which can then be used for lignin production. As a result, the lignin pathway could lead to S an G lignin production and CGA pathway could only produce CGA through HQT. In conclusion, we expect to gain more information to confirm CGA and lignin connection. (AU)