Generation of a recipient line for Rubisco engineering by multiplex genome editing in tobacco

Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase) is the primary CO₂-fixing enzyme on our planet. Its slow kinetics and poor discrimination between carbon dioxide and oxygen as substrates severely limit the efficiency of photosynthetic carbon fixation by plants. Attempts to improve Rubisco by genetic engineering have been hampered by the complexity of the Rubisco system, with a gene family for the enzyme's small subunit being encoded in the nuclear genome and the gene for the large subunit residing in the plastid (chloroplast) genome. Another nuclear gene family encodes the enzyme Rubisco activase, which is required to facilitate catalysis in the active site of the enzyme. The Rubisco subunits and the activase have co-evolved, making single-gene replacements largely fruitless and typically resulting in dysfunctional Rubiscos. Here, we have generated a dedicated plant line for Rubisco engineering that lacks nuclear genes for components of the Rubisco system. Using multiplex genome editing by CRISPR-Cas9 in the diploid tobacco species Nicotiana sylvestris, we have knocked out six loci encoding the small subunit of Rubisco and three loci encoding Rubisco activase. The generated mutants are incapable of autotrophic growth, and grafting experiments are underway to obtain transgene-free T₁ progeny. The recipient line produced here provides a clean genetic background, in which heterologous Rubisco systems from other organisms can be readily implemented and systematically tested in comparative functional studies. It greatly simplifies Rubisco engineering and creates new opportunities for future efforts to enhance photosynthetic carbon assimilation and increase crop yields.