CRISPR-Cas9-Mediated Mutagenesis of the Rubisco Small Subunit Family in Nicotiana tabacum

Abstract

Engineering the small subunit of the key CO2-fixing enzyme Rubisco (SSU, encoded by rbcS) in plants currently poses a significant challenge, as many plants have polyploid genomes and SSUs are encoded by large multigene families. Here, we used CRISPR-Cas9-mediated genome editing approach to simultaneously knock-out multiple rbcS homologs in the model tetraploid crop tobacco (Nicotiana tabacum cv. Petit Havana). The three rbcS homologs rbcS_S1a, rbcS_S1b and rbcS_T1 account for at least 80% of total rbcS expression in tobacco. In this study, two multiplexing guide RNAs (gRNAs) were designed to target homologous regions in these three genes. We generated tobacco mutant lines with indel mutations in all three genes, including one line with a 670 bp deletion in rbcS-T1. The Rubisco content of three selected mutant lines in the T1 generation was reduced by ca. 93% and mutant plants accumulated only 10% of the total biomass of wild-type plants. As a second goal, we developed a proof-of-principle approach to simultaneously introduce a non-native rbcS gene while generating the triple SSU knockout by co-transformation into a wild-type tobacco background. Our results show that CRISPR-Cas9 is a viable tool for the targeted mutagenesis of rbcS families in polyploid species and will contribute to efforts aimed at improving photosynthetic efficiency through expression of superior non-native Rubisco enzymes in plants.

Improving the efficiency of Rubisco by resurrecting its ancestors in the family Solanaceae

“…In plants and most algae, the L 8 S 8 Rubisco is assembled with the L subunit encoded from a single rbcL gene located in the chloroplast genome and the S subunits produced from the RBCS multigene family in the nucleus and imported into the chloroplast. Considerable progress has been made to engineer Rubisco with superior kinetics into plants by modifying the L subunit (11,13,14), the S subunit (15)(16)(17), or both subunits simultaneously (18)(19)(20). However, biogenesis of L 8 S 8 complexes in the chloroplast stroma of algae and plants is an elaborate process and involves the chaperonins and multiple chaperones (21)(22)(23)(24)(25).…”

Simultaneously induced mutations in eIF4E genes by CRISPR/Cas9 enhance PVY resistance in tobacco

“…Similarly, members of the rbcS gene family displaying high sequence homology were knocked out by a dual sgRNA-CRISPR/Cas9 vector in Nicotiana tabacum cv. Petit Havana 51 . Moreover, multiple sgRNA expressing constructs were also employed to simultaneously edit non-homologous genes (phytoene desaturase ( PDS ) and PDR-type transporter ( PDR6 )) in tobacco 52 .…”

CRISPR-Based Crop Improvements: A Way Forward to Achieve Zero Hunger

“…Researchers have improved the photosynthetic machinery of diploid crop plants and that of polyploid crops by using the CRISPR-Cas system. For instance, multiple rbcS homologues ( rbcS_S1a , rbcS_S1b , and rbcS_T1 ) were simultaneously knocked out in tobacco ( Nicotiana tabacum ), a tetraploid model crop, and the mutant plants showed a high photosynthetic rate as compared with wild-type plants . Hence, the CRISPR-based gene editing system can be used to engineer diploid and polyploid species with highly efficient photosynthetic systems.…”