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

Sophie Donovan,Alistair J Mccormick,Elizabete Carmo-Silva,Yuwei Mao,Douglas J Orr

doi:10.3389/fgeed.2020.605614

Sophie Donovan, Alistair J Mccormick + Show 3 more

Open Access

https://doi.org/10.3389/fgeed.2020.605614

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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.

Highlights

The assimilation of CO2 in photosynthetic organisms is primarily catalyzed by the bi-functional enzyme ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco)
Subsequent amplification of DNA from agro-infiltrated leaves showed that the expected amplicons for wild-type Rubisco small subunit (rbcS)-T1 and rbcS-S1a/b were absent or barely detectable compared to the wild-type control (Figure 1C)
We found mutations in the T1 generation for line 4 and line 9, FIGURE 3 | Molecular analyses of T1 plants with SpCas9-induced mutations in rbcS-T1. (A) RNA was extracted from 45-day-old plants and the abundance of transcripts for nine rbcS homologs was measured by quantitative PCR with gene-specific primers and the transcript level is shown relative to wild-type (Supplementary Information 1 in Supplementary Material). (B) Abundance of Rubisco large subunit transcripts and total rbcS transcripts

Summary

Introduction

The assimilation of CO2 in photosynthetic organisms is primarily catalyzed by the bi-functional enzyme ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco). A family of phylogenetically distinct rbcS homologs were identified in Nicotiana tabacum (tobacco), rice, and several other species that produce Rubisco with altered catalytic properties, including an increased kccat and decreased SC/O (Morita et al, 2014, 2016; Laterre et al, 2017; Pottier et al, 2018). These rbcS homologs are typically expressed in non-photosynthetic tissues, overexpression could lead to changes in the catalytic properties of the Rubisco pool in leaves, provided that the remaining rbcS family members are sufficiently suppressed (Morita et al, 2016)

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Conclusion