Abstract
The targeted genome editing technique, CRISPR/Cas9 system, has been widely used to modify genes of interest in a predictable and precise manner. In this study, we describe the CRISPR/Cas9-mediated efficient editing of representative SNF (symbiotic nitrogen fixation) related genes in the model legume Lotus japonicus via Agrobacterium-mediated stable or hairy root transformation. We first predicted nine endogenous U6 genes in Lotus and then demonstrated the efficacy of the LjU6-1 gene promoter in driving expression of single guide RNAs (sgRNAs) by using a split yellow fluorescence protein (YFP) reporter system to restore the fluorescence in Arabidopsis protoplasts. Next, we chose a customized sgRNA targeting SYMRK (symbiosis receptor-like kinase) loci and achieved ~35% mutagenic efficiency in 20 T0 transgenic plants, two of them containing biallelic homozygous mutations with a 2-bp deletion near the PAM region. We further designed two sgRNAs targeting three homologous leghemoglobin loci (LjLb1, LjLb2, LjLb3) for testing the possibility of generating multi-gene knockouts. 20 out of 70 hairy root transgenic plants exhibited white nodules, with at least two LjLbs disrupted in each plant. Compared with the constitutively active CaMV 35S promoter, the nodule-specific LjLb2 promoter was also effective in gene editing in nodules by hairy root transformation. Triple mutant knockout of LjLbs was also obtained by stable transformation using two sgRNAs. Collectively, these studies demonstrate that the CRISPR/Cas9 system should greatly facilitate functional analyses of SNF related genes in Lotus japonicus.
Highlights
The bioavailability of soil organic nitrogen is one of the most important limiting factors to plant growth and crop yield
We demonstrated the applicability of the clustered regularlyinterspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system to efficiently target single and multiple SNF genes in stable transgenic Lotus plants or by hairy root transformation
A BbsI recognition site in the LjU6-1 gene promoter was mutated (G→C) so that the guide RNA can be inserted between two BbsI sites with annealed oligonucleotides
Summary
The bioavailability of soil organic nitrogen is one of the most important limiting factors to plant growth and crop yield. Within the nitrogen-fixing nodules of legumes, bacteroids utilize carbohydrates derived from plant photosynthate and, in return, provide fixed nitrogen compounds to help host plants grow when the environmental nitrogen supply is limited (Oldroyd et al, 2011). Detailed investigation of the molecular mechanisms of rhizobia recognition and nodule organogenesis in legumes may provide promising targets for engineering cereal crops with the capability to accommodate nitrogen-fixing bacteria intracellularly to fix their own nitrogen and reduce the use of N fertilizer in the future (Charpentier and Oldroyd, 2010; Geurts et al, 2016)
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