Abstract
Rhizobia are widespread gram-negative soil bacteria and indispensable symbiotic partners of leguminous plants that facilitate the most highly efficient biological nitrogen fixation in nature. Although genetic studies in Sinorhizobium meliloti have advanced our understanding of symbiotic nitrogen fixation (SNF), the current methods used for genetic manipulations in Sinorhizobium meliloti are time-consuming and labor-intensive. In this study, we report the development of a few precise gene modification tools that utilize the CRISPR/Cas9 system and various deaminases. By fusing the Cas9 nickase to an adenine deaminase, we developed an adenine base editor (ABE) system that facilitated adenine-to-guanine transitions at one-nucleotide resolution without forming double-strand breaks (DSB). We also engineered a cytidine base editor (CBE) and a guanine base editor (GBE) that catalyze cytidine-to-thymine substitutions and cytidine-to-guanine transversions, respectively, by replacing adenine deaminase with cytidine deaminase and other auxiliary enzymes. All of these base editors are amenable to the assembly of multiple synthetic guide RNA (sgRNA) cassettes using Golden Gate Assembly to simultaneously achieve multigene mutations or disruptions. These CRISPR-mediated base editing tools will accelerate the functional genomics study and genome manipulation of rhizobia.
Highlights
Rhizobia are widespread gram-negative soil bacteria that are mainly classified into two clades, α- and β-proteobacteria
The adenine base editor (ABE) consists of a heterodimeric tRNA adenosine deaminase (TadA) (TadA-TadA∗) fused to the N-terminus of the CRISPRassociated protein 9 nuclease (Cas9) nickase with an XTEN linker (Figures 1A,B; Gaudelli et al, 2017) that was codon-optimized and synthesized
The ABEs and the synthetic guide RNA (sgRNA) are transcribed in a head-to-tail pattern and separated by the rrnBT1 terminator (Figure 1A)
Summary
Rhizobia are widespread gram-negative soil bacteria that are mainly classified into two clades, α- and β-proteobacteria. Rhizobia use nitrogenase to directly convert atmospheric nitrogen (N2) into ammonium (Masson-Boivin et al, 2009; Seefeldt et al, 2009; Poole et al, 2018). A number of rhizobial species establish symbiotic relationships with their leguminous hosts that drive the most efficient form of nitrogen fixation in nature. Symbiotic nitrogen fixation (SNF) plays an extremely important role in agriculture (Roy et al, 2020). Sinorhizobium meliloti has been reclassified as Ensifer meliloti and is one of the well-studied symbiotic nitrogen-fixing bacteria, which nodulates particular plant genera, such as Medicago truncatula. Most of the gene clusters involved in the synthesis of Nod factors (nod), nitrogen fixation (nif ), and nitrogen metabolism are located on pSymA (Galibert et al, 2001)
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