Single component CRISPR-mediated base- editors for Agrobacterium and their use to develop an improved suite of strains

Abstract

Agrobacterium mediated plant transformation largely depends on two distinct strain lineages – C58 and Ach5. To better serve the plant transformation community, we have created a suite of auxotrophic and auxotrophic recombinant deficient mutants of C58 derivatives EHA105, GV3101::pMP90, and Ach5 derivative LBA4404. While these derivatives are useful, having additional strain backgrounds available would help expand the repertoire for plant transformation even further. Toward that end, two underutilized hypervirulent strains are K599 (NCPPB 2659), and Chry5—but disarmed variants are not easily accessible. To improve availability, we produced disarmed versions of A. rhizogenes strain K599 and A. tumefaciens strain Chry5 and introduced the same desirable mutations as with the other lineages. Each thymidine auxotrophy and recombination deficiency were introduced to existing and newly disarmed Agrobacterium strains via loss of function mutations conferred to thyA and recA, respectively, through CRISPR-mediated base-editing of codons amenable to nonsense mutation. To streamline the editing process, we created a series of visually marked single component base-editor vectors and a corresponding guide-filtering Geneious Prime wrapper plugin for expedited guide filtering. These new strains, the simplified CRISPR-mediated base-editor plasmids, and streamlined workflow will improve the ease with which future Agrobacterium strain derivatives are created while also supporting plant transformation at large.