Fig. 3.

Schematic representation of the PHO2-B and PHO2-C targets and the reagent components. a) Three gRNA targets were designed for each of the 4 PHO2-B and PHO2-C haplotypes with 2 targets in the first exon and the third target in exon 6 for PHO2-B and exon 3 for PHO2-C. b) The reagent components include; the binary vector backbone [pTRANS_220] containing a 35S: nptII selectable marker for kanamycin selection, a Cas9 [pMOD_A] module, a guide RNA [pMOD_B] module that can utilize either the Csy4 or tRNA splicing mechanism for the release of multiple gRNAs, and the rolD: TREX2 exonuclease, in a [pMOD_C] module. All 3 modules are assembled into the binary vector by AarI-mediated golden gate reaction. c) The completed reagent is sequence confirmed and transformed into the Agrobacterium strain LBA4404 for alfalfa leaf explant transformation (Samac and Austin-Phillips 2006). The following nomenclature was used to indicate the type of reagent used for the gene editing. For example, “PhoM#” and “PhocM#” refer to either the pDIRECT or pTRANS reagents with the “c” indicating Csy4 splicing system. The “t” in “PhotM#” refers to the pTRANS reagent with the tRNA splicing system. Both the pTRANS reagents (PhocM# and PhotM#) harbor the TREX2 exonuclease cassette.