Fig. 5 Base substitution donor construct for gene targeting. (a) Target endogenous DME, sgRNA and P1633A substitution donor sequence. Red letters indicate
PAM sequence, blue highlight indicates P1633A amino acid substitution site,
green highlight indicates silent mutation, and black square indicates AccI site
that is used for genotyping of GT plants. (b) A flow chart for construction of base
substitution donor construct. First PCR round involves amplification of both
homology arms with primers harboring the intended mutations (orange circle).
In the second PCR round both these homology arms are fused together. (c) Base
substitution donor T-DNA construct harbors AtU6-sgRNA cassette, base substitution donor, and selection marker (bar) gene
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