Confirmation of inheritance of a single modified or nonmodified GFP gene in each of 7 T2 progeny from 6 individual T1 generation plants – and evidence of Cas9 gene and/or sgRNA gene silencing in T2 progeny.
DNA was isolated from each of 7 T2 progeny from each of 6 different progenitor T1 plants (T1 Plants #3 to #8). PCR was used to amplify a 250 bp DNA fragment containing the sgRNA target region of the nonfunctional, out-of-frame, GFP gene. DNA sequencing of the fragment provided the sequence of the 31 bp region displayed for each of the 42 T2 plants. The DNA sequence of the original GFP gene is provide as the top line in each column along with the sequence of one mutagenized GFP gene found in a leaf of the original progenitor T1 plant. A) DNA sequences of three groups of T2 plants in which there has been a Cas9/sgRNA-mediated gene modification including insertion of an A nucleotide (Plant #4 progeny), a T nucleotide (Plant #5 progeny), or deletion of an A nucleotide (Plant #6 progeny) that restored a proper reading frame and resulted in T2 progeny displaying a green fluorescence phenotype, B) DNA sequences of three groups of T2 plants (progeny of T1 Plants #3, #7 and #8) in which there was no inherited Cas9/sgRNA-mediated gene modification. (GF−), No green fluorescence phenotype; (GF+), Green fluorescence phenotype; (Mut+), Inherited mutagenized GFP gene; (Mut-), No inherited mutagenized GFP gene. C, D, E, and F) DNA sequencing traces from sequencing of PCR amplified Cas9/sgRNA target sites from a single leaf of an individual T2 progeny from T1 progenitor Plants #3, #7 and #8, respectively. G) A DNA sequencing trace from sequencing of the PCR amplified Cas9/sgRNA target sites isolated from a single leaf of T1 Plant #1 showing multiple overlapping DNA peaks caused by the presence of multiple different DNA sequences in the separate mutagenized GFP genes present in different patches of cells scattered throughout the leaf.