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Figure 1.

Design of a Cas9/sgRNA system for mutagenesis and restoration of activity of a non-functional (out-of-frame) mutant GFP gene in Arabidopsis.

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Figure 2.

Cas9/sgRNA-mediated mutagenesis in Arabidopsis.

Scheme for Cas9/sgRNA-mediated mutagenesis of a non-functional (out-of-frame) mutant GFP gene, detection of T1 Arabidopsis leaves with restored GFP gene function by confocal microscopy and documentation of target site DNA mutagenesis.

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Figure 3.

Cas9/sgRNA-mediated mutagenesis of nonfunctional mutant GFP genes in Arabidopsis leaves.

Expression in T1 Arabidopsis leaf cells of functional GFP genes produced by Cas9/sgRNA-mediated mutagenesis of nonfunctional mutant GFP genes introduced along with the Cas9 and sgRNA genes using A. tumefaciens and a floral dip transformation protocol. A and C) Detection of green fluorescence protein signals in transgenic leaves. B and D) Merged image of red chlorophyll fluorescence and GFP fluorescence. Leaves from hygromycin resistant seedlings were photographed ten days (A and B) and twenty days (C and D) after seed germination. Bar, 100 µm.

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Figure 4.

Robust expression in guard cells of functional GFP genes created by Cas9/sgRNA-mediated mutagenesis.

A) Detection of green fluorescence protein signals in guard cells of transgenic leaf stomata with lesser expression in surrounding leaf epidermal cells of T1 transgenic Arabidopsis plants. B) Merged image of red chlorophyll fluorescence and GFP fluorescence. Bar, 50 µm. Photographed twenty days after seed germination.

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Figure 5.

Efficiency of Cas9/sgRNA mutagenesis in Arabidopsis plants.

PCR/Restriction Enzyme (PCR/RE) analysis of total DNA extracts from individual hygromycin resistant T1 Arabidopsis plants (lanes 1–12) showing the relative proportion of nonfunctional GFP genes mutagenized by Cas9/sgRNA activity. Bottom arrow indicates the expected ∼125 bp DNA fragments resulting from ApaLI cleavage of the ∼250 bp PCR product amplified from nonfunctional, out-of-frame, nonmutagenized GFP genes that contains an intact ApaLI cleavage site in the sgRNA target region. Top arrow indicates the expected ∼250 bp size of PCR products from GFP genes mutagenized by the Cas9/sgRNA system in such a manner that they are no longer are susceptible to cleavage by ApaLI. NG (Nonfunctional GFPGene), the PCR products amplified from a sgRNA target site of a cloned nonfunctional, out-of-frame, GFP gene digested with ApaLI. % modified GFP Gene = (pixels in 250 bp band)/(pixels in 250 bp band+pixels in 125 bp band) ×100.

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Figure 6.

Confirmation by DNA sequencing of Cas9/sgRNA-mediated mutagenesis of the sgRNA target site within the nonfuctional, out-of-frame, GFP gene.

Five cloned DNA fragments of 250 bp containing DNA from PCR amplified sgRNA target regions of (previously) nonfunctional GFP genes from three different GFP-positive T1 Arabidopsis plants (Plants 21, 22 and 25) and two different GFP-negative plants (Plants 23 and 24) were subjected to DNA sequencing. DNA sequences of a segment of 48 nucleotides surrounding the sgRNA target site are shown for each clone with the sequence of the nonmutagenized DNA region shown as the top line of each group. The 20 nucleotide target sequence for the Cas9/sgRNA complex is depicted in blue, the PAM site in red and the ApaLI recognition site is underlined in blue. Brackets ([]) denote nondisplayed DNA sequences. For the Cas9/sgRNA-mutagenized DNA sequences, deleted nucleotides are depicted as red dots and inserted nucleotides are shown in green. The net length of insertions and/or deletions (In/Del) are presented in the column to the right.

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Figure 7.

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.

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