Emergence rate of mutated plants via embryo induction method.

<p>Emergence rate of mutated plants via embryo induction method.</p

CRISPR/Cas9-mediated targeted mutagenesis in grape

<div><p>RNA-guided genome editing using the CRISPR/Cas9 CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) system has been applied successfully in several plant species. However, to date, there are few reports on the use of any of the current genome editing approaches in grape—an important fruit crop with a large market not only for table grapes but also for wine. Here, we report successful targeted mutagenesis in grape (<i>Vitis vinifera</i> L., cv. Neo Muscat) using the CRISPR/Cas9 system. When a Cas9 expression construct was transformed to embryonic calli along with a synthetic sgRNA expression construct targeting the <i>Vitis vinifera</i> phytoene desaturase (VvPDS) gene, regenerated plants with albino leaves were obtained. DNA sequencing confirmed that the VvPDS gene was mutated at the target site in regenerated grape plants. Interestingly, the ratio of mutated cells was higher in lower, older, leaves compared to that in newly appearing upper leaves. This result might suggest either that the proportion of targeted mutagenized cells is higher in older leaves due to the repeated induction of DNA double strand breaks (DSBs), or that the efficiency of precise DSBs repair in cells of old grape leaves is decreased.</p></div

Selection of target sites in the <i>VvPDS</i> gene.

<p>(a) Locations of target sites of CRISPR/Cas9-mediated target mutagenesis in the ‘Neo Muscat’ (<i>V</i>. <i>vinifera</i> L.) PDS gene. (b) Sequences of target sites. The PAM sequence (NGG) is shown in blue, and the 20bp target sequence is shown in black. The red arrowhead indicates the expected cleavage site. The recognition sequences of the restriction enzymes used for CAPS analysis are underlined. (c) <i>In vitro</i> cleavage assay of four target sites.</p

Primer sequences.

<p>Primer sequences.</p

Detection of Cas9 expression in regenerated plants.

<p>Immunological detection of Cas9. PDS-t2-1^st-#1–2 and PDS-t2-1^st-#1–3 are regenerated plants obtained from same transgenic line.</p

Detection of mutations in PDS-t2 target locus in regenerated plant.

<p>(a) Chlorophyll-deficient variegated plant due to mutation in the PDS-t2 target locus. (b) Representative sequences of PDS-t2 target locus in leaves. The wild type sequence is shown at the top, with the PAM sequence highlighted in cyan, and the target sequence in red. Dashes, deleted bases. The net change in length is noted to the right of each sequence (+, insertion;—deletion). The number of clones representing each mutant allele is shown in the column on the right.</p

Emergence rate of mutated plants via callus induction method.

<p>Emergence rate of mutated plants via callus induction method.</p

Detection of mutations at the PDS-t3 target locus in regenerated plants.

<p>(a) Chlorophyll-deficient variegated plant due to mutation in the PDS-t3 target locus. (b) Representative sequences of the PDS-t3 target locus in leaves. The wild type sequence is shown at the top with the PAM sequence highlighted in cyan, and the target sequence in red. Dashes, deleted bases. The net change in length is noted to the right of each sequence (+, insertion;—deletion). The number of clones representing each mutant allele is shown in the column on the right.</p

Purification of rice RAD51A1 and RAD51A2.

<p>(A) The amino acid sequences of rice RAD51A1 and RAD51A2 from japonica cultivar group, cv. Nipponbare, rice RAD51 from indica cultivar group, cv. Pusa Basmati 1, and human RAD51, aligned with the ClustalX software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075451#pone.0075451-Thompson1" target="_blank">[50]</a>. Black and gray boxes indicate identical and similar amino acid residues, respectively. The L1 and L2 loops, which are important for DNA binding, are represented by red lines. (B) Purified rice RAD51A1, RAD51A2, and human RAD51. Lane 1 indicates the molecular mass markers, and lanes 2, 3, and 4 represent rice RAD51A1 (0.5 µg), RAD51A2 (0.5 µg), and human RAD51 (0.5 µg), respectively. (C) The ATPase activities of <i>Oryza sativa</i> RAD51A1 and RAD51A2. The reactions were conducted with φX174 circular ssDNA (left panel), linearized φX174 dsDNA (center panel), or without DNA (right panel), in the presence of 5 µM ATP. Blue circles and red squares represent the experiments with RAD51A1 and RAD51A2, respectively. The averages of three independent experiments are shown with the SD values.</p

The polymerization activities of rice RAD51A1 and RAD51A2.

<p>A 70 µg portion of RAD51A1 (A) or RAD51A2 (B) was analyzed by Superdex 200 gel filtration chromatography. The top, middle, and bottom panels represent the experiments without ATP, with ATP, and with ADP, respectively.</p