Establishment of <i>pten</i> knockout medaka with transcription activator–like effector nucleases (TALENs) as a model of PTEN deficiency disease

<div><p>Phosphatase and tensin homolog (PTEN) is a lipid and protein phosphatase that antagonizes signaling by the phosphatidylinositol 3-kinase (PI3K)–AKT signaling pathway. The <i>PTEN</i> gene is a major tumor suppressor, with mutations of this gene occurring frequently in tumors of humans and mice. We have now developed mutant medaka deficient in PTEN with the use of transcription activator–like effector nuclease (TALEN) technology. Medaka possesses two <i>pten</i> genes, <i>ptena</i> and <i>ptenb</i>, similar to zebrafish. We established 16 <i>ptena</i> mutant lines and two <i>ptenb</i> mutant lines. Homozygous single <i>pten</i> mutants were found to be viable and fertile. In contrast, <i>pten</i> double-knockout (dko) embryos manifested severe abnormalities in vasculogenesis, eye size, and tail development at 72 hours post fertilization(hpf) and died before hatching. Immunoblot analysis revealed that the ratio of phosphorylated to total forms of AKT (pAKT/AKT) in <i>pten</i> dko embryos was four times that in wild-type embryos, indicative of up-regulation of signaling by the PI3K-AKT pathway. Treatment of <i>pten</i> dko embryos with the PI3K inhibitor LY294002 reduced the pAKT/AKT ratio by about one-half and partially rescued the defect in vasculogenesis. Additional inhibitors of the PI3K-AKT pathway, including rapamycin and <i>N</i>-α-tosyl-L-phenylalanyl chloromethyl ketone, also partially restored vasculogenesis in the dko embryos. Our model system thus allows <i>pten</i> dko embryos to be readily distinguished from wild-type embryos at an early stage of development and is suitable for the screening of drugs able to compensate for PTEN deficiency.</p></div

PI3K-AKT signaling pathway activity in adult <i>pten</i> mutant medaka.

<p>Extracts (20 μg of protein) derived from the dorsal muscle of 7- to 10-mpf fish were subjected to immunoblot analysis with antibodies to phosphorylated (p) or total forms of AKT as well as with those to α-tubulin (loading control). Each lane corresponds to an individual. The pAKT/AKT ratio for individual fish of the indicated <i>pten</i> genotypes was determined by densitometry. There were another two independent replications that showed much the same results.</p

Effects of rapamycin on <i>pten</i> dko embryos.

<p>(<b>A</b>) A total of 41 embryos obtained from <i>ptena</i>^+/−<i>ptenb</i>^−/− parents was treated with 5 μM rapamycin for 96 h beginning at 26 hpf. (<b>B</b>) At 7 dpf, the embryos were photographed and genotyped. Eight of the 41 embryos had the <i>ptena</i>^−/−<i>ptenb</i>^−/− genotype (images 1–8), nine were <i>ptena</i>^+/−<i>ptenb</i>^−/−(image 9), and 10 were <i>ptena</i>^+/+<i>ptenb</i>^−/−(image 10). Partial Cuvierian ducts developed in seven of the eight dko embryos. X, no duct. (<b>C</b>) Snapshots from movies of the dko embryos shown in images 2 (upper) and 4 (lower) in (B). Blood cells can be seen flowing through the Cuvierian duct (upper) and the tail vascular duct (lower) indicated by the arrows. Times are in seconds.</p

Mutation efficiency for <i>pten</i> TALEN constructs revealed by analysis of G0 medaka embryos.

<p>Mutation efficiency for <i>pten</i> TALEN constructs revealed by analysis of G0 medaka embryos.</p

Characterization of medaka PTEN genes and design of TALEN constructs.

<p><b>(A</b>) RT-PCR analysis of total RNA isolated from whole embryos of medaka was performed with two sets of primers designed to amplify almost the entire open reading frames of <i>ptena</i> (lane 1) and <i>ptenb</i> (lane 2). The arrow indicates the specific amplification products, with the faster-migrating bands being found to represent technical artifacts by sequencing analysis. Lane M, molecular size markers. (<b>B</b>) Products of medaka <i>ptena</i> and <i>ptenb</i> predicted from the sequences of the amplified cDNAs and database information. Arrowheads labeled #1 and #2 indicate the localization of the TALEN target sequences shown in (C). (<b>C</b>) TALEN target sites in <i>ptena</i> and <i>ptenb</i>.</p

Effects of LY294002 on vascular development and blood cell flow in <i>pten</i> dko embryos.

<p>(<b>A</b>) Wild-type or <i>pten</i> dko embryos were exposed (or not) to 15 μM LY294002 for 48 h beginning at 30 or 48 hpf. (<b>B</b>) At 7 dpf, the embryos were photographed and genotyped. The dko embryos treated with LY294002 developed partial Cuvierian ducts (4 of 4 treated at 30 hpf, and 2 of 3 treated at 48 hpf). The dko embryos not exposed to the drug (2/2) did not manifest vasculogenesis. X, no duct. (<b>C</b>) Snapshots from a movie of a 4-dpf dko embryo that had been treated with LY294002 for 48 h beginning at 30 hpf. Blood cells can be seen flowing through the Cuvierian duct (arrow). Times are indicated in seconds.</p

Plasmid map of pBACi, CRISPRi plasmid for <i>S</i>. <i>aureus</i>.

<p>Plasmid map of pBACi, CRISPRi plasmid for <i>S</i>. <i>aureus</i>.</p

Repression of various virulence and antibiotic resistant factors in MW2 using CRISPRi.

<p>Knockdown strain names were as follows. C: MW2/pBACi (Vector control), 1. MW2/pYS69 (<i>icaA</i> silenced knockdown strain 1), 2: MW2/pYS70 (<i>icaA</i> silenced knockdown strain 2), 3: MW2/pYS106 (<i>sec</i> silenced knockdown strain 1), 4: MW2/pYS107 (<i>sec</i> silenced knockdown strain 2), 5: MW2/pYS108 (<i>sec</i> silenced knockdown strain 3), 6: MW2/pYS109 (<i>coa</i> silenced knockdown strain 1) 7: MW2/pYS110 (<i>coa</i> silenced knockdown strain 2), 8: MW2/pYS111 (<i>coa</i> silenced knockdown strain 3), 9: MW2/pYS112 (<i>blaZ</i> silenced knockdown strain 1), and 10: MW2/pYS113 (<i>blaZ</i> silenced knockdown strain 2). All phenotype assays were repeated at least three times (qPCR and ELISA: three independent cultures and three independent measurements. Other assays: three independent assays). Average and SE of each assay are shown in graphs. Statistics: Student’s <i>t</i>-test. 4A. crRNA binding regions. 100bp upstream sequences of four genes in MW2 and nucleotide sequences corresponding to spacer sequences designed in this study. Underline: spacer sequences, red boxes: PAM sequence (NGG), start codon: start codon of gene, capital letters: putative -35b, -10b and ribosome binding sites of genes. 4B. Biofilm formation on plastic surfaces. After 24 h culture, the amount of biofilm on surface was assayed. G-: without additional glucose, G+: with additional glucose. 4C. OD₅₉₀ value of extracts from G+ wells as shown in Fig 4B. 4D. Repression of <i>icaA</i> mRNA. 4E. SDS-PAGE of supernatants. Culture media were subjected to electrophoresis. Arrow head: Protein corresponding to SEC (SEC from MW2: 28kDa). M: molecular weight marker (kDa), 4F: Sandwich ELISA of supernatants. SEC production was quantified. 4G. Repression of <i>sec</i> mRNA. 4H. Coagulase test. White arrow indicates a fibrin clot. 4I. Repression of <i>coa</i> mRNA. 4J. Measurement of β-lactamase activity. OD₄₉₀ of nitrocephin degraded product was measured. The value of control was compared with silencing vectors (%, vertical line). 4K. Repression of <i>blaZ</i> mRNA.</p

Staphylococcal protein A (Spa) silenced using CRISPRi.

<p>No significant growth inhibition was observed between the control and the silenced knockdown strains. pBACi (C): Vector control, pYS103 [103]: <i>spa</i> silenced knockdown vector 1, pYS104 [104]: <i>spa</i> silenced knockdown vector 2, pYS105 [105]: <i>spa</i> silenced knockdown vector 3. 3A. The upstream sequence of <i>spa</i> in MW2 and nucleotide sequences corresponding to spacer sequences. The partial sequence between <i>spa</i> (MW0084) and <i>sarS</i> (MW0085) in MW2 (Accession number: BA000033) is shown (101,008 bp-101,207 bp in MW2). Underline: spacer sequence [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185987#pone.0185987.ref001" target="_blank">1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185987#pone.0185987.ref003" target="_blank">3</a>], red boxes: PAM sequence (NGG), start codon: start codon of <i>spa</i> (ttg), capital letters: putative -35b, -10b and SD sequence (ribosome binding site) of <i>spa</i> in MW2. 3B. Western blotting to detect Spa in vector controls and silenced knockdown strains. Seven strains were analyzed. Three independent cultures from a single colony were performed and representative data are shown. Brackets indicate Protein A. Due to posttranslational processing of the cell surface-exposed ProteinA, some strains show double bands. “Plasmid No.” indicates serial pYS number plasmid used in this study (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185987#pone.0185987.s001" target="_blank">S1 File</a>). M: molecular marker. 75, 50 and 37 mean 75kDa, 50kDa and 37kDa bands, respectively. C: pBACi control vector (No crRNA coding region for <i>spa</i> silencing), pYS103-105: silencing CRISPRi vector containing the nucleotide sequence 1–3 corresponding to Fig 3A. Arrow heads: Spa. Two bands were found. This might result from processed/non-processed bands [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185987#pone.0185987.ref052" target="_blank">52</a>]. 3C. Repression of <i>spa</i> mRNA in MW2 using CRISPRi. Relative <i>spa</i> gene expression (/<i>gyrB</i>) is shown. MW2/pBACi, MW2/pYS103, MW2/pYS104 and MW2/pYS105 were independently cultured three times. Three independent qPCR were performed (n = 9/strain). The average and SE are shown. Statistics: Student’s <i>t</i>-test.</p

Tailor-made gene silencing of <i>Staphylococcus aureus</i> clinical isolates by CRISPR interference

<div><p>Preparing the genetically modified organisms have required much time and labor, making it the rate-limiting step but CRISPR/Cas9 technology appearance has changed this difficulty. Although reports on CRISPR/Cas9 technology such as genome editing and CRISPR interference (CRISPRi) in eukaryotes increased, those in prokaryotes especially in Staphylococci were limited. Thus, its potential in the bacteriology remains unexplored. This is attributed to ecological difference between eukaryotes and prokaryotes. Here, we constructed a novel CRISPRi plasmid vector, pBACi for <i>Staphylococcus aureus</i>. The transformation efficiency of <i>S</i>. <i>aureus</i> was ~10⁴ CFU/μg DNA using a vector extracted from <i>dcm</i> negative, which encoded one of DNA modification genes, <i>E</i>. <i>coli</i>. Further, pBACi was introduced into various clinical isolates including that not accepting the conventional temperature-sensitive vector. <i>dcas9</i> in the vector was expressed throughout the growth phases of <i>S</i>. <i>aureus</i> and this vector decreased various gene mRNA expressions based on the crRNA targeting sequences and altered the knockdown strains’ phenotypes. The targeted genes included various virulence and antibiotic resistant genes. Bioinformatics suggest this vector can be introduced into wide range of low-GC Gram-positive bacteria. Because this new CRISPR/Cas9-based vector can easily prepare knockdown strains, we believe the novel vector will facilitate the characterization of the function of genes from <i>S</i>. <i>aureus</i> and other Gram-positive bacteria.</p></div