Application of STAgR.

<p>(A) Colony PCR of a 6xSTAgR reaction using two different promoters as well as both, the canonical and the SAM loop gRNA scaffold. The gel shows a colony PCR of 22 bacterial colonies, of which seven showed the amplicon indicative of the full length STAgR reaction (2444bp). (B) Exemplary colony PCR of STAgR constructs with 0 to 8 gRNA expression cassettes. (C) A STAgR plasmid containing four gRNAs or a mixture of four single gRNA plasmids have been transfected into P19 Cells expressing dCas9-VPR. (D) After 7 days mRNA was extracted and transcript levels of target genes have been compared via qPCR. Error bars depict standard errors of the mean.</p

The STAgR protocol.

<p>(A) An Overview over STAgR procedure. STAgR allows simple and fast generation of multiplexing vectors in one overnight reaction. STAgR is also highly customizable as diverse strings and vectors can be used to assemble expression cassettes with different promoters and gRNA scaffolds. (B) Sequences of overhang primers used for generation of STAgR vectors.</p

Functional validation of STAgR.

<p>(A) Colony PCR of a 4xSTAgR reaction (using a string sequence containing a hU6 promoter and a canonical gRNA scaffold). 24 bacterial colonies are shown, of which six present the amplicon size indicative of the full length reaction (1596 bp). Additionally marked are amplicon sizes indicative of two (823 bp) and single gRNAs (458 bp). (B) Quantification of cloning efficiencies from three different 4xSTAgR reactions (n = 130). (C) A schematic showing constructs used for functional validation of STAgR gRNAs. A gRNA targeting the GFP ORF was either delivered in a single gRNA expression vector or on each of four different positions in STAgR vectors. (D) Functional validation of STAgR vectors shown in Fig 2C. HeLa cells stably expressing d2GFP and Cas9 have been transfected with vectors depicted above. Flow cytometry indicates that STAgR constructs are similarly efficient in mutating the ORF of GFP compared to a single gRNA vector. (E) Colony PCR of a 4xSTAgR reaction using four different promoters and SAM loop scaffolds. 24 bacterial colonies are shown, of which seven colonies incorporated the amplicon size indicative of the full length reaction (2043 bp). Shorter amplicons are indicative of gRNA subsets, which vary in size, depending on the incorporated promoter.</p

Verification of microarray results by qRT-PCR and qChIP.

<p>(A) Relative expression levels of <i>Gaa</i>, <i>Has2</i>, <i>Isl1</i>, <i>Kif1b</i>, <i>Mtmr2</i>, <i>Pax6</i>, <i>Pcsk1n</i>, <i>and Snca</i> in wild type (WT, shown in black) and Pax6^+/− (shown in gray) lenses were determined using qRT-PCR as described in Methods. B2m, Hprt and Ccni transcripts were tested as internal references, and all were found unchanged between the WT and Pax6^+/− lenses. The data are expressed relative to the unchanged expression level of B2m transcripts. For statistical evaluation of the results, p-values were calculated from paired Student t-tests. (B) Validation of Pax6-binding regions in lens chromatin by qChIPs. A and B are distal regions with Pax6 binding identified in ChIP-Chip experiments and P regions are binding regions around the proximal promoters. At each of these gene loci, a non-specific region (negative signals in ChIP-Chip experiments and no candidate Pax6 binding sites predicted) was also included as a negative control. In addition, Cryaa promoter (Cryaa-P) and +6 kb region serve as positive and negative controls respectively. The specific enrichments of Pax6 binding were detected at Isl1-A, Mtmr2-A, Snca-A, Gaa-P, Kif1b-P, Pcsk1n-B and Pcsk1n-A regions. The calculation of the cutoff value (0.100 of 1% input) for background signals and specific binding signals is described in Materials and Methods.</p

Pax6 regulates expression of Snca.

<p>(A) Identification of Pax6-binding region by ChIP-Chip in lens chromatin and corresponding luciferase reporter constructs for transfection assays. (B) Pax6 regulates <i>Snca</i> promoter/distal region in cultured cells. Transient transfections were performed in P19 embryonic carcinoma and in αTN4-1 lens cell as described in Methods. (C) Prediction of Pax6 binding sites with novel Pax6 DNA binding motifs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054507#pone.0054507-Xie1" target="_blank">[48]</a>. (D) EMSA validation of Pax6 binding to the probes identified by motif 1-1, 1-2 and 3-3. PD/HD, recombinant Pax6 protein containing both Pax6 paired domain (PD) and homeodomain (HD). P6CON, DNA-binding concensus for Pax6 paired domain.</p

Twenty-seven genes relevant to lens placode formation and lens morphogenesis show differential expression in Pax6^−/− E9.5 mutated lens placodes.

<p>(A) A list of 27 genes includes a combination of well-characterized genes in lens biology and selected differentially expressed genes in Pax6 null (<i>Sey</i>) cortex <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054507#pone.0054507-Holm1" target="_blank">[45]</a>. The differentially expressed genes in Pax6^−/− E9.5 wild type and mutated lens placodes were identified using the Illumina Mouse6 bead microarrays as described elsewhere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054507#pone.0054507-Huang1" target="_blank">[96]</a>. Twenty-four of the 27 genes were differentially expressed in at least 50% of experiments. (B) Relative expression levels of <i>Fat4</i>, <i>Trpm3</i>, <i>Pax6</i>, <i>Has2</i>, <i>Efnb2</i>, and <i>Nav1</i> in wild type (WT, black bars) and Pax6^−/− (open bars) lens placode and mutated ectoderm were determined using qRT-PCR as described in Methods.</p

Pax6 regulates expression of Kif1b.

<p>(A) Identification of Pax6-binding regions by ChIP-Chip in lens chromatin and a corresponding luciferase reporter construct for transfection assays. (B) A diagrammatic summary of Pax6 mutants, N50K, R128C, R242T and R317X. β, N-terminal β-turn unit; PD, paird domain; PAI, N-termianl subdomian of PD; RED, C-terminal subdomian of PD; L, linker region; HD, homeodomain; PST, proline-serine-threonine rich transactivation domain. (C) Pax6 activates <i>Kif1b</i> promoter in cultured cells. Transient transfections were performed in P19 embryonic carcinoma and in αTN4-1 lens cell as described in Methods. (D) Prediction of Pax6 binding sites with novel Pax6 DNA binding motifs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054507#pone.0054507-Xie1" target="_blank">[48]</a>. (E) EMSA validation of Pax6 binding to two probes identified by motifs 2-2 and 4-1. PD/HD, recombinant Pax6 protein containing both Pax6 paired domain (PD) and homeodomain (HD). P6CON, DNA-binding concensus for Pax6 paired domain.</p

Diagrammatic summary of novel functions of Pax6 during lens development.

<p>The connections between genes regulated by Pax6 in lens placode were identified via Ingenuity Pathway Analysis (IPA) (Ingenuity Systems, Mountain View, CA). Expression of Stat3 is reduced in the Pax6 mutated E9.5 embryonic tissues. The Tnf promoter region is occupied by Pax6 in lens chromatin.</p

Identification of genes regulated by Pax6 in newborn lens.

<p>Venn diagram showing identification of 76 genes both bound and regulated by Pax6 by intersecting two genome-wide data sets: RNA expression profiling (559 transcripts, P1 lens, Pax6^+/− versus wild type) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054507#pone.0054507-Wolf1" target="_blank">[26]</a> and present ChIP-chip studies (2,335 peaks) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054507#pone.0054507.s005" target="_blank">Table S1</a>) in lens chromatin. The table summarized the known function of six important and validated Pax6 direct target genes from this group of 76 genes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054507#pone-0054507-g004" target="_blank">Figure 4</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054507#pone.0054507-Zhao1" target="_blank">[75]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054507#pone.0054507-Bijvoet1" target="_blank">[121]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054507#pone.0054507-Bolino1" target="_blank">[122]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054507#pone.0054507-Fricker1" target="_blank">[123]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054507#pone.0054507-Masliah1" target="_blank">[124]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054507#pone.0054507-Pan1" target="_blank">[125]</a>.</p

Down-regulation of Kif1b and Snca in Sye/Sey forebrains.

<p>In situ hybridization analysis of E14 wild type (WT) and Pax6^Sey/Sey mouse embryos. Ganglionic eminence, GE.</p