Visual evaluation of targeting specificity of selected sgRNAs.

<p>Exclusive and homogenous expression of eGFP in the domains of <i>cryaa</i> (A, A’), <i>rx2</i> (B) and <i>actb</i> (D) was evident already in the injected generation (F0). The integrations were transmitted to the next generation (F1; C, E).</p

CCTop: An Intuitive, Flexible and Reliable CRISPR/Cas9 Target Prediction Tool

<div><p>Engineering of the CRISPR/Cas9 system has opened a plethora of new opportunities for site-directed mutagenesis and targeted genome modification. Fundamental to this is a stretch of twenty nucleotides at the 5’ end of a guide RNA that provides specificity to the bound Cas9 endonuclease. Since a sequence of twenty nucleotides can occur multiple times in a given genome and some mismatches seem to be accepted by the CRISPR/Cas9 complex, an efficient and reliable <i>in silico</i> selection and evaluation of the targeting site is key prerequisite for the experimental success. Here we present the CRISPR/Cas9 target online predictor (CCTop, <a href="http://crispr.cos.uni-heidelberg.de" target="_blank">http://crispr.cos.uni-heidelberg.de</a>) to overcome limitations of already available tools. CCTop provides an intuitive user interface with reasonable default parameters that can easily be tuned by the user. From a given query sequence, CCTop identifies and ranks all candidate sgRNA target sites according to their off-target quality and displays full documentation. CCTop was experimentally validated for gene inactivation, non-homologous end-joining as well as homology directed repair. Thus, CCTop provides the bench biologist with a tool for the rapid and efficient identification of high quality target sites.</p></div

Experimental verification of sgRNA-1.

<p>(A) <i>In vitro</i> cleavage depending on sgRNA-1/Cas9 occurred on linearized plasmids containing <i>eGFP</i> but not <i>eGFP</i>^<i>var</i>. Successful cleavage of <i>eGFP</i> plasmid (4128bp) resulted in a 2052bp and 2076bp fragment. The absence of expected fragments (627bp, 4025bp) demonstrated that <i>eGFP</i>^<i>var</i> (4652bp) was not digested by sgRNA-1/Cas9. (B) A faint double band (2154bp, 2198bp, asterisk) indicated inefficient digestion of off-target 1 (OT#1) while OT#2 and OT#3 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124633#pone.0124633.s003" target="_blank">S1 Table</a>) were not cleaved. Note: contrast was enhanced for better visualization. (C) Silent mutations of sgRNA-1 target site in <i>eGFP</i>^<i>var</i>. (D-E) Injections of sgRNA-1 and Cas9 mRNA into <i>wimb</i>^<i>-/+</i> embryos (D) resulted in strong inactivation of eGFP (E).</p

CCTop web interface.

<p>(A) Main page containing the input fields to customize the identification of sgRNA target sites and the off-target prediction. (B) Results page providing detailed information of all identified sgRNA target sites.</p

Figure S2: Establishment of a TGF signalling reporter in zebrafish from TGFβ-facilitated optic fissure fusion and the role of bone morphogenetic protein antagonism

(A) Construct for the TGFβ signalling reporter. Multimerised Smad Binding Elements (SBEs) in combination with a minimal promoter (MP) drive membrane localised GFP (GFPcaax). (B) Bright field, fluorescent and merged images of a zebrafish larva expressing the TGFβ signalling reporter construct (21.5 hpf). Note the expression domains in the forebrain (encircled) and the tail. (C) SB431542 reduces TGFβ reporter activity, DMSO treated fish as control. Treatment was administered from 24 hpf to 48 hpf. The inhibitor was renewed during the time of the treatment

Figure S1: Supplemental images of TGFβ2 KO coloboma phenotypes from TGFβ-facilitated optic fissure fusion and the role of bone morphogenetic protein antagonism

(A) Serial frontal sections (E18.5, H&E) of a TGFβ2 KO embryo from mixed genetic background. The optic fissure margins did not fuse but grew inwards. Scale bars represent 200µm. (B) Serial frontal sections (E18.5, H&E) of a TGFβ2 KO embryo from mixed genetic background. In addition to the usual coloboma phenotype, there is a dorsal coloboma. Scale bars represent 200µm. (C) Serial frontal sections (E18.5, H&E) of a TGFβ2/GDNF double KO embryo. In addition to the usual coloboma phenotype, there is a dorsal coloboma. Scale bars represent 200µm.ted fish as control

Selected NASCAR candidates.

<p>The first 8 entries correspond to the candidates that showed expression in medaka.</p><p>* = partial motif co-linearity, DF = “double flanked”, SF = “single flanked”</p><p>Selected NASCAR candidates.</p

Deletion of conserved motifs (grey area) from the predicted fish regions results in change of enhancer activity in both tested constructs.

<p><b>Schematic on the right shows the motif configuration in the human and medaka locus for hs1344 and hs865, respectively. The full grey area is deleted from the medaka enhancer and the remaining sequence tested for reporter expression. Images on the left show the reporter activity of the medaka constructs prior to and after the deletion.</b> Hs1344 ol2-1delta gains two symmetrical domains in the midbrain (red arrowheads), while hs865 ol2-1delta shows a loss of expression in the central part of the original domain.</p

NASCAR workflow.

<p>(1) Seeds perfectly matching between query (i.e. enhancer) and target (e.g. genomic window) sequence (small black segments) are extended up- and downstream (red segments) using a match/mismatch scoring scheme to generate a raw motif profile. Motifs that overlap the predefined window boundaries are also taken into account and virtually extend the window (grey areas). (2) As a next step, overlapping regions of the extracted raw motifs in the target sequence are determined (grey areas) and the smaller motif truncated whenever it overlaps a larger one (2 to 3). Motifs smaller than the initial seed size after truncation are discarded in this step. (3) Same filtering procedure is repeated in the query sequence for the processed profile (3 to 4). (4) Motifs below the noise threshold (bright blue segment) are discarded and the basic similarity (“PURE”) score calculated from the fully filtered motif profile (dark blue). (5) In addition, a pattern detection method searches for co-linear arrangements in the profile (grey area). Panel shows the same motif composition as (4) but in a co-linear configuration. This time, the motif below the noise threshold (bright pink) is kept as it is contained in a pattern. The score of the full pattern (all pink motifs) is subsequently added to the previously calculated basic score, resulting in the “COMB” score. For a given enhancer, the whole process is repeated window by window until the last window in the target sequence is reached.</p

Handling Permutation in Sequence Comparison: Genome-Wide Enhancer Prediction in Vertebrates by a Novel Non-Linear Alignment Scoring Principle

<div><p>Enhancers have been described to evolve by permutation without changing function. This has posed the problem of how to predict enhancer elements that are hidden from alignment-based approaches due to the loss of co-linearity. Alignment-free algorithms have been proposed as one possible solution. However, this approach is hampered by several problems inherent to its underlying working principle. Here we present a new approach, which combines the power of alignment and alignment-free techniques into one algorithm. It allows the prediction of enhancers based on the query and target sequence only, no matter whether the regulatory logic is co-linear or reshuffled. To test our novel approach, we employ it for the prediction of enhancers across the evolutionary distance of ~450Myr between human and medaka. We demonstrate its efficacy by subsequent <i>in vivo</i> validation resulting in 82% (9/11) of the predicted medaka regions showing reporter activity. These include five candidates with partially co-linear and four with reshuffled motif patterns. Orthology in flanking genes and conservation of the detected co-linear motifs indicates that those candidates are likely functionally equivalent enhancers. In sum, our results demonstrate that the proposed principle successfully predicts mutated as well as permuted enhancer regions at an encouragingly high rate.</p></div