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 in silico selection and evaluation of the targeting site is key prerequisite for the experimental success. Here we present the CRISPR/Cas9 target online predictor (CCTop, http://crispr.cos.uni-heidelberg.de) 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.

Morphological asymmetry is a common feature of animal body plans, from shell coiling in snails to organ placement in humans. The signaling protein Nodal is key for determining this laterality. Many vertebrates, including humans, use cilia for breaking symmetry during embryonic development: rotating cilia produce a leftward flow of extracellular fluids that induces the asymmetric expression of Nodal. By contrast, Nodal asymmetry can be induced flow-independently in invertebrates. Here, we ask when and why flow evolved. We propose that flow was present at the base of the deuterostomes and that it is required to maintain organ asymmetry in otherwise perfectly bilaterally symmetrical vertebrates.

CRISPR/Cas9 efficiently induces targeted mutations via non-homologous-end-joining but for genome editing, precise, homology-directed repair (HDR) of endogenous DNA stretches is a prerequisite. To favor HDR, many approaches interfere with the repair machinery or manipulate Cas9 itself. Using Medaka we show that the modification of 5’ ends of long dsDNA donors strongly enhances HDR, favors efficient single-copy integration by retaining a monomeric donor conformation thus facilitating successful gene replacement or tagging.

The embryonic skin of Xenopus tadpoles serves as an experimental model system for mucociliary epithelia (MCE) such as the human airway epithelium. MCEs are characterized by the presence of mucus-secreting goblet and multiciliated cells (MCCs). A third cell type, ion-secreting cells (ISCs), is present in the larval skin as well. Synchronized beating of MCC cilia is required for directional transport of mucus. Here we describe a novel cell type in the Xenopus laevis larval epidermis, characterized by serotonin synthesis and secretion. It is termed small secretory cell (SSC). SSCs are detectable at early tadpole stages, unlike MCCs and ISCs, which are specified at early neurulation. Subcellularly, serotonin was found in large, apically localized vesicle-like structures, which were entirely shed into the surrounding medium. Pharmacological inhibition of serotonin synthesis decreased the velocity of cilia-driven fluid flow across the skin epithelium. This effect was mediated by serotonin type 3 receptor (Htr3), which was expressed in ciliated cells. Knockdown of Htr3 compromised flow velocity by reducing the ciliary motility of MCCs. SSCs thus represent a distinct and novel entity of the frog tadpole MCE, required for ciliary beating and mucus transport across the larval skin. The identification and characterization of SSCs consolidates the value of the Xenopus embryonic skin as a model system for human MCEs, which have been known for serotonin-dependent regulation of ciliary beat frequency.

Most vertebrate embryos break symmetry by a cilia-driven leftward flow during neurulation. In the frog Xenopus asymmetric expression of the ion pump ATP4a was reported at the 4-cell stage. The "ion-flux" model postulates that symmetry is broken flow-independently through an ATP4-generated asymmetric voltage gradient that drives serotonin through gap junctions to one side of the embryo. Here, we show that ATP4a is symmetrically expressed. Gene knockdown or pharmacological inhibition compromised organ situs, asymmetric marker gene expression, and leftward flow. The gastrocoel roof plate (GRP), where flow in frog occurs, revealed fewer, shortened, and misaligned cilia. Foxj1, a master control gene of motile cilia, was downregulated in the superficial mesoderm, from which the GRP develops. Specifically, ATP4 was required for Wnt/β-catenin-regulated Foxj1 induction and Wnt/PCP-dependent cilia polarization. Our work argues for evolutionary conservation of symmetry breakage in the vertebrates.