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.

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.

The inner surfaces of the human heart are covered by a complex network of muscular strands that is thought to be a vestige of embryonic development. 1,2 The function of these trabeculae in adults and their genetic architecture are unknown. To investigate this we performed a genome-wide association study using fractal analysis of trabecular morphology as an image-derived phenotype in 18,096 UK Biobank participants. We identified 16 significant loci containing genes associated with haemodynamic phenotypes and regulation of cytoskeletal arborisation. 3,4 Using biomechanical simulations and human observational data, we demonstrate that trabecular morphology is an important determinant of cardiac performance. Through genetic association studies with cardiac disease phenotypes and Mendelian randomisation, we find a causal relationship between trabecular morphology and cardiovascular disease risk. These findings suggest an unexpected role for myocardial trabeculae in the function of the adult heart, identify conserved pathways that regulate structural complexity, and reveal their influence on susceptibility to disease. MainThe chambers of the mature human heart have a complex inner surface whose function is unknown. Unlike the smooth endothelium of the great vessels, the endocardial surfaces of both ventricles are lined by a fenestrated network of muscular trabeculae which extend into the cavity. Their embryological development is driven by highly-conserved signalling pathways involving the endocardium-myocardium and extra-cellular matrix that regulate myocardial proliferation during cardiac morphogenesis. 2,[5][6][7][8][9] 1 Cell lineage tracing suggests that trabeculae have a molecular and developmental identity which is distinct from the compact myocardium. 10 The high surface area of trabeculae enables nutrient and oxygen diffusion from blood pool to myocardium before the coronary circulation is established. 1 Trabeculae are also vital to formation of the conduction system. 11 Theoretical analyses have proposed that their complex structure may contribute to efficient intra-ventricular flow patterns. 12-14 While hypertrabeculation is observed as a feature of some genetically-characterised cardiomyopathies, 15 the physiological function of trabeculae in adult hearts, their genetic architecture, and potential role in common disease have not been determined.The distinguishing trait of trabeculae is their branching morphology and the degree of such biological complexity in the heart can be quantified by fractal dimension (FD) analysis of cardiac magnetic resonance (CMR) imaging. 8 In a replicated genomewide association study (GWAS), using FD as an image-derived phenotype, we identify loci linked with trabecular morphology. Knockout models of loci-associated genes showed a marked decrease in trabecular complexity. Using biomechanical modelling and human observational data, we find a causal relationship between myocardial trabeculation and ventricular performance, with Mendelian randomisation showing that reduced trabecula...

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.