Natural genetic variation quantitatively regulates heart rate and dimension.

The polygenic contribution to heart development and function along the health-disease continuum remains unresolved. To gain insight into the genetic basis of quantitative cardiac phenotypes, we utilize highly inbred Japanese rice fish models, Oryzias latipes, and Oryzias sakaizumii. Employing automated quantification of embryonic heart rates as core metric, we profiled phenotype variability across five inbred strains. We observed maximal phenotypic contrast between individuals of the HO5 and the HdrR strain. HO5 showed elevated heart rates associated with embryonic ventricular hypoplasia and impaired adult cardiac function. This contrast served as the basis for genome-wide mapping. In a segregation population of 1192 HO5 x HdrR F2 embryos, we mapped 59 loci (173 genes) associated with heart rate. Experimental validation of the top 12 candidate genes in loss-of-function models revealed their causal and distinct impact on heart rate, development, ventricle size, and arrhythmia. Our study uncovers new diagnostic and therapeutic targets for developmental and electrophysiological cardiac diseases and provides a novel scalable approach to investigate the intricate genetic architecture of the vertebrate heart.

CRISPR-based knockout and base editing confirm the role of MYRF in heart development and congenital heart disease.

High-throughput DNA sequencing studies increasingly associate DNA variants with congenital heart disease (CHD). However, functional modeling is a crucial prerequisite for translating genomic data into clinical care. We used CRISPR-Cas9-mediated targeting of 12 candidate genes in the vertebrate model medaka (Oryzias latipes), five of which displayed a novel cardiovascular phenotype spectrum in F0 (crispants): mapre2, smg7, cdc42bpab, ankrd11 and myrf, encoding a transcription factor recently linked to cardiac-urogenital syndrome. Our myrf mutant line showed particularly prominent embryonic cardiac defects recapitulating phenotypes of pediatric patients, including hypoplastic ventricle. Mimicking human mutations, we edited three sites to generate specific myrf single-nucleotide variants via cytosine and adenine base editors. The Glu749Lys missense mutation in the conserved intramolecular chaperon autocleavage domain fully recapitulated the characteristic myrf mutant phenotype with high penetrance, underlining the crucial function of this protein domain. The efficiency and scalability of base editing to model specific point mutations accelerate gene validation studies and the generation of human-relevant disease models.

De novo PAM generation to reach initially inaccessible target sites for base editing.

Base editing by CRISPR crucially depends on the presence of a protospacer adjacent motif (PAM) at the correct distance from the editing site. Here, we present and validate an efficient one-shot approach termed 'inception' that expands the editing range. This is achieved by sequential, combinatorial base editing: de novo generated synonymous, non-synonymous or intronic PAM sites facilitate subsequent base editing at nucleotide positions that were initially inaccessible, further opening the targeting range of highly precise editing approaches. We demonstrate the applicability of the inception concept in medaka (Oryzias latipes) in three settings: loss of function, by introducing a pre-termination STOP codon in the open reading frame of oca2; locally confined multi-codon changes to generate allelic variants with different phenotypic severity in kcnh6a; and the removal of a splice acceptor site by targeting intronic sequences of rx3. Using sequentially acting base editors in the described combinatorial approach expands the number of accessible target sites by 65% on average. This allows the use of well-established tools with NGG PAM recognition for the establishment of thus far unreachable disease models, for hypomorphic allele studies and for efficient targeted mechanistic investigations in a precise and predictable manner.

Efficient single copy integration via homology-directed repair (scHDR) by 5'modification of large DNA donor fragments in mice.

CRISPR/Cas-based approaches have largely replaced conventional gene targeting strategies. However, homology-directed repair (HDR) in the mouse genome is not very efficient, and precisely inserting longer sequences using HDR remains challenging given that donor constructs preferentially integrate as concatemers. Here, we showed that injecting 5' biotinylated donor DNA into mouse embryos at the two-cell stage led to efficient single-copy HDR (scHDR) allele generation. Our dedicated genotyping strategy showed that these alleles occurred with frequencies of 19%, 20%, and 26% at three independent gene loci, indicating that scHDR was dramatically increased by 5' biotinylation. Thus, we suggest that the combination of a 5' biotinylated donor and diligent analysis of concatemer integration are prerequisites for efficiently and reliably generating conditional alleles or other large fragment knock-ins in the mouse genome.

Precise in vivo functional analysis of DNA variants with base editing using ACEofBASEs target prediction.

Single nucleotide variants (SNVs) are prevalent genetic factors shaping individual trait profiles and disease susceptibility. The recent development and optimizations of base editors, rubber and pencil genome editing tools now promise to enable direct functional assessment of SNVs in model organisms. However, the lack of bioinformatic tools aiding target prediction limits the application of base editing in vivo. Here, we provide a framework for adenine and cytosine base editing in medaka (Oryzias latipes) and zebrafish (Danio rerio), ideal for scalable validation studies. We developed an online base editing tool ACEofBASEs (a careful evaluation of base-edits), to facilitate decision-making by streamlining sgRNA design and performing off-target evaluation. We used state-of-the-art adenine (ABE) and cytosine base editors (CBE) in medaka and zebrafish to edit eye pigmentation genes and transgenic GFP function with high efficiencies. Base editing in the genes encoding troponin T and the potassium channel ERG faithfully recreated known cardiac phenotypes. Deep-sequencing of alleles revealed the abundance of intended edits in comparison to low levels of insertion or deletion (indel) events for ABE8e and evoBE4max. We finally validated missense mutations in novel candidate genes of congenital heart disease (CHD) dapk3, ube2b, usp44, and ptpn11 in F0 and F1 for a subset of these target genes with genotype-phenotype correlation. This base editing framework applies to a wide range of SNV-susceptible traits accessible in fish, facilitating straight-forward candidate validation and prioritization for detailed mechanistic downstream studies.

DNA contains sequences of four different molecules known as bases that represent our genetic code. In a mutation called a single nucleotide variant (or SNV for short), a single base in the sequence is swapped for another base. This can lead the individual carrying this SNV to produce a slightly different version of a protein to that found in other people. This slightly different protein may not work properly, or may perform a different task. In recent years, researchers have identified thousands of SNVs in humans linked with congenital heart diseases, but the roles of many of these SNVs remain unclear. Tools known as base editors allow researchers to efficiently modify single bases in DNA. Base editors use molecules known as short guide RNAs (or sgRNAs for short) to direct enzymes to specific positions in the DNA to swap, delete or insert a base. The sgRNAs need to be carefully designed to target the correct bases, however, which is a time consuming process. Furthermore, base editors were developed in cells grown in laboratories and so far only a few studies have demonstrated how they could be used in living animals. To overcome these limitations, Cornean, Gierten, Welz et al. developed a framework for base editing in two species of fish that are often used as models in research, namely medaka and zebrafish. The framework uses existing base editors that swap individual target bases and a new online tool ­ referred to as ACEofBASEs ­ to help design the required sgRNAs. The team were able to use the framework to characterize the medaka equivalents of four SNVs that have been previously associated with congenital heart disease in humans. The new framework developed here will help researchers to investigate the roles of SNVs in fish and other animals and validate human disease candidates. This approach could also be used to study the various ways that cells modify proteins by changing the specific bases involved in such modifications.

Boosting targeted genome editing using the hei-tag.

Precise, targeted genome editing by CRISPR/Cas9 is key for basic research and translational approaches in model and non-model systems. While active in all species tested so far, editing efficiencies still leave room for improvement. The bacterial Cas9 needs to be efficiently shuttled into the nucleus as attempted by fusion with nuclear localization signals (NLSs). Additional peptide tags such as FLAG- or myc-tags are usually added for immediate detection or straightforward purification. Immediate activity is usually granted by administration of preassembled protein/RNA complexes. We present the 'hei-tag (high efficiency-tag)' which boosts the activity of CRISPR/Cas genome editing tools already when supplied as mRNA. The addition of the hei-tag, a myc-tag coupled to an optimized NLS via a flexible linker, to Cas9 or a C-to-T (cytosine-to-thymine) base editor dramatically enhances the respective targeting efficiency. This results in an increase in bi-allelic editing, yet reduction of allele variance, indicating an immediate activity even at early developmental stages. The hei-tag boost is active in model systems ranging from fish to mammals, including tissue culture applications. The simple addition of the hei-tag allows to instantly upgrade existing and potentially highly adapted systems as well as to establish novel highly efficient tools immediately applicable at the mRNA level.

The genetic code stored within DNA provides cells with the instructions they need to carry out their role in the body. Any changes to these genes, or the DNA sequence around them, has the potential to completely alter how a cell behaves. Scientists have developed various tools that allow them to experimentally modify the genome of cells or even entire living organisms. This includes the popular Cas9 enzyme which cuts DNA at specific sites, and base editors which can precisely change bits of genetic code without cutting DNA. While there are lots of Cas9 enzymes and base editors currently available, these often differ greatly in their activity depending on which cell type or organism they are applied to. Finding a tool that can effectively modify the genome of an organism at the right time during development also poses a challenge. All the cells in an organism arise from a single fertilized cell. If this cell is genetically edited, all its subsequent daughter cells (which make up the entire organism) will contain the genetic modification. However, most genome editing tools only work efficiently later in development, resulting in an undesirable mosaic organism composed of both edited and non-edited cells. Here, Thumberger et al. have developed a new 'high efficiency-tag' (also known as hei-tag for short) that can enhance the activity of gene editing tools and overcome this barrier. The tag improves the efficiency of gene editing by immediately shuttling a Cas9 enzyme to the nucleus, the cellular compartment that stores DNA. In all cases, gene editing tools with hei-tag worked better than those without in fish embryos and mouse cells grown in the laboratory. When Cas9 enzymes connected to a hei-tag were injected into the first fertilized cell of a fish embryo, this resulted in an even distribution of edited genes spread throughout the whole organism. To understand how a gene affects an organism, researchers need to be able to edit it as early in development as possible. Attaching the 'hei-tag' to already available tools could help boost their activity and make them more efficient. It could also allow advances in medical research aimed at replacing faulty genes with fully functioning ones.

The Medaka Inbred Kiyosu-Karlsruhe (MIKK) panel.

BACKGROUND: Unraveling the relationship between genetic variation and phenotypic traits remains a fundamental challenge in biology. Mapping variants underlying complex traits while controlling for confounding environmental factors is often problematic. To address this, we establish a vertebrate genetic resource specifically to allow for robust genotype-to-phenotype investigations. The teleost medaka (Oryzias latipes) is an established genetic model system with a long history of genetic research and a high tolerance to inbreeding from the wild. RESULTS: Here we present the Medaka Inbred Kiyosu-Karlsruhe (MIKK) panel: the first near-isogenic panel of 80 inbred lines in a vertebrate model derived from a wild founder population. Inbred lines provide fixed genomes that are a prerequisite for the replication of studies, studies which vary both the genetics and environment in a controlled manner, and functional testing. The MIKK panel will therefore enable phenotype-to-genotype association studies of complex genetic traits while allowing for careful control of interacting factors, with numerous applications in genetic research, human health, drug development, and fundamental biology. CONCLUSIONS: Here we present a detailed characterization of the genetic variation across the MIKK panel, which provides a rich and unique genetic resource to the community by enabling large-scale experiments for mapping complex traits.

Genomic variations and epigenomic landscape of the Medaka Inbred Kiyosu-Karlsruhe (MIKK) panel.

BACKGROUND: The teleost medaka (Oryzias latipes) is a well-established vertebrate model system, with a long history of genetic research, and multiple high-quality reference genomes available for several inbred strains. Medaka has a high tolerance to inbreeding from the wild, thus allowing one to establish inbred lines from wild founder individuals. RESULTS: We exploit this feature to create an inbred panel resource: the Medaka Inbred Kiyosu-Karlsruhe (MIKK) panel. This panel of 80 near-isogenic inbred lines contains a large amount of genetic variation inherited from the original wild population. We use Oxford Nanopore Technologies (ONT) long read data to further investigate the genomic and epigenomic landscapes of a subset of the MIKK panel. Nanopore sequencing allows us to identify a large variety of high-quality structural variants, and we present results and methods using a pan-genome graph representation of 12 individual medaka lines. This graph-based reference MIKK panel genome reveals novel differences between the MIKK panel lines and standard linear reference genomes. We find additional MIKK panel-specific genomic content that would be missing from linear reference alignment approaches. We are also able to identify and quantify the presence of repeat elements in each of the lines. Finally, we investigate line-specific CpG methylation and performed differential DNA methylation analysis across these 12 lines. CONCLUSIONS: We present a detailed analysis of the MIKK panel genomes using long and short read sequence technologies, creating a MIKK panel-specific pan genome reference dataset allowing for investigation of novel variation types that would be elusive using standard approaches.

A rapid and simple procedure for the isolation and cultivation of fibroblast-like cells from medaka and zebrafish embryos and fin clip biopsies.

In many human diseases, the molecular pathophysiological mechanisms are not understood, which makes the development and testing of new therapeutic approaches difficult. The generation and characterization of animal models such as mice, rats, fruit flies, worms or fish offers the possibility for in detail studies of a disease's development, its course and potential therapies in an organismal context, which considerably minimizes the risk of therapeutic side effects for patients. Nevertheless, due to the high numbers of experimental animals used in research worldwide, attempts to develop alternative test systems will help in reducing their count. In this regard, the cell culture system displays a suitable option due to its potential of delivering nearly unlimited material and the good opportunities for high-throughput studies such as drug testing. Here, we describe a quick and simple method to isolate and cultivate vital fibroblast-like cells from embryos and adults of two popular teleost model organisms, the Japanese rice fish medaka (Oryzias latipes) and the zebrafish (Danio rerio).

In vivo identification and validation of novel potential predictors for human cardiovascular diseases.

Genetics crucially contributes to cardiovascular diseases (CVDs), the global leading cause of death. Since the majority of CVDs can be prevented by early intervention there is a high demand for the identification of predictive causative genes. While genome wide association studies (GWAS) correlate genes and CVDs after diagnosis and provide a valuable resource for such causative candidate genes, often preferentially those with previously known or suspected function are addressed further. To tackle the unaddressed blind spot of understudied genes, we particularly focused on the validation of human heart phenotype-associated GWAS candidates with little or no apparent connection to cardiac function. Building on the conservation of basic heart function and underlying genetics from fish to human we combined CRISPR/Cas9 genome editing of the orthologs of human GWAS candidates in isogenic medaka with automated high-throughput heart rate analysis. Our functional analyses of understudied human candidates uncovered a prominent fraction of heart rate associated genes from adult human patients impacting on the heart rate in embryonic medaka already in the injected generation. Following this pipeline, we identified 16 GWAS candidates with potential diagnostic and predictive power for human CVDs.

αß/γδ T cell lineage outcome is regulated by intrathymic cell localization and environmental signals.

αß and γδ T cells are two distinct sublineages that develop in the vertebrate thymus. Thus far, their differentiation from a common progenitor is mostly understood to be regulated by intrinsic mechanisms. However, the proportion of αß/γδ T cells varies in different vertebrate taxa. How this process is regulated in species that tend to produce a high frequency of γδ T cells is unstudied. Using an in vivo teleost model, the medaka, we report that progenitors first enter a thymic niche where their development into γδ T cells is favored. Translocation from this niche, mediated by chemokine receptor Ccr9b, is a prerequisite for their differentiation into αß T cells. On the other hand, the thymic niche also generates opposing gradients of the cytokine interleukin-7 and chemokine Ccl25a, and, together, they influence the lineage outcome. We propose a previously unknown mechanism that determines the proportion of αß/γδ lineages within species.

Fish primary embryonic pluripotent cells assemble into retinal tissue mirroring in vivo early eye development.

Organoids derived from pluripotent stem cells promise the solution to current challenges in basic and biomedical research. Mammalian organoids are however limited by long developmental time, variable success, and lack of direct comparison to an in vivo reference. To overcome these limitations and address species-specific cellular organization, we derived organoids from rapidly developing teleosts. We demonstrate how primary embryonic pluripotent cells from medaka and zebrafish efficiently assemble into anterior neural structures, particularly retina. Within 4 days, blastula-stage cell aggregates reproducibly execute key steps of eye development: retinal specification, morphogenesis, and differentiation. The number of aggregated cells and genetic factors crucially impacted upon the concomitant morphological changes that were intriguingly reflecting the in vivo situation. High efficiency and rapid development of fish-derived organoids in combination with advanced genome editing techniques immediately allow addressing aspects of development and disease, and systematic probing of impact of the physical environment on morphogenesis and differentiation.

A patient-based medaka alg2 mutant as a model for hypo-N-glycosylation.

Defects in the evolutionarily conserved protein-glycosylation machinery during embryonic development are often fatal. Consequently, congenital disorders of glycosylation (CDG) in human are rare. We modelled a putative hypomorphic mutation described in an alpha-1,3/1,6-mannosyltransferase (ALG2) index patient (ALG2-CDG) to address the developmental consequences in the teleost medaka (Oryzias latipes). We observed specific, multisystemic, late-onset phenotypes, closely resembling the patient's syndrome, prominently in the facial skeleton and in neuronal tissue. Molecularly, we detected reduced levels of N-glycans in medaka and in the patient's fibroblasts. This hypo-N-glycosylation prominently affected protein abundance. Proteins of the basic glycosylation and glycoprotein-processing machinery were over-represented in a compensatory response, highlighting the regulatory topology of the network. Proteins of the retinal phototransduction machinery, conversely, were massively under-represented in the alg2 model. These deficiencies relate to a specific failure to maintain rod photoreceptors, resulting in retinitis pigmentosa characterized by the progressive loss of these photoreceptors. Our work has explored only the tip of the iceberg of N-glycosylation-sensitive proteins, the function of which specifically impacts on cells, tissues and organs. Taking advantage of the well-described human mutation has allowed the complex interplay of N-glycosylated proteins and their contribution to development and disease to be addressed.

The C-Mannosylome of Human Induced Pluripotent Stem Cells Implies a Role for ADAMTS16 C-Mannosylation in Eye Development.

C-mannosylation is a modification of tryptophan residues with a single mannose and can affect protein folding, secretion, and/or function. To date, only a few proteins have been demonstrated to be C-mannosylated, and studies that globally assess protein C-mannosylation are scarce. To interrogate the C-mannosylome of human induced pluripotent stem cells, we compared the secretomes of CRISPR-Cas9 mutants lacking either the C-mannosyltransferase DPY19L1 or DPY19L3 to WT human induced pluripotent stem cells using MS-based quantitative proteomics. The secretion of numerous proteins was reduced in these mutants, including that of A Disintegrin And Metalloproteinase with ThromboSpondin Motifs 16 (ADAMTS16), an extracellular protease that was previously reported to be essential for optic fissure fusion in zebrafish eye development. To test the functional relevance of this observation, we targeted dpy19l1 or dpy19l3 in embryos of the Japanese rice fish medaka (Oryzias latipes) by CRISPR-Cas9. We observed that targeting of dpy19l3 partially caused defects in optic fissure fusion, called coloboma. We further showed in a cellular model that DPY19L1 and DPY19L3 mediate C-mannosylation of a recombinantly expressed thrombospondin type 1 repeat of ADAMTS16 and thereby support its secretion. Taken together, our findings imply that DPY19L3-mediated C-mannosylation is involved in eye development by assisting secretion of the extracellular protease ADAMTS16.

A complementary study approach unravels novel players in the pathoetiology of Hirschsprung disease.

Hirschsprung disease (HSCR, OMIM 142623) involves congenital intestinal obstruction caused by dysfunction of neural crest cells and their progeny during enteric nervous system (ENS) development. HSCR is a multifactorial disorder; pathogenetic variants accounting for disease phenotype are identified only in a minority of cases, and the identification of novel disease-relevant genes remains challenging. In order to identify and to validate a potential disease-causing relevance of novel HSCR candidate genes, we established a complementary study approach, combining whole exome sequencing (WES) with transcriptome analysis of murine embryonic ENS-related tissues, literature and database searches, in silico network analyses, and functional readouts using candidate gene-specific genome-edited cell clones. WES datasets of two patients with HSCR and their non-affected parents were analysed, and four novel HSCR candidate genes could be identified: ATP7A, SREBF1, ABCD1 and PIAS2. Further rare variants in these genes were identified in additional HSCR patients, suggesting disease relevance. Transcriptomics revealed that these genes are expressed in embryonic and fetal gastrointestinal tissues. Knockout of these genes in neuronal cells demonstrated impaired cell differentiation, proliferation and/or survival. Our approach identified and validated candidate HSCR genes and provided further insight into the underlying pathomechanisms of HSCR.

Genetic and functional insights into the fractal structure of the heart.

The inner surfaces of the human heart are covered by a complex network of muscular strands that is thought to be a remnant of embryonic development1,2. The function of these trabeculae in adults and their genetic architecture are unknown. Here we performed a genome-wide association study to investigate image-derived phenotypes of trabeculae using the fractal analysis of trabecular morphology in 18,096 participants of the UK Biobank. We identified 16 significant loci that contain genes associated with haemodynamic phenotypes and regulation of cytoskeletal arborization3,4. Using biomechanical simulations and observational data from human participants, we demonstrate that trabecular morphology is an important determinant of cardiac performance. Through genetic association studies with cardiac disease phenotypes and Mendelian randomization, we find a causal relationship between trabecular morphology and risk of cardiovascular disease. These findings suggest a previously unknown role for myocardial trabeculae in the function of the adult heart, identify conserved pathways that regulate structural complexity and reveal the influence of the myocardial trabeculae on susceptibility to cardiovascular disease.

Pcdh18a regulates endocytosis of E-cadherin during axial mesoderm development in zebrafish.

The notochord defines the axial structure of all vertebrates during development. Notogenesis is a result of major cell reorganization in the mesoderm, the convergence and the extension of the axial cells. However, it is currently not fully understood how these processes act together in a coordinated way during notochord formation. The prechordal plate is an actively migrating cell population in the central mesoderm anterior to the trailing notochordal plate cells. We show that prechordal plate cells express Protocadherin 18a (Pcdh18a), a member of the cadherin superfamily. We find that Pcdh18a-mediated recycling of E-cadherin adhesion complexes transforms prechordal plate cells into a cohesive and fast migrating cell group. In turn, the prechordal plate cells subsequently instruct the trailing mesoderm. We simulated cell migration during early mesoderm formation using a lattice-based mathematical framework and predicted that the requirement for an anterior, local motile cell cluster could guide the intercalation and extension of the posterior, axial cells. Indeed, a grafting experiment validated the prediction and local Pcdh18a expression induced an ectopic prechordal plate-like cell group migrating towards the animal pole. Our findings indicate that the Pcdh18a is important for prechordal plate formation, which influences the trailing mesodermal cell sheet by orchestrating the morphogenesis of the notochord.

Swift Large-scale Examination of Directed Genome Editing.

In the era of CRISPR gene editing and genetic screening, there is an increasing demand for quick and reliable nucleic acid extraction pipelines for rapid genotyping of large and diverse sample sets. Despite continuous improvements of current workflows, the handling-time and material costs per sample remain major limiting factors. Here we present a robust method for low-cost DIY-pipet tips addressing these needs; i.e. using a cellulose filter disc inserted into a regular pipet tip. These filter-in-tips allow for a rapid, stand-alone four-step genotyping workflow by simply binding the DNA contained in the primary lysate to the cellulose filter, washing it in water and eluting it directly into the buffer for the downstream application (e.g. PCR). This drastically cuts down processing time to maximum 30 seconds per sample, with the potential for parallelizing and automation. We show the ease and sensitivity of our procedure by genotyping genetically modified medaka (Oryzias latipes) and zebrafish (Danio rerio) embryos (targeted by CRISPR/Cas9 knock-out and knock-in) in a 96-well plate format. The robust isolation and detection of multiple alleles of various abundancies in a mosaic genetic background allows phenotype-genotype correlation already in the injected generation, demonstrating the reliability and sensitivity of the protocol. Our method is applicable across kingdoms to samples ranging from cells to tissues i. e. plant seedlings, adult flies, mouse cell culture and tissue as well as adult fish fin-clips.

An Early Function of Polycystin-2 for Left-Right Organizer Induction in Xenopus.

Nodal signaling controls asymmetric organ placement during vertebrate embryogenesis. Nodal is induced by a leftward fluid flow at the ciliated left-right organizer (LRO). The mechanism of flow sensing, however, has remained elusive. pkd2 encodes the calcium channel Polycystin-2, which is required for kidney development and laterality, and may act in flow perception. Here, we have studied the role of Polycystin-2 in Xenopus and show that pkd2 is indispensable for left-right (LR) asymmetry. Knockdown of pkd2 prevented left-asymmetric nodal cascade induction in the lateral plate mesoderm. Defects were due to failure of LRO specification, morphogenesis, and, consequently, absence of leftward flow. Polycystin-2 synergizes with the unconventional nodal-type signaling molecule Xnr3 to induce the LRO precursor tissue before gastrulation, upstream of symmetry breakage. Our data uncover an unknown function of pkd2 in LR axis formation, which we propose represents an ancient role of Polycystin-2 during LRO induction in lower vertebrates.

Efficient single-copy HDR by 5' modified long dsDNA donors.

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.