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.

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.