Efficient identification of CRISPR/Cas9-induced insertions/deletions by direct germline screening in zebrafish.

BACKGROUND: The CRISPR/Cas9 system is a prokaryotic immune system that infers resistance to foreign genetic material and is a sort of 'adaptive immunity'. It has been adapted to enable high throughput genome editing and has revolutionised the generation of targeted mutations. RESULTS: We have developed a scalable analysis pipeline to identify CRISPR/Cas9 induced mutations in hundreds of samples using next generation sequencing (NGS) of amplicons. We have used this system to investigate the best way to screen mosaic Zebrafish founder individuals for germline transmission of induced mutations. Screening sperm samples from potential founders provides much better information on germline transmission rates and crucially the sequence of the particular insertions/deletions (indels) that will be transmitted. This enables us to combine screening with archiving to create a library of cryopreserved samples carrying known mutations. It also allows us to design efficient genotyping assays, making identifying F1 carriers straightforward. CONCLUSIONS: The methods described will streamline the production of large numbers of knockout alleles in selected genes for phenotypic analysis, complementing existing efforts using random mutagenesis.

High-throughput and quantitative genome-wide messenger RNA sequencing for molecular phenotyping.

BACKGROUND: We present a genome-wide messenger RNA (mRNA) sequencing technique that converts small amounts of RNA from many samples into molecular phenotypes. It encompasses all steps from sample preparation to sequence analysis and is applicable to baseline profiling or perturbation measurements. RESULTS: Multiplex sequencing of transcript 3' ends identifies differential transcript abundance independent of gene annotation. We show that increasing biological replicate number while maintaining the total amount of sequencing identifies more differentially abundant transcripts. CONCLUSIONS: This method can be implemented on polyadenylated RNA from any organism with an annotated reference genome and in any laboratory with access to Illumina sequencing.

Xenopus mutant reveals necessity of rax for specifying the eye field which otherwise forms tissue with telencephalic and diencephalic character.

The retinal anterior homeobox (rax) gene encodes a transcription factor necessary for vertebrate eye development. rax transcription is initiated at the end of gastrulation in Xenopus, and is a key part of the regulatory network specifying anterior neural plate and retina. We describe here a Xenopus tropicalis rax mutant, the first mutant analyzed in detail from a reverse genetic screen. As in other vertebrates, this nonsense mutation results in eyeless animals, and is lethal peri-metamorphosis. Tissue normally fated to form retina in these mutants instead forms tissue with characteristics of diencephalon and telencephalon. This implies that a key role of rax, in addition to defining the eye field, is in preventing alternative forebrain identities. Our data highlight that brain and retina regions are not determined by the mid-gastrula stage but are by the neural plate stage. An RNA-Seq analysis and in situ hybridization assays for early gene expression in the mutant revealed that several key eye field transcription factors (e.g. pax6, lhx2 and six6) are not dependent on rax activity through neurulation. However, these analyses identified other genes either up- or down-regulated in mutant presumptive retinal tissue. Two neural patterning genes of particular interest that appear up-regulated in the rax mutant RNA-seq analysis are hesx1 and fezf2. These genes were not previously known to be regulated by rax. The normal function of rax is to partially repress their expression by an indirect mechanism in the presumptive retina region in wildtype embryos, thus accounting for the apparent up-regulation in the rax mutant. Knock-down experiments using antisense morpholino oligonucleotides directed against hesx1 and fezf2 show that failure to repress these two genes contributes to transformation of presumptive retinal tissue into non-retinal forebrain identities in the rax mutant.

A systematic genome-wide analysis of zebrafish protein-coding gene function.

Since the publication of the human reference genome, the identities of specific genes associated with human diseases are being discovered at a rapid rate. A central problem is that the biological activity of these genes is often unclear. Detailed investigations in model vertebrate organisms, typically mice, have been essential for understanding the activities of many orthologues of these disease-associated genes. Although gene-targeting approaches and phenotype analysis have led to a detailed understanding of nearly 6,000 protein-coding genes, this number falls considerably short of the more than 22,000 mouse protein-coding genes. Similarly, in zebrafish genetics, one-by-one gene studies using positional cloning, insertional mutagenesis, antisense morpholino oligonucleotides, targeted re-sequencing, and zinc finger and TAL endonucleases have made substantial contributions to our understanding of the biological activity of vertebrate genes, but again the number of genes studied falls well short of the more than 26,000 zebrafish protein-coding genes. Importantly, for both mice and zebrafish, none of these strategies are particularly suited to the rapid generation of knockouts in thousands of genes and the assessment of their biological activity. Here we describe an active project that aims to identify and phenotype the disruptive mutations in every zebrafish protein-coding gene, using a well-annotated zebrafish reference genome sequence, high-throughput sequencing and efficient chemical mutagenesis. So far we have identified potentially disruptive mutations in more than 38% of all known zebrafish protein-coding genes. We have developed a multi-allelic phenotyping scheme to efficiently assess the effects of each allele during embryogenesis and have analysed the phenotypic consequences of over 1,000 alleles. All mutant alleles and data are available to the community and our phenotyping scheme is adaptable to phenotypic analysis beyond embryogenesis.

A comparative survey of the frequency and distribution of polymorphism in the genome of Xenopus tropicalis.

Naturally occurring DNA sequence variation within a species underlies evolutionary adaptation and can give rise to phenotypic changes that provide novel insight into biological questions. This variation exists in laboratory populations just as in wild populations and, in addition to being a source of useful alleles for genetic studies, can impact efforts to identify induced mutations in sequence-based genetic screens. The Western clawed frog Xenopus tropicalis (X. tropicalis) has been adopted as a model system for studying the genetic control of embryonic development and a variety of other areas of research. Its diploid genome has been extensively sequenced and efforts are underway to isolate mutants by phenotype- and genotype-based approaches. Here, we describe a study of genetic polymorphism in laboratory strains of X. tropicalis. Polymorphism was detected in the coding and non-coding regions of developmental genes distributed widely across the genome. Laboratory strains exhibit unexpectedly high frequencies of genetic polymorphism, with alleles carrying a variety of synonymous and non-synonymous codon substitutions and nucleotide insertions/deletions. Inter-strain comparisons of polymorphism uncover a high proportion of shared alleles between Nigerian and Ivory Coast strains, in spite of their distinct geographical origins. These observations will likely influence the design of future sequence-based mutation screens, particularly those using DNA mismatch-based detection methods which can be disrupted by the presence of naturally occurring sequence variants. The existence of a significant reservoir of alleles also suggests that existing laboratory stocks may be a useful source of novel alleles for mapping and functional studies.

Genetic screens for mutations affecting development of Xenopus tropicalis.

We present here the results of forward and reverse genetic screens for chemically-induced mutations in Xenopus tropicalis. In our forward genetic screen, we have uncovered 77 candidate phenotypes in diverse organogenesis and differentiation processes. Using a gynogenetic screen design, which minimizes time and husbandry space expenditures, we find that if a phenotype is detected in the gynogenetic F2 of a given F1 female twice, it is highly likely to be a heritable abnormality (29/29 cases). We have also demonstrated the feasibility of reverse genetic approaches for obtaining carriers of mutations in specific genes, and have directly determined an induced mutation rate by sequencing specific exons from a mutagenized population. The Xenopus system, with its well-understood embryology, fate map, and gain-of-function approaches, can now be coupled with efficient loss-of-function genetic strategies for vertebrate functional genomics and developmental genetics.

Genetic and genomic prospects for Xenopus tropicalis research.

Research using Xenopus laevis has made enormous contributions to our understanding of vertebrate development, control of the eukaryotic cell cycle and the cytoskeleton. One limitation, however, has been the lack of systematic genetic studies in Xenopus to complement molecular and cell biological investigations. Work with the closely related diploid frog Xenopus tropicalis is beginning to address this limitation. Here, we review the resources that will make genetic studies using X. tropicalis a reality.

Depletion of the cell-cycle inhibitor p27(Xic1) impairs neuronal differentiation and increases the number of ElrC(+) progenitor cells in Xenopus tropicalis.

The Xenopus p27(Xic1) gene encodes a cyclin dependent kinase (CDK) inhibitor of the Cip/Kip family. We have previously shown that p27(Xic1) is expressed in the cells of the neural plate as they become post-mitotic (Development 127 (2000) 1303). To investigate whether p27(Xic1) is necessary for cell cycle exit and/or neuronal differentiation, we used antisense morpholino oligos (MO) to knockdown the protein levels in vivo. For such knockdown studies, Xenopus tropicalis is a better model system than Xenopus laevis, since it has a diploid genome. Indeed, while X. laevis has two p27(Xic1) paralogs, p27(Xic1) and p28(Kix1), we have found only one ortholog in X. tropicalis, equidistant from the X. laevis genes. The X. tropicalis p27(Xic1) was expressed in a similar pattern to the X. laevis gene. Depletion of p27(Xic1) in X. tropicalis caused an increase in proliferation and a suppression of the neuronal differentiation marker, N-tubulin. At the same time, we found an increase in the expression of ElrC, a marker of cells as they undergo a transition from proliferation to differentiation. We conclude that p27(Xic1) is necessary for cells to exit the cell cycle and differentiate; in its absence, cells accumulate in a progenitor state. The expression of p27(Xic1) in the embryo is regionalised but the transcriptional regulation of p27(Xic1) is not well understood. We report the isolation of a p27(Xic1) genomic clone and we identify a 5' region capable of driving reporter gene expression specifically in the neural tube and the eye.

Molecular components of the endoderm specification pathway in Xenopus tropicalis.

Xenopus laevis has been instrumental in elucidating a conserved molecular pathway that regulates vertebrate endoderm specification. However, loss-of-function analysis is required to resolve the precise function of the genes involved. For such analysis, antisense oligos and possibly forward genetics are likely to be more effective in the diploid species Xenopus tropicalis than in the pseudotetraploid Xenopus laevis. Here we have isolated most of the tropicalis genes in the endoderm specification pathway, specifically, tVegT, tMixer, tMix, tBix, tGata6, tSox17alpha, tSox17beta, tFoxA1, tHex, and tCerberus, which lack the redundant copies that are found in laevis. In situ hybridization analysis has revealed identical expression patterns between the orthologous tropicalis and laevis endoderm genes, thus suggesting conserved genetic functions. Furthermore, we noted that the smaller tropicalis embryos gave better probe penetration than in laevis whole-mount in situ hybridizations-allowing us to visualize transcripts in the deep endoderm in tropicalis, which is difficult in laevis. This study illustrates how an entire genetic pathway can be quickly transferred from laevis to tropicalis due to high sequence conservation between the sister species and the large number of tropicalis-expressed sequence tags that are now available.