Injury-induced cooperation of InhibinßA and JunB is essential for cell proliferation in Xenopus tadpole tail regeneration.

In animal species that have the capability of regenerating tissues and limbs, cell proliferation is enhanced after wound healing and is essential for the reconstruction of injured tissue. Although the ability to induce cell proliferation is a common feature of such species, the molecular mechanisms that regulate the transition from wound healing to regenerative cell proliferation remain unclear. Here, we show that upon injury, InhibinßA and JunB cooperatively function for this transition during Xenopus tadpole tail regeneration. We found that the expression of inhibin subunit beta A (inhba) and junB proto-oncogene (junb) is induced by injury-activated TGF-ß/Smad and MEK/ERK signaling in regenerating tails. Similarly to junb knockout (KO) tadpoles, inhba KO tadpoles show a delay in tail regeneration, and inhba/junb double KO (DKO) tadpoles exhibit severe impairment of tail regeneration compared with either inhba KO or junb KO tadpoles. Importantly, this impairment is associated with a significant reduction of cell proliferation in regenerating tissue. Moreover, JunB regulates tail regeneration via FGF signaling, while InhibinßA likely acts through different mechanisms. These results demonstrate that the cooperation of injury-induced InhibinßA and JunB is critical for regenerative cell proliferation, which is necessary for re-outgrowth of regenerating Xenopus tadpole tails.

Sf3b4 mutation in Xenopus tropicalis causes RNA splicing defects followed by massive gene dysregulation that disrupt cranial neural crest development.

Nager syndrome is a rare craniofacial and limb disorder characterized by midface retrusion, micrognathia, absent thumbs, and radial hypoplasia. This disorder results from haploinsufficiency of SF3B4 (splicing factor 3b, subunit 4) a component of the pre-mRNA spliceosomal machinery. The spliceosome is a complex of RNA and proteins that function together to remove introns and join exons from transcribed pre-mRNA. While the spliceosome is present and functions in all cells of the body, most spliceosomopathies - including Nager syndrome - are cell/tissue-specific in their pathology. In Nager syndrome patients, it is the neural crest (NC)-derived craniofacial skeletal structures that are primarily affected. To understand the pathomechanism underlying this condition, we generated a Xenopus tropicalis sf3b4 mutant line using the CRISPR/Cas9 gene editing technology. Here we describe the sf3b4 mutant phenotype at neurula, tail bud, and tadpole stages, and performed temporal RNA-sequencing analysis to characterize the splicing events and transcriptional changes underlying this phenotype. Our data show that while loss of one copy of sf3b4 is largely inconsequential in Xenopus tropicalis, homozygous deletion of sf3b4 causes major splicing defects and massive gene dysregulation, which disrupt cranial NC cell migration and survival, thereby pointing at an essential role of Sf3b4 in craniofacial development.

Do organisms need an impact factor? Citations of key biological resources including model organisms reveal usage patterns and impact.

Research resources like transgenic animals and antibodies are the workhorses of biomedicine, enabling investigators to relatively easily study specific disease conditions. As key biological resources, transgenic animals and antibodies are often validated, maintained, and distributed from university based stock centers. As these centers heavily rely largely on grant funding, it is critical that they are cited by investigators so that usage can be tracked. However, unlike systems for tracking the impact of papers, the conventions and systems for tracking key resource usage and impact lag behind. Previous studies have shown that about 50% of the resources are not findable, making the studies they are supporting irreproducible, but also makes tracking resources difficult. The RRID project is filling this gap by working with journals and resource providers to improve citation practices and to track the usage of these key resources. Here, we reviewed 10 years of citation practices for five university based stock centers, characterizing each reference into two broad categories: findable (authors could use the RRID, stock number, or full name) and not findable (authors could use a nickname or a common name that is not unique to the resource). The data revealed that when stock centers asked their communities to cite resources by RRID, in addition to helping stock centers more easily track resource usage by increasing the number of RRID papers, authors shifted from citing resources predominantly by nickname (~50% of the time) to citing them by one of the findable categories (~85%) in a matter of several years. In the case of one stock center, the MMRRC, the improvement in findability is also associated with improvements in the adherence to NIH rigor criteria, as determined by a significant increase in the Rigor and Transparency Index for studies using MMRRC mice. From this data, it was not possible to determine whether outreach to authors or changes to stock center websites drove better citation practices, but findability of research resources and rigor adherence was improved.

Functional dissection and assembly of a small, newly evolved, W chromosome-specific genomic region of the African clawed frog Xenopus laevis.

Genetic triggers for sex determination are frequently co-inherited with other linked genes that may also influence one or more sex-specific phenotypes. To better understand how sex-limited regions evolve and function, we studied a small W chromosome-specific region of the frog Xenopus laevis that contains only three genes (dm-w, scan-w, ccdc69-w) and that drives female differentiation. Using gene editing, we found that the sex-determining function of this region requires dm-w but that scan-w and ccdc69-w are not essential for viability, female development, or fertility. Analysis of mesonephros+gonad transcriptomes during sexual differentiation illustrates masculinization of the dm-w knockout transcriptome, and identifies mostly non-overlapping sets of differentially expressed genes in separate knockout lines for each of these three W-specific gene compared to wildtype sisters. Capture sequencing of almost all Xenopus species and PCR surveys indicate that the female-determining function of dm-w is present in only a subset of species that carry this gene. These findings map out a dynamic evolutionary history of a newly evolved W chromosome-specific genomic region, whose components have distinctive functions that frequently degraded during Xenopus diversification, and evidence the evolutionary consequences of recombination suppression.

Photoreceptor disc incisures form as an adaptive mechanism ensuring the completion of disc enclosure.

The first steps of vision take place within a stack of tightly packed disc-shaped membranes, or 'discs', located in the outer segment compartment of photoreceptor cells. In rod photoreceptors, discs are enclosed inside the outer segment and contain deep indentations in their rims called 'incisures'. The presence of incisures has been documented in a variety of species, yet their role remains elusive. In this study, we combined traditional electron microscopy with three-dimensional electron tomography to demonstrate that incisures are formed only after discs become completely enclosed. We also observed that, at the earliest stage of their formation, discs are not round as typically depicted but rather are highly irregular in shape and resemble expanding lamellipodia. Using genetically manipulated mice and frogs and measuring outer segment protein abundances by quantitative mass spectrometry, we further found that incisure size is determined by the molar ratio between peripherin-2, a disc rim protein critical for the process of disc enclosure, and rhodopsin, the major structural component of disc membranes. While a high perpherin-2 to rhodopsin ratio causes an increase in incisure size and structural complexity, a low ratio precludes incisure formation. Based on these data, we propose a model whereby normal rods express a modest excess of peripherin-2 over the amount required for complete disc enclosure in order to ensure that this important step of disc formation is accomplished. Once the disc is enclosed, the excess peripherin-2 incorporates into the rim to form an incisure.

Advances in the Xenopus immunome: Diversification, expansion, and contraction.

Xenopus is a genus of African clawed frogs including two species, X. tropicalis and X. laevis that are extensively used in experimental biology, immunology, and biomedical studies. The availability of fully sequenced and annotated Xenopus genomes is strengthening genome-wide analyses of gene families and transgenesis to model human diseases. However, inaccuracies in genome annotation for genes involved in the immune system (i.e., immunome) hamper immunogenetic studies. Furthermore, advanced genome technologies (e.g., single-cell and RNA-Seq) rely on well-annotated genomes. The annotation problems of Xenopus immunome include a lack of established orthology across taxa, merged gene models, poor representation in gene pages on Xenbase, misannotated genes and missing gene IDs. The Xenopus Research Resource for Immunobiology in collaboration with Xenbase and a group of investigators are working to resolve these issues in the latest versions of genome browsers. In this review, we summarize the current problems of previously misannotated gene families that we have recently resolved. We also highlight the expansion, contraction, and diversification of previously misannotated gene families.

Age-associated DNA methylation changes in Xenopus frogs.

Age-associated changes in DNA methylation have been characterized across various animals, but not yet in amphibians, which are of particular interest because they include widely studied model organisms. In this study, we present clear evidence that the aquatic vertebrate species Xenopus tropicalis displays patterns of age-associated changes in DNA methylation. We have generated whole-genome bisulfite sequencing (WGBS) profiles from skin samples of nine frogs representing young, mature, and old adults and characterized the gene- and chromosome-scale DNA methylation changes with age. Many of the methylation features and changes we observe are consistent with what is known in mammalian species, suggesting that the mechanism of age-related changes is conserved. Moreover, we selected a few thousand age-associated CpG sites to build an assay based on targeted DNA methylation analysis (TBSseq) to expand our findings in future studies involving larger cohorts of individuals. Preliminary results of a pilot TBSeq experiment recapitulate the findings obtained with WGBS setting the basis for the development of an epigenetic clock assay. The results of this study will allow us to leverage the unique resources available for Xenopus to study how DNA methylation relates to other hallmarks of ageing.

Photoreceptor disc incisures form as an adaptive mechanism ensuring the completion of disc enclosure.

The first steps of vision take place within a stack of tightly packed disc-shaped membranes, or "discs", located in the outer segment compartment of photoreceptor cells. In rod photoreceptors, discs are enclosed inside the outer segment and contain deep indentations in their rims called "incisures". The presence of incisures has been documented in a variety of species, yet their role remains elusive. In this study, we combined traditional electron microscopy with three-dimensional electron tomography to demonstrate that incisures are formed only after discs become completely enclosed. We also observed that, at the earliest stage of their formation, discs are not round as typically depicted but rather are highly irregular in shape and resemble expanding lamellipodia. Using genetically manipulated mice and frogs and measuring outer segment protein abundances by quantitative mass spectrometry, we further found that incisure size is determined by the molar ratio between peripherin-2, a disc rim protein critical for the process of disc enclosure, and rhodopsin, the major structural component of disc membranes. While a high perpherin-2 to rhodopsin ratio causes an increase in incisure size and structural complexity, a low ratio precludes incisure formation. Based on these data, we propose a model whereby normal rods express a modest excess of peripherin-2 over the amount required for complete disc enclosure in order to ensure that this important step of disc formation is accomplished. Once the disc is enclosed, the excess peripherin-2 incorporates into the rim to form an incisure.

Development and metamorphosis in frogs deficient in the thyroid hormone transporter MCT8.

Precisely regulated thyroid hormone (TH) signaling within tissues during frog metamorphosis gives rise to the organism-wide coordination of developmental events among organs required for survival. This TH signaling is controlled by multiple cellular mechanisms, including TH transport across the plasma membrane. A highly specific TH transporter has been identified, namely monocarboxylate transporter 8 (MCT8), which facilitates uptake and efflux of TH and is differentially and dynamically expressed among tissues during metamorphosis. We hypothesized that loss of MCT8 would alter tissue sensitivity to TH and affect the timing of tissue transformation. To address this, we used CRISPR/Cas9 to introduce frameshift mutations inslc16a2, the gene encoding MCT8, inXenopus laevis. We produced homozygous mutant tadpoles with a 29-bp mutation in the l-chromosome and a 20-bp mutation in the S-chromosome. We found that MCT8 mutants survive metamorphosis with normal growth and development of external morphology throughout the larval period. Consistent with this result, the expression of the pituitary hormone regulating TH plasma levels (tshb) was similar among genotypes as was TH response gene expression in brain at metamorphic climax. Further, delayed initiation of limb outgrowth during natural metamorphosis and reduced hindlimb and tail TH sensitivity were not observed in MCT8 mutants. In sum, we did not observe an effect on TH-dependent development in MCT8 mutants, suggesting compensatory TH transport occurs in tadpole tissues, as seen in most tissues in all model organisms examined.

Maternal Wnt11b regulates cortical rotation during Xenopus axis formation: analysis of maternal-effect wnt11b mutants.

Asymmetric signalling centres in the early embryo are essential for axis formation in vertebrates. These regions (e.g. amphibian dorsal morula, mammalian anterior visceral endoderm) require stabilised nuclear ß-catenin, but the role of localised Wnt ligand signalling activity in their establishment remains unclear. In Xenopus, dorsal ß-catenin is initiated by vegetal microtubule-mediated symmetry breaking in the fertilised egg, known as 'cortical rotation'. Localised wnt11b mRNA and ligand-independent activators of ß-catenin have been implicated in dorsal ß-catenin activation, but the extent to which each contributes to axis formation in this paradigm remains unclear. Here, we describe a CRISPR-mediated maternal-effect mutation in Xenopus laevis wnt11b.L. We find that wnt11b is maternally required for robust dorsal axis formation and for timely gastrulation, and zygotically for left-right asymmetry. Importantly, we show that vegetal microtubule assembly and cortical rotation are reduced in wnt11b mutant eggs. In addition, we show that activated Wnt coreceptor Lrp6 and Dishevelled lack behaviour consistent with roles in early ß-catenin stabilisation, and that neither is regulated by Wnt11b. This work thus implicates Wnt11b in the distribution of putative dorsal determinants rather than in comprising the determinants themselves. This article has an associated 'The people behind the papers' interview.

Developing immortal cell lines from Xenopus embryos, four novel cell lines derived from Xenopus tropicalis.

The diploid anuran Xenopus tropicalis has emerged as a key research model in cell and developmental biology. To enhance the usefulness of this species, we developed methods for generating immortal cell lines from Nigerian strain (NXR_1018, RRID:SCR_013731) X. tropicalis embryos. We generated 14 cell lines that were propagated for several months. We selected four morphologically distinct lines, XTN-6, XTN-8, XTN-10 and XTN-12 for further characterization. Karyotype analysis revealed that three of the lines, XTN-8, XTN-10 and XTN-12 were primarily diploid. XTN-6 cultures showed a consistent mixed population of diploid cells, cells with chromosome 8 trisomy, and cells containing a tetraploid content of chromosomes. The lines were propagated using conventional culture methods as adherent cultures at 30°C in a simple, diluted L-15 medium containing fetal bovine serum without use of a high CO2 incubator. Transcriptome analysis indicated that the four lines were distinct lineages. These methods will be useful in the generation of cell lines from normal and mutant strains of X. tropicalis as well as other species of Xenopus.

Normal Table of Xenopus development: a new graphical resource.

Normal tables of development are essential for studies of embryogenesis, serving as an important resource for model organisms, including the frog Xenopus laevis. Xenopus has long been used to study developmental and cell biology, and is an increasingly important model for human birth defects and disease, genomics, proteomics and toxicology. Scientists utilize Nieuwkoop and Faber's classic 'Normal Table of Xenopus laevis (Daudin)' and accompanying illustrations to enable experimental reproducibility and reuse the illustrations in new publications and teaching. However, it is no longer possible to obtain permission for these copyrighted illustrations. We present 133 new, high-quality illustrations of X. laevis development from fertilization to metamorphosis, with additional views that were not available in the original collection. All the images are available on Xenbase, the Xenopus knowledgebase (http://www.xenbase.org/entry/zahn.do), for download and reuse under an attributable, non-commercial creative commons license. Additionally, we have compiled a 'Landmarks Table' of key morphological features and marker gene expression that can be used to distinguish stages quickly and reliably (https://www.xenbase.org/entry/landmarks-table.do). This new open-access resource will facilitate Xenopus research and teaching in the decades to come.

Endogenous Retroviruses Augment Amphibian (Xenopus laevis) Tadpole Antiviral Protection.

The global amphibian declines are compounded by infections with members of the Ranavirus genus such as Frog Virus 3 (FV3). Premetamorphic anuran amphibians are believed to be significantly more susceptible to FV3 while this pathogen targets the kidneys of both pre- and postmetamorphic animals. Paradoxically, FV3-challenged Xenopus laevis tadpoles exhibit lower kidney viral loads than adult frogs. Presently, we demonstrate that X. laevis tadpoles are intrinsically more resistant to FV3 kidney infections than cohort-matched metamorphic and postmetamorphic froglets and that this resistance appears to be epigenetically conferred by endogenous retroviruses (ERVs). Using a X. laevis kidney-derived cell line, we show that enhancing ERV gene expression activates cellular double-stranded RNA-sensing pathways, resulting in elevated mRNA levels of antiviral interferon (IFN) cytokines and thus greater anti-FV3 protection. Finally, our results indicate that large esterase-positive myeloid-lineage cells, rather than renal cells, are responsible for the elevated ERV/IFN axis seen in the tadpole kidneys. This conclusion is supported by our observation that CRISPR-Cas9 ablation of colony-stimulating factor-3 results in abolished homing of these myeloid cells to tadpole kidneys, concurrent with significantly abolished tadpole kidney expression of both ERVs and IFNs. We believe that the manuscript marks an important step forward in understanding the mechanisms controlling amphibian antiviral defenses and thus susceptibility and resistance to pathogens like FV3. IMPORTANCE Global amphibian biodiversity is being challenged by pathogens like the Frog Virus 3 (FV3) ranavirus, underlining the need to gain a greater understanding of amphibian antiviral defenses. While it was previously believed that anuran (frog/toad) amphibian tadpoles are more susceptible to FV3, we demonstrated that tadpoles are in fact more resistant to this virus than metamorphic and postmetamorphic froglets. We showed that this resistance is conferred by large myeloid cells within the tadpole kidneys (central FV3 target), which possess an elevated expression of endogenous retroviruses (ERVs). In turn, these ERVs activate cellular double-stranded RNA-sensing pathways, resulting in a greater expression of antiviral interferon cytokines, thereby offering the observed anti-FV3 protection.

Deep learning is widely applicable to phenotyping embryonic development and disease.

Genome editing simplifies the generation of new animal models for congenital disorders. However, the detailed and unbiased phenotypic assessment of altered embryonic development remains a challenge. Here, we explore how deep learning (U-Net) can automate segmentation tasks in various imaging modalities, and we quantify phenotypes of altered renal, neural and craniofacial development in Xenopus embryos in comparison with normal variability. We demonstrate the utility of this approach in embryos with polycystic kidneys (pkd1 and pkd2) and craniofacial dysmorphia (six1). We highlight how in toto light-sheet microscopy facilitates accurate reconstruction of brain and craniofacial structures within X. tropicalis embryos upon dyrk1a and six1 loss of function or treatment with retinoic acid inhibitors. These tools increase the sensitivity and throughput of evaluating developmental malformations caused by chemical or genetic disruption. Furthermore, we provide a library of pre-trained networks and detailed instructions for applying deep learning to the reader's own datasets. We demonstrate the versatility, precision and scalability of deep neural network phenotyping on embryonic disease models. By combining light-sheet microscopy and deep learning, we provide a framework for higher-throughput characterization of embryonic model organisms. This article has an associated 'The people behind the papers' interview.

Generation of a new six1-null line in Xenopus tropicalis for study of development and congenital disease.

The vertebrate Six (Sine oculis homeobox) family of homeodomain transcription factors plays critical roles in the development of several organs. Six1 plays a central role in cranial placode development, including the precursor tissues of the inner ear, as well as other cranial sensory organs and the kidney. In humans, mutations in SIX1 underlie some cases of Branchio-oto-renal (BOR) syndrome, which is characterized by moderate-to-severe hearing loss. We utilized CRISPR/Cas9 technology to establish a six1 mutant line in Xenopus tropicalis that is available to the research community. We demonstrate that at larval stages, the six1-null animals show severe disruptions in gene expression of putative Six1 target genes in the otic vesicle, cranial ganglia, branchial arch, and neural tube. At tadpole stages, six1-null animals display dysmorphic Meckel's, ceratohyal, and otic capsule cartilage morphology. This mutant line will be of value for the study of the development of several organs as well as congenital syndromes that involve these tissues.

TGF-ß1 signaling is essential for tissue regeneration in the Xenopus tadpole tail.

Amphibians such as Xenopus tropicalis exhibit a remarkable capacity for tissue regeneration after traumatic injury. Although transforming growth factor-ß (TGF-ß) receptor signaling is known to be essential for tissue regeneration in fish and amphibians, the role of TGF-ß ligands in this process is not well understood. Here, we show that inhibition of TGF-ß1 function prevents tail regeneration in Xenopus tropicalis tadpoles. We found that expression of tgfb1 is present before tail amputation and is sustained throughout the regeneration process. CRISPR-mediated knock-out (KO) of tgfb1 retards tail regeneration; the phenotype of tgfb1 KO tadpoles can be rescued by injection of tgfb1 mRNA. Cell proliferation, a critical event for the success of tissue regeneration, is downregulated in tgfb1 KO tadpoles. In addition, tgfb1 KO reduces the expression of phosphorylated Smad2/3 (pSmad2/3) which is important for TGF-ß signal-mediated cell proliferation. Collectively, our results show that TGF-ß1 regulates cell proliferation through the activation of Smad2/3. We therefore propose that TGF-ß1 plays a critical role in TGF-ß receptor-dependent tadpole tail regeneration in Xenopus.

Obtaining Xenopus laevis Embryos.

The embryos of the African clawed frog, Xenopus laevis, are a powerful substrate for the study of complex fundamental biological and disease mechanisms in neurobiology, physiology, molecular biology, cell biology, and developmental biology. A simple and straightforward technique for generating a large number of developmentally synchronized embryos is in vitro fertilization (IVF). IVF permits simultaneous fertilization of thousands of eggs but requires the death of the parental male, which may not be feasible if the male comes from a stock of precious animals. An alternative to euthanizing a precious male is to use a natural mating, which allows for the collection of many embryos with minimal preparation but with the potential loss of the experimental advantage of developmental synchronization. Here we present both strategies for obtaining X. laevis embryos.

Obtaining Xenopus laevis Eggs.

Nearly a century ago, studies by Lancelot Hogben and others demonstrated that ovulation in female Xenopus laevis can be induced via injection of mammalian gonadotropins into the dorsal lymph sac, allowing for egg production throughout the year independent of the normal reproductive cycles. Hormonally induced females are capable of producing thousands of eggs in a single spawning, which can then be fertilized to generate embryos or used as a substrate for generation of egg extracts. The protocol for induction of ovulation and subsequent egg collection is straightforward and robust, yet some of its details may vary among laboratories based on prior training, availability of necessary reagents, or the experimental objectives. As the goal of this protocol is not to describe every single variation possible for acquiring eggs but to provide a simple and clear description that can be easily applied by researchers with no prior working experience with X. laevis, we focus on describing the method we use at the National Xenopus Resource-that is, inducing ovulation in X. laevis via dorsal lymph sac injection of gonadotropic hormones and the stimulation of egg laying through application of gentle pressure to the females.

A frog with three sex chromosomes that co-mingle together in nature: Xenopus tropicalis has a degenerate W and a Y that evolved from a Z chromosome.

In many species, sexual differentiation is a vital prelude to reproduction, and disruption of this process can have severe fitness effects, including sterility. It is thus interesting that genetic systems governing sexual differentiation vary among-and even within-species. To understand these systems more, we investigated a rare example of a frog with three sex chromosomes: the Western clawed frog, Xenopus tropicalis. We demonstrate that natural populations from the western and eastern edges of Ghana have a young Y chromosome, and that a male-determining factor on this Y chromosome is in a very similar genomic location as a previously known female-determining factor on the W chromosome. Nucleotide polymorphism of expressed transcripts suggests genetic degeneration on the W chromosome, emergence of a new Y chromosome from an ancestral Z chromosome, and natural co-mingling of the W, Z, and Y chromosomes in the same population. Compared to the rest of the genome, a small sex-associated portion of the sex chromosomes has a 50-fold enrichment of transcripts with male-biased expression during early gonadal differentiation. Additionally, X. tropicalis has sex-differences in the rates and genomic locations of recombination events during gametogenesis that are similar to at least two other Xenopus species, which suggests that sex differences in recombination are genus-wide. These findings are consistent with theoretical expectations associated with recombination suppression on sex chromosomes, demonstrate that several characteristics of old and established sex chromosomes (e.g., nucleotide divergence, sex biased expression) can arise well before sex chromosomes become cytogenetically distinguished, and show how these characteristics can have lingering consequences that are carried forward through sex chromosome turnovers.

Maximizing CRISPR/Cas9 phenotype penetrance applying predictive modeling of editing outcomes in Xenopus and zebrafish embryos.

CRISPR/Cas9 genome editing has revolutionized functional genomics in vertebrates. However, CRISPR/Cas9 edited F0 animals too often demonstrate variable phenotypic penetrance due to the mosaic nature of editing outcomes after double strand break (DSB) repair. Even with high efficiency levels of genome editing, phenotypes may be obscured by proportional presence of in-frame mutations that still produce functional protein. Recently, studies in cell culture systems have shown that the nature of CRISPR/Cas9-mediated mutations can be dependent on local sequence context and can be predicted by computational methods. Here, we demonstrate that similar approaches can be used to forecast CRISPR/Cas9 gene editing outcomes in Xenopus tropicalis, Xenopus laevis, and zebrafish. We show that a publicly available neural network previously trained in mouse embryonic stem cell cultures (InDelphi-mESC) is able to accurately predict CRISPR/Cas9 gene editing outcomes in early vertebrate embryos. Our observations can have direct implications for experiment design, allowing the selection of guide RNAs with predicted repair outcome signatures enriched towards frameshift mutations, allowing maximization of CRISPR/Cas9 phenotype penetrance in the F0 generation.