Evaluating the association of biallelic OGDHL variants with significant phenotypic heterogeneity.

BACKGROUND: Biallelic variants in OGDHL, encoding part of the α-ketoglutarate dehydrogenase complex, have been associated with highly heterogeneous neurological and neurodevelopmental disorders. However, the validity of this association remains to be confirmed. A second OGDHL patient cohort was recruited to carefully assess the gene-disease relationship. METHODS: Using an unbiased genotype-first approach, we screened large, multiethnic aggregated sequencing datasets worldwide for biallelic OGDHL variants. We used CRISPR/Cas9 to generate zebrafish knockouts of ogdhl, ogdh paralogs, and dhtkd1 to investigate functional relationships and impact during development. Functional complementation with patient variant transcripts was conducted to systematically assess protein functionality as a readout for pathogenicity. RESULTS: A cohort of 14 individuals from 12 unrelated families exhibited highly variable clinical phenotypes, with the majority of them presenting at least one additional variant, potentially accounting for a blended phenotype and complicating phenotypic understanding. We also uncovered extreme clinical heterogeneity and high allele frequencies, occasionally incompatible with a fully penetrant recessive disorder. Human cDNA of previously described and new variants were tested in an ogdhl zebrafish knockout model, adding functional evidence for variant reclassification. We disclosed evidence of hypomorphic alleles as well as a loss-of-function variant without deleterious effects in zebrafish variant testing also showing discordant familial segregation, challenging the relationship of OGDHL as a conventional Mendelian gene. Going further, we uncovered evidence for a complex compensatory relationship among OGDH, OGDHL, and DHTKD1 isoenzymes that are associated with neurodevelopmental disorders and exhibit complex transcriptional compensation patterns with partial functional redundancy. CONCLUSIONS: Based on the results of genetic, clinical, and functional studies, we formed three hypotheses in which to frame observations: biallelic OGDHL variants lead to a highly variable monogenic disorder, variants in OGDHL are following a complex pattern of inheritance, or they may not be causative at all. Our study further highlights the continuing challenges of assessing the validity of reported disease-gene associations and effects of variants identified in these genes. This is particularly more complicated in making genetic diagnoses based on identification of variants in genes presenting a highly heterogenous phenotype such as "OGDHL-related disorders".

Clinical, genetic, epidemiologic, evolutionary, and functional delineation of TSPEAR-related autosomal recessive ectodermal dysplasia 14.

TSPEAR variants cause autosomal recessive ectodermal dysplasia (ARED) 14. The function of TSPEAR is unknown. The clinical features, the mutation spectrum, and the underlying mechanisms of ARED14 are poorly understood. Combining data from new and previously published individuals established that ARED14 is primarily characterized by dental anomalies such as conical tooth cusps and hypodontia, like those seen in individuals with WNT10A-related odontoonychodermal dysplasia. AlphaFold-predicted structure-based analysis showed that most of the pathogenic TSPEAR missense variants likely destabilize the ß-propeller of the protein. Analysis of 100000 Genomes Project (100KGP) data revealed multiple founder TSPEAR variants across different populations. Mutational and recombination clock analyses demonstrated that non-Finnish European founder variants likely originated around the end of the last ice age, a period of major climatic transition. Analysis of gnomAD data showed that the non-Finnish European population TSPEAR gene-carrier rate is ∼1/140, making it one of the commonest AREDs. Phylogenetic and AlphaFold structural analyses showed that TSPEAR is an ortholog of drosophila Closca, an extracellular matrix-dependent signaling regulator. We, therefore, hypothesized that TSPEAR could have a role in enamel knot, a structure that coordinates patterning of developing tooth cusps. Analysis of mouse single-cell RNA sequencing (scRNA-seq) data revealed highly restricted expression of Tspear in clusters representing enamel knots. A tspeara -/-;tspearb -/- double-knockout zebrafish model recapitulated the clinical features of ARED14 and fin regeneration abnormalities of wnt10a knockout fish, thus suggesting interaction between tspear and wnt10a. In summary, we provide insights into the role of TSPEAR in ectodermal development and the evolutionary history, epidemiology, mechanisms, and consequences of its loss of function variants.

WARS1 and SARS1: Two tRNA synthetases implicated in autosomal recessive microcephaly.

Aminoacylation of transfer RNA (tRNA) is a key step in protein biosynthesis, carried out by highly specific aminoacyl-tRNA synthetases (ARSs). ARSs have been implicated in autosomal dominant and autosomal recessive human disorders. Autosomal dominant variants in tryptophanyl-tRNA synthetase 1 (WARS1) are known to cause distal hereditary motor neuropathy and Charcot-Marie-Tooth disease, but a recessively inherited phenotype is yet to be clearly defined. Seryl-tRNA synthetase 1 (SARS1) has rarely been implicated in an autosomal recessive developmental disorder. Here, we report five individuals with biallelic missense variants in WARS1 or SARS1, who presented with an overlapping phenotype of microcephaly, developmental delay, intellectual disability, and brain anomalies. Structural mapping showed that the SARS1 variant is located directly within the enzyme's active site, most likely diminishing activity, while the WARS1 variant is located in the N-terminal domain. We further characterize the identified WARS1 variant by showing that it negatively impacts protein abundance and is unable to rescue the phenotype of a CRISPR/Cas9 wars1 knockout zebrafish model. In summary, we describe two overlapping autosomal recessive syndromes caused by variants in WARS1 and SARS1, present functional insights into the pathogenesis of the WARS1-related syndrome and define an emerging disease spectrum: ARS-related developmental disorders with or without microcephaly.

Biallelic variants in WARS1 cause a highly variable neurodevelopmental syndrome and implicate a critical exon for normal auditory function.

Aminoacyl-tRNA synthetases (ARSs) are essential enzymes for faithful assignment of amino acids to their cognate tRNA. Variants in ARS genes are frequently associated with clinically heterogeneous phenotypes in humans and follow both autosomal dominant or recessive inheritance patterns in many instances. Variants in tryptophanyl-tRNA synthetase 1 (WARS1) cause autosomal dominantly inherited distal hereditary motor neuropathy and Charcot-Marie-Tooth disease. Presently, only one family with biallelic WARS1 variants has been described. We present three affected individuals from two families with biallelic variants (p.Met1? and p.(Asp419Asn)) in WARS1, showing varying severities of developmental delay and intellectual disability. Hearing impairment and microcephaly, as well as abnormalities of the brain, skeletal system, movement/gait, and behavior were variable features. Phenotyping of knocked down wars-1 in a Caenorhabditis elegans model showed depletion is associated with defects in germ cell development. A wars1 knockout vertebrate model recapitulates the human clinical phenotypes, confirms variant pathogenicity, and uncovers evidence implicating the p.Met1? variant as potentially impacting an exon critical for normal hearing. Together, our findings provide consolidating evidence for biallelic disruption of WARS1 as causal for an autosomal recessive neurodevelopmental syndrome and present a vertebrate model that recapitulates key phenotypes observed in patients.

Chondroitin/dermatan sulfate glycosyltransferase genes are essential for craniofacial development.

Chondroitin/dermatan sulfate (CS/DS) proteoglycans are indispensable for animal development and homeostasis but the large number of enzymes involved in their biosynthesis have made CS/DS function a challenging problem to study genetically. In our study, we generated loss-of-function alleles in zebrafish genes encoding CS/DS biosynthetic enzymes and characterized the effect on development in single and double mutants. Homozygous mutants in chsy1, csgalnact1a, csgalnat2, chpfa, ust and chst7, respectively, develop to adults. However, csgalnact1a-/- fish develop distinct craniofacial defects while the chsy1-/- skeletal phenotype is milder and the remaining mutants display no gross morphological abnormalities. These results suggest a high redundancy for the CS/DS biosynthetic enzymes and to further reduce CS/DS biosynthesis we combined mutant alleles. The craniofacial phenotype is further enhanced in csgalnact1a-/-;chsy1-/- adults and csgalnact1a-/-;csgalnact2-/- larvae. While csgalnact1a-/-;csgalnact2-/- was the most affected allele combination in our study, CS/DS is still not completely abolished. Transcriptome analysis of chsy1-/-, csgalnact1a-/- and csgalnact1a-/-;csgalnact2-/- larvae revealed that the expression had changed in a similar way in the three mutant lines but no differential expression was found in any of fifty GAG biosynthesis enzymes identified. Thus, zebrafish larvae do not increase transcription of GAG biosynthesis genes as a consequence of decreased CS/DS biosynthesis. The new zebrafish lines develop phenotypes similar to clinical characteristics of several human congenital disorders making the mutants potentially useful to study disease mechanisms and treatment.

Large-scale generation and phenotypic characterization of zebrafish CRISPR mutants of DNA repair genes.

A systematic knowledge of the roles of DNA repair genes at the level of the organism has been limited due to the lack of appropriate experimental approaches using animal model systems. Zebrafish has become a powerful vertebrate genetic model system with availability due to the ease of genome editing and large-scale phenotype screening. Here, we generated zebrafish mutants for 32 DNA repair and replication genes through multiplexed CRISPR/Cas9-mediated mutagenesis. Large-scale phenotypic characterization of our mutant collection revealed that three genes (atad5a, ddb1, pcna) are essential for proper embryonic development and hematopoiesis; seven genes (apex1, atrip, ino80, mre11a, shfm1, telo2, wrn) are required for growth and development during juvenile stage and six genes (blm, brca2, fanci, rad51, rad54l, rtel1) play critical roles in sex development. Furthermore, mutation in six genes (atad5a, brca2, polk, rad51, shfm1, xrcc1) displayed hypersensitivity to DNA damage agents. Our zebrafish mutant collection provides a unique resource for understanding of the roles of DNA repair genes at the organismal level.

Cnr2 Is Important for Ribbon Synapse Maturation and Function in Hair Cells and Photoreceptors.

The role of the cannabinoid receptor 2 (CNR2) is still poorly described in sensory epithelia. We found strong cnr2 expression in hair cells (HCs) of the inner ear and the lateral line (LL), a superficial sensory structure in fish. Next, we demonstrated that sensory synapses in HCs were severely perturbed in larvae lacking cnr2. Appearance and distribution of presynaptic ribbons and calcium channels (Cav1.3) were profoundly altered in mutant animals. Clustering of membrane-associated guanylate kinase (MAGUK) in post-synaptic densities (PSDs) was also heavily affected, suggesting a role for cnr2 for maintaining the sensory synapse. Furthermore, vesicular trafficking in HCs was strongly perturbed suggesting a retrograde action of the endocannabinoid system (ECs) via cnr2 that was modulating HC mechanotransduction. We found similar perturbations in retinal ribbon synapses. Finally, we showed that larval swimming behaviors after sound and light stimulations were significantly different in mutant animals. Thus, we propose that cnr2 is critical for the processing of sensory information in the developing larva.

Lupus Susceptibility Region Containing CDKN1B rs34330 Mechanistically Influences Expression and Function of Multiple Target Genes, Also Linked to Proliferation and Apoptosis.

OBJECTIVE: In a recent genome-wide association study, a significant genetic association between rs34330 of CDKN1B and risk of systemic lupus erythematosus (SLE) in Han Chinese was identified. This study was undertaken to validate the reported association and elucidate the biochemical mechanisms underlying the effect of the variant. METHODS: We performed an allelic association analysis in patients with SLE, followed by a meta-analysis assessing genome-wide association data across 11 independent cohorts (n = 28,872). In silico bioinformatics analysis and experimental validation in SLE-relevant cell lines were applied to determine the functional consequences of rs34330. RESULTS: We replicated a genetic association between SLE and rs34330 (meta-analysis P = 5.29 × 10-22 , odds ratio 0.84 [95% confidence interval 0.81-0.87]). Follow-up bioinformatics and expression quantitative trait locus analysis suggested that rs34330 is located in active chromatin and potentially regulates several target genes. Using luciferase and chromatin immunoprecipitation-real-time quantitative polymerase chain reaction, we demonstrated substantial allele-specific promoter and enhancer activity, and allele-specific binding of 3 histone marks (H3K27ac, H3K4me3, and H3K4me1), RNA polymerase II (Pol II), CCCTC-binding factor, and a critical immune transcription factor (interferon regulatory factor 1 [IRF-1]). Chromosome conformation capture revealed long-range chromatin interactions between rs34330 and the promoters of neighboring genes APOLD1 and DDX47, and effects on CDKN1B and the other target genes were directly validated by clustered regularly interspaced short palindromic repeat (CRISPR)-based genome editing. Finally, CRISPR/dead CRISPR-associated protein 9-based epigenetic activation/silencing confirmed these results. Gene-edited cell lines also showed higher levels of proliferation and apoptosis. CONCLUSION: Collectively, these findings suggest a mechanism whereby the rs34330 risk allele (C) influences the presence of histone marks, RNA Pol II, and IRF-1 transcription factor to regulate expression of several target genes linked to proliferation and apoptosis. This process could potentially underlie the association of rs34330 with SLE.

A biallelic variant in CLRN2 causes non-syndromic hearing loss in humans.

Deafness, the most frequent sensory deficit in humans, is extremely heterogeneous with hundreds of genes involved. Clinical and genetic analyses of an extended consanguineous family with pre-lingual, moderate-to-profound autosomal recessive sensorineural hearing loss, allowed us to identify CLRN2, encoding a tetraspan protein, as a new deafness gene. Homozygosity mapping followed by exome sequencing identified a 14.96 Mb locus on chromosome 4p15.32p15.1 containing a likely pathogenic missense variant in CLRN2 (c.494C > A, NM_001079827.2) segregating with the disease. Using in vitro RNA splicing analysis, we show that the CLRN2 c.494C > A variant leads to two events: (1) the substitution of a highly conserved threonine (uncharged amino acid) to lysine (charged amino acid) at position 165, p.(Thr165Lys), and (2) aberrant splicing, with the retention of intron 2 resulting in a stop codon after 26 additional amino acids, p.(Gly146Lysfs*26). Expression studies and phenotyping of newly produced zebrafish and mouse models deficient for clarin 2 further confirm that clarin 2, expressed in the inner ear hair cells, is essential for normal organization and maintenance of the auditory hair bundles, and for hearing function. Together, our findings identify CLRN2 as a new deafness gene, which will impact future diagnosis and treatment for deaf patients.

Identification of the Wallenda JNKKK as an Alk suppressor reveals increased competitiveness of Alk-expressing cells.

Anaplastic lymphoma kinase (Alk) is a receptor tyrosine kinase of the insulin receptor super-family that functions as oncogenic driver in a range of human cancers such as neuroblastoma. In order to investigate mechanisms underlying Alk oncogenic signaling, we conducted a genetic suppressor screen in Drosophila melanogaster. Our screen identified multiple loci important for Alk signaling, including members of Ras/Raf/ERK-, Pi3K-, and STAT-pathways as well as tailless (tll) and foxo whose orthologues NR2E1/TLX and FOXO3 are transcription factors implicated in human neuroblastoma. Many of the identified suppressors were also able to modulate signaling output from activated oncogenic variants of human ALK, suggesting that our screen identified targets likely relevant in a wide range of contexts. Interestingly, two misexpression alleles of wallenda (wnd, encoding a leucine zipper bearing kinase similar to human DLK and LZK) were among the strongest suppressors. We show that Alk expression leads to a growth advantage and induces cell death in surrounding cells. Our results suggest that Alk activity conveys a competitive advantage to cells, which can be reversed by over-expression of the JNK kinase kinase Wnd.

Building the vertebrate codex using the gene breaking protein trap library.

One key bottleneck in understanding the human genome is the relative under-characterization of 90% of protein coding regions. We report a collection of 1200 transgenic zebrafish strains made with the gene-break transposon (GBT) protein trap to simultaneously report and reversibly knockdown the tagged genes. Protein trap-associated mRFP expression shows previously undocumented expression of 35% and 90% of cloned genes at 2 and 4 days post-fertilization, respectively. Further, investigated alleles regularly show 99% gene-specific mRNA knockdown. Homozygous GBT animals in ryr1b, fras1, tnnt2a, edar and hmcn1 phenocopied established mutants. 204 cloned lines trapped diverse proteins, including 64 orthologs of human disease-associated genes with 40 as potential new disease models. Severely reduced skeletal muscle Ca2+ transients in GBT ryr1b homozygous animals validated the ability to explore molecular mechanisms of genetic diseases. This GBT system facilitates novel functional genome annotation towards understanding cellular and molecular underpinnings of vertebrate biology and human disease.

The human genome counts over 20,000 genes, which can be turned on and off to create the proteins required for most of life processes. Once produced, proteins need move to specific locations in the cell, where they are able to perform their jobs. Despite striking scientific advances, 90% of human genes are still under-studied; where the proteins they code for go, and what they do remains unknown. Zebrafish share many genes with humans, but they are much easier to manipulate genetically. Here, Ichino et al. used various methods in zebrafish to create a detailed 'catalogue' of previously poorly understood genes, focusing on where the proteins they coded for ended up and the biological processes they were involved with. First, a genetic tool called gene-breaking transposons (GBTs) was used to create over 1,200 strains of genetically altered fish in which a specific protein was both tagged with a luminescent marker and unable to perform its role. Further analysis of 204 of these strains revealed new insight into the role of each protein, with many having unexpected roles and localisations. For example, in one zebrafish strain, the affected gene was similar to a human gene which, when inactivated, causes severe muscle weakness. These fish swam abnormally slowly and also had muscle problems, suggesting that the GBT fish strains could 'model' the human disease. This work sheds new light on the role of many previously poorly understood genes. In the future, similar collections of GBT fish strains could help researchers to study both normal human biology and disease. They could especially be useful in cases where the genes responsible for certain conditions are still difficult to identify.

Amyloid precursor protein-b facilitates cell adhesion during early development in zebrafish.

Understanding the biological function of amyloid beta (Aß) precursor protein (APP) beyond its role in Alzheimer's disease is emerging. Yet, its function during embryonic development is poorly understood. The zebrafish APP orthologue, Appb, is strongly expressed during early development but thus far has only been studied via morpholino-mediated knockdown. Zebrafish enables analysis of cellular processes in an ontogenic context, which is limited in many other vertebrates. We characterized zebrafish carrying a homozygous mutation that introduces a premature stop in exon 2 of the appb gene. We report that appb mutants are significantly smaller until 2 dpf and display perturbed enveloping layer (EVL) integrity and cell protrusions at the blastula stage. Moreover, appb mutants surviving beyond 48 hpf exhibited no behavioral defects at 6 dpf and developed into healthy and fertile adults. The expression of the app family member, appa, was also found to be altered in appb mutants. Taken together, we show that appb is involved in the initial development of zebrafish by supporting the integrity of the EVL, likely by mediating cell adhesion properties. The loss of Appb might then be compensated for by other app family members to maintain normal development.

MultiFRAGing: Rapid and Simultaneous Genotyping of Multiple Alleles in a Single Reaction.

Powerful and simple, RNA-guided CRISPR/Cas9 technology is a versatile genome editing tool that has revolutionized targeted mutagenesis. CRISPR-based genome editing has enabled large-scale functional genetic studies through the generation of gene knockouts in a variety of model organisms including zebrafish, and can be used to target multiple genes simultaneously. One of the challenges associated with the large scale application of this technique to zebrafish is the lack of a cost-effective method by which to identify mutants. To address this, we optimized the high-throughput, high-resolution fluorescent PCR-based fragment analysis method to develop MultiFRAGing - a robust and cost-effective method to genotype multiple targets in a single reaction. Our approach can identify indels in up to four targets from a single reaction, which represents a four-fold increase in genotyping throughput. This method can be used by any laboratory with access to capillary electrophoresis-based sequencing equipment.

Small fish, big prospects: using zebrafish to unravel the mechanisms of hereditary hearing loss.

Over the past decade, advancements in high-throughput sequencing have greatly enhanced our knowledge of the mutational signatures responsible for hereditary hearing loss. In its present state, the field has a largely uncensored view of protein coding changes in a growing number of genes that have been associated with hereditary hearing loss, and many more that have been proposed as candidate genes. Sequencing data can now be generated using methods that have become widespread and affordable. The greatest hurdles facing the field concern functional validation of uncharacterized genes and rapid application to human diseases, including hearing and balance disorders. To date, over 30 hearing-related disease models exist in zebrafish. New genome editing technologies, including CRISPR/Cas9 will accelerate the functional validation of hearing loss genes and variants in zebrafish. Here, we discuss current progress in the field and recent advances in genome editing approaches.

Transcriptomic Analyses of Inner Ear Sensory Epithelia in Zebrafish.

Analysis of gene expression has the potential to assist in the understanding of multiple cellular processes including proliferation, cell-fate specification, senesence, and activity in both healthy and disease states. Zebrafish model has been increasingly used to understand the process of hearing and the development of the vertebrate auditory system. Within the zebrafish inner ear, there are three otolith organs, each containing a sensory macula of hair cells. The saccular macula is primarily involved in hearing, the utricular macula is primarily involved in balance and the function of the lagenar macula is not completely understood. The goal of this study is to understand the transcriptional differences in the sensory macula associated with different otolith organs with the intention of understanding the genetic mechanisms responsible for the distinct role each organ plays in sensory perception. The sensory maculae of the saccule, utricle, and lagena were dissected out of adult Et(krt4:GFP)sqet4 zebrafish expressing green fluorescent protein in hair cells for transcriptional analysis. The total RNAs of the maculae were isolated and analyzed by RNA GeneChip microarray. Several of the differentially expressed genes are known to be involved in deafness, otolith development and balance. Gene expression among these otolith organs was very well conserved with less than 10% of genes showing differential expression. Data from this study will help to elucidate which genes are involved in hearing and balance. Furthermore, the findings of this study will assist in the development of the zebrafish model for human hearing and balance disorders. Anat Rec, 303:527-543, 2020. © 2019 American Association for Anatomy.

Altered Swimming Behaviors in Zebrafish Larvae Lacking Cannabinoid Receptor 2.

Background and Objectives: The cannabinoid receptor 2 (CB2) was previously implicated in brain functions, including complex behaviors. Here, we assessed the role of CB2 in selected swimming behaviors in zebrafish larvae and developed an in vivo upscalable whole-organism approach for CB2 ligand screening. Experimental Approach: Using CRISPR-Cas9 technology, we generated a novel null allele (cnr2upr1 ) and a stable homozygote-viable loss-of-function (CB2-KO) line. We measured in untreated wild-type and cnr2upr1/upr1 larvae, photo-dependent (swimming) responses (PDR) and center occupancy (CO) to establish quantifiable anxiety-like parameters. Next, we measured PDR alteration and CO variation while exposing wild-type and mutant animals to an anxiolytic drug (valproic acid [VPA]) or to an anxiogenic drug (pentylenetetrazol [PTZ]). Finally, we treated wild-type and mutant larvae with two CB2-specific agonists (JWH-133 and HU-308) and two CB2-specific antagonists, inverse agonists (AM-630 and SR-144528). Results: Untreated CB2-KO showed a different PDR than wild-type larvae as well as a decreased CO. VPA treatments diminished swimming activity in all animals but to a lesser extend in mutants. CO was strongly diminished and even more in mutants. PTZ-induced inverted PDR was significantly stronger in light and weaker in dark periods and the CO lower in PTZ-treated mutants. Finally, two of four tested CB2 ligands had a detectable activity in the assay. Conclusions: We showed that larvae lacking CB2 behave differently in complex behaviors that can be assimilated to anxiety-like behaviors. Mutant larvae responded differently to VPA and PTZ treatments, providing in vivo evidence of CB2 modulating complex behaviors. We also established an upscalable combined genetic/behavioral approach in a whole organism that could be further developed for high-throughput drug discovery platforms.

Expanding the CRISPR Toolbox in Zebrafish for Studying Development and Disease.

The study of model organisms has revolutionized our understanding of the mechanisms underlying normal development, adult homeostasis, and human disease. Much of what we know about gene function in model organisms (and its application to humans) has come from gene knockouts: the ability to show analogous phenotypes upon gene inactivation in animal models. The zebrafish (Danio rerio) has become a popular model organism for many reasons, including the fact that it is amenable to various forms of genetic manipulation. The RNA-guided CRISPR/Cas9-mediated targeted mutagenesis approaches have provided powerful tools to manipulate the genome toward developing new disease models and understanding the pathophysiology of human diseases. CRISPR-based approaches are being used for the generation of both knockout and knock-in alleles, and also for applications including transcriptional modulation, epigenome editing, live imaging of the genome, and lineage tracing. Currently, substantial effort is being made to improve the specificity of Cas9, and to expand the target coverage of the Cas9 enzymes. Novel types of naturally occurring CRISPR systems [Cas12a (Cpf1); engineered variants of Cas9, such as xCas9 and SpCas9-NG], are being studied and applied to genome editing. Since the majority of pathogenic mutations are single point mutations, development of base editors to convert C:G to T:A or A:T to G:C has further strengthened the CRISPR toolbox. In this review, we provide an overview of the increasing number of novel CRISPR-based tools and approaches, including lineage tracing and base editing.

Guided genetic screen to identify genes essential in the regeneration of hair cells and other tissues.

Regenerative medicine holds great promise for both degenerative diseases and traumatic tissue injury which represent significant challenges to the health care system. Hearing loss, which affects hundreds of millions of people worldwide, is caused primarily by a permanent loss of the mechanosensory receptors of the inner ear known as hair cells. This failure to regenerate hair cells after loss is limited to mammals, while all other non-mammalian vertebrates tested were able to completely regenerate these mechanosensory receptors after injury. To understand the mechanism of hair cell regeneration and its association with regeneration of other tissues, we performed a guided mutagenesis screen using zebrafish lateral line hair cells as a screening platform to identify genes that are essential for hair cell regeneration, and further investigated how genes essential for hair cell regeneration were involved in the regeneration of other tissues. We created genetic mutations either by retroviral insertion or CRISPR/Cas9 approaches, and developed a high-throughput screening pipeline for analyzing hair cell development and regeneration. We screened 254 gene mutations and identified 7 genes specifically affecting hair cell regeneration. These hair cell regeneration genes fell into distinct and somewhat surprising functional categories. By examining the regeneration of caudal fin and liver, we found these hair cell regeneration genes often also affected other types of tissue regeneration. Therefore, our results demonstrate guided screening is an effective approach to discover regeneration candidates, and hair cell regeneration is associated with other tissue regeneration.

Highly Efficient Cpf1-Mediated Gene Targeting in Mice Following High Concentration Pronuclear Injection.

Cpf1 has emerged as an alternative to the Cas9 RNA-guided nuclease. Here we show that gene targeting rates in mice using Cpf1 can meet, or even surpass, Cas9 targeting rates (approaching 100% targeting), but require higher concentrations of mRNA and guide. We also demonstrate that coinjecting two guides with close targeting sites can result in synergistic genomic cutting, even if one of the guides has minimal cutting activity.