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Nanophthalmos is characterised by shorter posterior and anterior segments of the eye, with a predisposition towards high hyperopia and primary angle-closure glaucoma. Variants in TMEM98 have been associated with autosomal dominant nanophthalmos in multiple kindreds, but definitive evidence for causation has been limited. Here we used CRISPR/Cas9 mutagenesis to recreate the human nanophthalmos-associated TMEM98 p.Ala193Pro (A193P) variant in mice. The A193P variant was associated with ocular phenotypes in both mice and humans, with dominant inheritance in humans and recessive inheritance in mice. Unlike their human counterparts, A193P homozygous mutant mice had normal axial length, normal intraocular pressure, and structurally normal scleral collagen. However, in both homozygous mice and heterozygous humans, the A193P variant was associated with discrete white spots throughout the retinal fundus, with corresponding retinal folds on histology. This direct comparison of a TMEM98 variant in mouse and human suggests that certain nanophthalmos-associated phenotypes are not only a consequence of a smaller eye, but that TMEM98 may itself play a primary role in retinal and scleral structure and integrity.

Refereed/Peer-reviewed Transcriptional enhancer elements are responsible for orchestrating the temporal and spatial control over gene expression that is crucial for programming cell identity during development. Here we describe a novel enhancer element that is important for regulating the expression of Prox1 in lymphatic endothelial cells. This evolutionarily conserved enhancer is bound by key lymphatic transcriptional regulators including GATA2, FOXC2, NFATC1 and PROX1. Genome editing of the enhancer to remove five nucleotides encompassing the GATA2-binding site resulted in perinatal death of homozygous mutant mice due to profound lymphatic vascular defects. Lymphatic endothelial cells in enhancer mutant mice exhibited reduced expression of genes characteristic of lymphatic endothelial cell identity and increased expression of genes characteristic of haemogenic endothelium, and acquired the capacity to generate haematopoietic cells. These data not only reveal a transcriptional enhancer element important for regulating Prox1 expression and lymphatic endothelial cell identity but also demonstrate that the lymphatic endothelium has haemogenic capacity, ordinarily repressed by Prox1.

Refereed/Peer-reviewed The genetic basis of the predisposition for Down Syndrome (DS) patients to develop cytokine receptor-like factor 2 rearranged (CRLF2r) acute lymphoblastic leukemia (ALL) is currently unknown. Genes located on chromosome 21 and expressed in hematopoietic cells are likely candidates for investigation of CRLF2r DS-ALL pathogenesis. We explored the high-mobility group nucleosome-binding protein 1 (HMGN1), located in the DS critical region, in an inducible CRISPR/Cas9 knockout (KO) xenograft model to assess the effect of HMGN1 loss of function on the leukemic burden. We demonstrated HMGN1 KO-mitigated leukemic phenotypes including hepatosplenomegaly, thrombocytopenia, and anemia, commonly observed in leukemia patients, and significantly increased survival in vivo. HMGN1 overexpression in murine stem cells and Ba/F3 cells in vitro, in combination with P2RY8-CRLF2, resulted in cytokine-independent transformation and upregulation of cell signaling pathways associated with leukemic development. Finally, in vitro screening demonstrated successful targeting of P2RY8-CRLF2 and HMGN1 co-expressing cell lines and patient samples with fedratinib (JAK2 inhibitor), and GSK-J4 (demethylase inhibitor) in combination. Together, these data provide critical insight into the development and persistence of CRLF2r DS-ALL and identify HMGN1 as a potential therapeutic target to improve outcomes and reduce toxicity in this high-risk cohort of young patients.

Invasive rodents, including house mice, are a major cause of environmental damage and biodiversity loss, particularly in island ecosystems. Eradication can be achieved through the distribution of rodenticide, but this approach is expensive to apply at scale, can have negative impacts (e.g. on non-target species, or through contamination), has animal ethics concerns, and has restrictions on where it can be used. Gene drives, which exhibit biased inheritance, have been proposed as a next generation strategy to control invasive alien pests and disease vectors. However, synthetic gene drives including CRISPR homing drives have proven to be technically challenging to develop in mice. Thethaplotype is a naturally-occurring segregation distortion locus with highly biased transmission from heterozygous males. Here we propose a novel gene drive strategy for population suppression,tCRISPR, that leveragesthaplotype bias and an embedded SpCas9/gRNA transgene to spread inactivating mutations in a haplosufficient female fertility gene. Using spatially explicit individual-basedin silicomodelling, we show that polyandry, sperm competition, dispersal, and transmission bias are critical factors fortCRISPR-mediated population suppression. Modelling of realistic parameter values indicates thattCRISPRcan eradicate an island population of 200,000 mice while the unmodifiedthaplotype fails under the same conditions. We also demonstrate feasibility of this approach by engineeringtCRISPRmice in a safe split drive format.tCRISPRmice exhibit high transmission of the modifiedthaplotype, and efficient generation and transmission of inactivating mutations in a recessive female fertility gene, crucially, at levels for which the modelling predicts that population eradication can occur. This is the first example of a feasible gene drive system for invasive alien rodent population control.

CRISPR gene-editing technology creates precise and permanent modifications to DNA. It has significantly advanced our ability to generate animal disease models for use in biomedical research and also has potential to revolutionize the treatment of genetic disorders. Duchenne muscular dystrophy (DMD) is a monogenic muscle-wasting disease that could potentially benefit from the development of CRISPR therapy. It is commonly associated with mutations that disrupt the reading frame of the DMD gene that encodes dystrophin, an essential scaffolding protein that stabilizes striated muscles and protects them from contractile-induced damage. CRISPR enables the rapid generation of various animal models harboring mutations that closely simulates the wide variety of mutations observed in DMD patients. These models provide a platform for the testing of sequence-specific interventions like CRISPR therapy that aim to reframe or skip DMD mutations to restore functional dystrophin expression. This article is categorized under: Congenital Diseases > Genetics/Genomics/Epigenetics

Gene drives are genetic elements that are transmitted to greater than 50% of offspring and have potential for population modification or suppression. While gene drives are known to occur naturally, the recent emergence of CRISPR-Cas9 genome-editing technology has enabled generation of synthetic gene drives in a range of organisms including mosquitos, flies, and yeast. For example, studies in Anopheles mosquitos have demonstrated95% transmission of CRISPR-engineered gene drive constructs, providing a possible strategy for malaria control. Recently published studies have also indicated that it may be possible to develop gene drive technology in invasive rodents such as mice. Here, we discuss the prospects for gene drive development in mice, including synthetic "homing drive" and X-shredder strategies as well as modifications of the naturally occurring t haplotype. We also provide detailed protocols for generation of gene drive mice through incorporation of plasmid-based transgenes in a targeted and non-targeted manner. Importantly, these protocols can be used for generating transgenic mice for any project that requires insertion of kilobase-scale transgenes such as knock-in of fluorescent reporters, gene swaps, overexpression/ectopic expression studies, and conditional "floxed" alleles.

Animal models are needed to develop interventions to prevent or treat intrauterine growth restriction (IUGR). Foetal growth rates and effects of in utero exposures differ between sexes, but little is known about sex-specific effects of increasing litter size. We established a murine IUGR model using pregnancies generated by multiple embryo transfers, and evaluated sex-specific responses to increasing litter size. CBAF1 embryos were collected at gestation day 0.5 (GD0.5) and 6, 8, 10 or 12 embryos were transferred into each uterine horn of pseudopregnant female CD1 mice (n = 32). Foetal and placental outcomes were measured at GD18.5. In the main experiment, foetuses were genotyped (Sry) for analysis of sex-specific outcomes. The number of implantation sites (P = 0.033) and litter size (number of foetuses, P = 0.008) correlated positively with the number of embryos transferred, while placental weight correlated negatively with litter size (both P < 0.01). The relationship between viable litter size and foetal weight differed between sexes (interaction P = 0.002), such that foetal weights of males (P = 0.002), but not females (P = 0.233), correlated negatively with litter size. Placental weight decreased with increasing litter size (P < 0.001) and was lower in females than males (P = 0.020). Our results suggest that male foetuses grow as fast as permitted by nutrient supply, whereas the female maintains placental reserve capacity. This strategy reflecting sex-specific gene expression is likely to place the male foetus at greater risk of death in the event of a ‘second hit’.

PCDH19-Clustering Epilepsy (PCDH19-CE) is an infantile onset disorder caused by mutation of the X-linked PCDH19 gene. Intriguingly, heterozygous females are affected while hemizygous males are not. While there is compelling evidence that this disorder stems from the coexistence of WT and PCDH19-null cells, the cellular mechanism underpinning the neurological phenotype remains unclear. Here, we investigate the impact of Pcdh19 WT and KO neuron mosaicism on synaptogenesis and network activity. Using our previously established knock-in and knock-out mouse models, together with CRISPR-Cas9 genome editing technology, we demonstrate a reduction in excitatory synaptic contacts between PCDH19-expressing and PCDH19-null neurons. Significantly altered neuronal morphology and neuronal network activities were also identified in the mixed populations. In addition, we show that in Pcdh19 heterozygous mice, where the coexistence of WT and KO neurons naturally occurs, aberrant contralateral axonal branching is present. Overall, our data show that mosaic expression of PCDH19 disrupts physiological neurite communication leading to abnormal neuronal activity, a hallmark of PCDH19-CE. Refereed/Peer-reviewed

Central conducting lymphatic anomaly (CCLA), characterized by the dysfunction of core collecting lymphatic vessels including the thoracic duct and cisterna chyli, and presenting as chylothorax, pleural effusions, chylous ascites, and lymphedema, is a severe disorder often resulting in fetal or perinatal demise. Although pathogenic variants in RAS/mitogen activated protein kinase (MAPK) signaling pathway components have been documented in some patients with CCLA, the genetic etiology of the disorder remains uncharacterized in most cases. Here, we identified biallelic pathogenic variants in

CRISPR-based synthetic gene drives have the potential to deliver a more effective and humane method of invasive vertebrate pest control than current strategies. Relatively efficient CRISPR gene drive systems have been developed in insects and yeast but not in mammals. Here, we investigated the efficiency of CRISPR-Cas9-based gene drives in

SOX3 is a transcription factor expressed within the developing and adult nervous system where it mostly functions to help maintain neural precursors. Sox3 is also expressed in other locations, notably within the spermatogonial stem/progenitor cell population in postnatal testis. Independent studies have shown that Sox3 null mice exhibit a spermatogenic block as young adults, the mechanism of which remains poorly understood. Using a panel of spermatogonial cell marker genes, we demonstrate that Sox3 is expressed within the committed progenitor fraction of the undifferentiated spermatogonial pool. Additionally, we use a Sox3 null mouse model to define a potential role for this factor in progenitor cell function. We demonstrate that Sox3 expression is required for transition of undifferentiated cells from a GFRα1+ self-renewing state to the NGN3 + transit-amplifying compartment. Critically, using chromatin immunoprecipitation, we demonstrate that SOX3 binds to a highly conserved region in the Ngn3 promoter region in vivo, indicating that Ngn3 is a direct target of SOX3. Together these studies indicate that SOX3 functions as a pro-commitment factor in spermatogonial stem/progenitor cells.

Pathogenic variants in the gene encoding the small GTPase Ras analogue in Brain 39b (RAB39B) are associated with early-onset parkinsonism. In this study we investigated the expression and localization of RAB39B (RNA and protein) in mouse brain tissue to gain a better understanding of its normal physiological function(s) and role in disease.We developed novel resources, including monoclonal antibodies directed against RAB39B and mice with Rab39b knockout, and performed real-time PCR and western blot analysis on whole brain lysates. To determine the spatial localization of Rab39b RNA and protein, we performed in-situ hybridization and immunohistochemistry on fresh frozen and fixed brain tissue. Our results show that RAB39B is localized throughout the cortex, hippocampus and substantia nigra of mice throughout postnatal life. We found high levels of RAB39B within MAP2 positive cortical and hippocampal neurons, and TH positive dopaminergic neurons in the substantia nigra pars compacta.Our studies support and extend current knowledge of the localization of RAB39B. We validate RAB39B as a neuron-enriched protein and demonstrate that it is present throughout the mouse cortex and hippocampus. Further, we observe high levels in the substantia nigra pars compacta, the brain region most affected in Parkinson’s disease pathology. The distribution of Rab39b is consistent with human disease associations with parkinsonism and cognitive impairment. We also describe and validate novel resources, including monoclonal antibodies directed against RAB39B and mice with Rab39b knockout, both of which are valuable tools for future studies of the molecular function of RAB39B.

Central conducting lymphatic anomaly (CCLA), characterized by the dysfunction of core collecting lymphatic vessels including the thoracic duct and cisterna chyli, and presenting as chylothorax, pleural effusions, chylous ascites, and lymphedema, is a severe disorder often resulting in fetal or perinatal demise. Although pathogenic variants in RAS/mitogen activated protein kinase (MAPK) signaling pathway components have been documented in some patients with CCLA, the genetic etiology of the disorder remains uncharacterized in most cases. Here, we identified biallelic pathogenic variants in MDFIC , encoding the MyoD family inhibitor domain containing protein, in seven individuals with CCLA from six independent families. Clinical manifestations of affected fetuses and children included nonimmune hydrops fetalis (NIHF), pleural and pericardial effusions, and lymphedema. Generation of a mouse model of human MDFIC truncation variants revealed that homozygous mutant mice died perinatally exhibiting chylothorax. The lymphatic vasculature of homozygous Mdfic mutant mice was profoundly mispatterned and exhibited major defects in lymphatic vessel valve development. Mechanistically, we determined that MDFIC controls collective cell migration, an important early event during the formation of lymphatic vessel valves, by regulating integrin β 1 activation and the interaction between lymphatic endothelial cells and their surrounding extracellular matrix. Our work identifies MDFIC variants underlying human lymphatic disease and reveals a crucial, previously unrecognized role for MDFIC in the lymphatic vasculature. Ultimately, understanding the genetic and mechanistic basis of CCLA will facilitate the development and implementation of new therapeutic approaches to effectively treat this complex disease.

Understanding the dynamic relationship between viral pathogens and cellular host factors is critical to furthering our knowledge of viral replication, disease mechanisms and development of anti-viral therapeutics. CRISPR genome editing technology has enhanced this understanding, by allowing identification of pro-viral and anti-viral cellular host factors for a wide range of viruses, most recently the cause of the COVID-19 pandemic, SARS-CoV-2. This review will discuss how CRISPR knockout and CRISPR activation genome-wide screening methods are a robust tool to investigate the viral life cycle and how other class 2 CRISPR systems are being repurposed for diagnostics.

Precise genomic modification using prime editing (PE) holds enormous potential for research and clinical applications. In this study, we generated all-in-one prime editing (PEA1) constructs that carry all the components required for PE, along with a selection marker. We tested these constructs (with selection) in HEK293T, K562, HeLa and mouse embryonic stem (ES) cells. We discovered that PE efficiency in HEK293T cells was much higher than previously observed, reaching up to 95% (mean 67%). The efficiency in K562 and HeLa cells, however, remained low. To improve PE efficiency in K562 and HeLa, we generated a nuclease prime editor and tested this system in these cell lines as well as mouse ES cells. PE-nuclease greatly increased prime editing initiation, however, installation of the intended edits was often accompanied by extra insertions derived from the repair template. Finally, we show that zygotic injection of the nuclease prime editor can generate correct modifications in mouse fetuses with up to 100% efficiency.

Bone morphogenetic protein (BMP) signaling is required for early forebrain development and cortical formation. How the endogenous modulators of BMP signaling regulate the structural and functional maturation of the developing brain remains unclear. Here, we show that expression of the BMP antagonist Grem1 marks committed layer V and VI glutamatergic neurons in the embryonic mouse brain. Lineage tracing of Grem1-expressing cells in the embryonic brain was examined by administration of tamoxifen to pregnant Grem1creERT; Rosa26LSLTdtomato mice at 13.5 days post coitum (dpc), followed by collection of embryos later in gestation. In addition, at 14.5 dpc, bulk mRNA-seq analysis of differentially expressed transcripts between FACS-sorted Grem1-positive and -negative cells was performed. We also generated Emx1-cre-mediated Grem1 conditional knockout mice (Emx1-Cre;Grem1flox/flox) in which the Grem1 gene was deleted specifically in the dorsal telencephalon. Grem1Emx1cKO animals had reduced cortical thickness, especially layers V and VI, and impaired motor balance and fear sensitivity compared with littermate controls. This study has revealed new roles for Grem1 in the structural and functional maturation of the developing cortex., Summary: The BMP antagonist Grem1 is expressed by committed deep-layer glutamatergic neurons in the embryonic mouse cortex. Grem1 conditional knockout mice display cortical and behavioral abnormalities.

Precise genomic modification using prime editing (PE) holds enormous potential for research and clinical applications. Currently, the delivery of PE components to mammalian cell lines requires multiple plasmid vectors. To overcome this limitation, we generated all-in-one prime editing (PEA1) constructs that carry all the components required for PE, along with a selection marker. We tested these constructs (with selection) in HEK293T, K562, HeLa and mouse embryonic stem (ES) cells. We discovered that PE efficiency in HEK293T cells was much higher than previously observed, reaching up to 95% (mean 67%). The efficiency in K562 and HeLa cells, however, remained low. To improve PE efficiency in K562 and HeLa, we generated a nuclease prime editor and tested this system in these cell lines as well as mouse ES cells. PE-nuclease greatly increased prime editing initiation, however, installation of the intended edits was often accompanied by extra insertions derived from the repair template. Finally, we show that zygotic injection of the nuclease prime editor can generate correct modifications in mouse fetuses with up to 100% efficiency. In summary, PE-nuclease and the PEA1 plasmids provide new tools to generate intended edits with high efficiency.

The RNA-guided endonuclease CRISPR-Cas system from Streptococcus pyogenes (SpCas9) is widely used for generating genetically modified mice via zygotic microinjection. Although SpCas9 is a ...

Enhancers are vitally important during embryonic development to control the spatial and temporal expression of genes. Recently, large scale genome projects have identified a vast number of putative developmental regulatory elements. However, the proportion of these that have been functionally assessed is relatively low. While enhancers have traditionally been studied using reporter assays, this approach does not characterise their contribution to endogenous gene expression. We have studied the murine Nestin (Nes) intron 2 enhancer, which is widely used to direct exogenous gene expression within neural progenitor cells in cultured cells and in vivo. We generated CRISPR deletions of the enhancer region in mice and assessed their impact on Nes expression during embryonic development. Loss of the Nes neural enhancer significantly reduced Nes expression in the developing CNS by as much as 82%. By assessing NES protein localization, we also show that this enhancer region contains repressor element(s) that inhibit Nes expression within the vasculature. Previous reports have stated that Nes is an essential gene, and its loss causes embryonic lethality. We also generated 2 independent Nes null lines and show that both develop without any obvious phenotypic effects. Finally, through crossing of null and enhancer deletion mice we provide evidence of trans-chromosomal interaction of the Nes enhancer and promoter.

Developmental and epileptic encephalopathies (DEE) are characterized by pharmacoresistant seizures with concomitant intellectual disability. Epilepsy of infancy with migrating focal seizures (EIMFS) is one of the most severe of these syndromes. De novo mutations in ion channels, including gain-of-function variants in KCNT1, have been found to play a major role in the etiology of EIMFS. Here, we test a potential precision therapeutic approach in KCNT1-associated DEE using a gene silencing antisense oligonucleotide (ASO) approach. The homozygous p.P924L (L/L) mouse model recapitulates the frequent, debilitating seizures and developmental compromise that are seen in patients. After a single intracerebroventricular bolus injection of a Kcnt1 gapmer ASO in symptomatic mice at postnatal day 40, seizure frequency was significantly reduced, behavioral abnormalities improved, and overall survival was extended compared to mice treated with a control ASO (non-hybridizing sequence). ASO administration at neonatal age was also well-tolerated and effective in controlling seizures and extending the lifespan of treated animals. The data presented here provides a proof of concept for ASO-based gene silencing as a promising therapeutic approach in KCNT1-associated epilepsies.

Bone morphogenetic protein (BMP) signaling is required for early forebrain development and cortical formation. How the endogenous modulators of BMP signaling regulate the structural and functional maturation of the developing brain remains unclear. Here we show that expression of the BMP antagonist,Grem1, marks a neuroprogenitor that gives rise to layer V and VI glutamatergic neurons in the embryonic mouse brain. Lineage tracing ofGrem1-expressing cells in the embryonic brain was examined by administration of tamoxifen to pregnantGrem1creERT Rosa26LSLTdtomatomice at 13.5 days post coitum (dpc), followed by collection of embryos later in gestation. In addition, at 14.5 dpc, bulk mRNA seq analysis of differentially expressed transcripts between FACS sortedGrem1positive and negative cells was performed. We also generatedEmx1-cremediatedGrem1conditional knockout mice (Emx1-Cre;Grem1flox/flox) in which theGrem1gene was deleted specifically in the dorsal telencephalon.Grem1Emx1cKOanimals had reduced cortical thickness, especially layers V and VI and impaired motor balance and fear sensitivity compared to littermate controls. This study has revealed new roles for Grem1 in the structural and functional maturation of the developing cortex.Summary statementThe BMP antagonist,Grem1, marks neuroprogenitors that give rise to deep layer glutamatergic neurons in the embryonic mouse brain.Grem1conditional knockout mice display cortical and behavioural abnormalities.

Introduced rodent populations pose significant threats worldwide, with particularly severe impacts on islands. Advancements in genome editing have motivated interest in synthetic gene drives that could potentially provide efficient and localized suppression of invasive rodent populations. Application of such technologies will require rigorous population genomic surveys to evaluate population connectivity, taxonomic identification, and to inform design of gene drive localization mechanisms. One proposed approach leverages the predicted shifts in genetic variation that accompany island colonization, wherein founder effects, genetic drift, and island‐specific selection are expected to result in locally fixed alleles (LFA) that are variable in neighboring nontarget populations. Engineering of guide RNAs that target LFA may thus yield gene drives that spread within invasive island populations, but would have limited impacts on nontarget populations in the event of an escape. Here we used pooled whole‐genome sequencing of invasive mouse (Mus musculus) populations on four islands along with paired putative source populations to test genetic predictions of island colonization and characterize locally fixed Cas9 genomic targets. Patterns of variation across the genome reflected marked reductions in allelic diversity in island populations and moderate to high degrees of differentiation from nearby source populations despite relatively recent colonization. Locally fixed Cas9 sites in female fertility genes were observed in all island populations, including a small number with multiplexing potential. In practice, rigorous sampling of presumptive LFA will be essential to fully assess risk of resistance alleles. These results should serve to guide development of improved, spatially limited gene drive design in future applications.

CRISPR-based synthetic gene drives have the potential to deliver a more effective and humane method of invasive vertebrate pest control than current strategies. Relatively efficient CRISPR gene drives have been developed in insects and yeast but not in mammals. Here we investigated the efficiency of CRISPR/Cas9-based gene drives in Mus musculus by constructing “split drive” systems with Cas9 under the control of zygotic (CAG) or germline (Vasa) promoters. While both systems generated double stranded breaks at their intended target site in vivo, no homing was detectable. Our data indicate that robust and specific Cas9 expression during meiosis is a critical requirement for the generation of efficient CRISPR-based synthetic gene drives in rodents.

PCDH19-Clustering Epilepsy (PCDH19-CE) is an infantile onset disorder caused by mutation of the X-linked PCDH19 gene. Intriguingly, heterozygous females are affected while hemizygous males are not. While there is compelling evidence that this disorder stems from the coexistence of WT and PCDH19-null cells, the cellular mechanism underpinning the neurological phenotype remains unclear. Here, we investigate the impact of Pcdh19 WT and KO neuron mosaicism on synaptogenesis and network activity. Using our previously established knock-in and knock-out mouse models, together with CRISPR-Cas9 genome editing technology, we demonstrate a reduction in excitatory synaptic contacts between PCDH19-expressing and PCDH19-null neurons. Significantly altered neuronal morphology and neuronal network activities were also identified in the mixed populations. In addition, we show that in Pcdh19 heterozygous mice, where the coexistence of WT and KO neurons naturally occurs, aberrant contralateral axonal branching is present. Overall, our data show that mosaic expression of PCDH19 disrupts physiological neurite communication leading to abnormal neuronal activity, a hallmark of PCDH19-CE.

PCDH19 Clustering Epilepsy (CE) is an intriguing early-onset seizure, autism and neurocognitive disorder with unique inheritance. The causative gene, PCDH19, is on the X-chromosome and encodes a cell-cell adhesion protein with restricted expression during brain development. Unlike other X-linked disorders, PCDH19-CE manifests in heterozygous females. Strikingly, hemizygous males are not affected. However, males with postzygotic somatic mutation in PCDH19 are affected and clinically similar to the affected females. PCDH19-CE is a disorder of cellular mosaicism. The coexistence of two different, but otherwise 'normal' cells in a PCDH19-CE individual, that is the wild type and the variant PCDH19 cells, has been proposed as the driving force of the disorder. This 'cellular interference' hypothesis could and has now been tested using sophisticated mouse models.

Invasive species are increasingly affecting agriculture, food, fisheries, and forestry resources throughout the world. As a result of global trade, invasive species are often introduced into new environments where they become established and cause harm to human health, agriculture, and the environment. Prevention of new introductions is a high priority for addressing the harm caused by invasive species, but unfortunately efforts to prevent new introductions do not address the economic harm that is presently manifested where invasive species have already become established. Genetic biocontrol can be defined as the release of organisms with genetic methods designed to disrupt the reproduction of invasive populations. While these methods offer the potential to control or even eradicate invasive species, there is a need to ensure that genetic biocontrol methods can be deployed in a way that minimizes potential harm to the environment. This review provides an overview of the state of genetic biocontrol, focusing on several approaches that were the subject of presentations at the Genetic Biocontrol for Invasive Species Workshop in Tarragona, Spain, March 31st, 2019, a workshop sponsored by the OECD's Co-operative Research Program on Biological Resource Management for Sustainable Agricultural Systems. The review considers four different approaches to genetic biocontrol for invasive species; sterile-release, YY Males, Trojan Female Technique, and gene drive. The different approaches will be compared with respect to the efficiency each affords as a genetic biocontrol tool, the practical utility and cost/benefits associated with implementation of the approach, and the regulatory considerations that will need to be addressed for each. The opinions expressed and arguments employed in this publication are the sole responsibility of the authors and do not necessarily reflect those of the OECD or of the governments of its Member countries.

Sotos syndrome is a developmental disorder characterized by a suite of clinical features. In children, the three cardinal features of Sotos syndrome are a characteristic facial appearance, learning disability and overgrowth (height and/or head circumference > 2 SDs above average). These features are also evident in adults with this syndrome. Over 90% of Sotos syndrome patients are haploinsufficient for the gene encoding nuclear receptor-binding Su(var)3-9, Enhancer-of-zesteand Trithorax domain-containing protein 1 (NSD1). NSD1 is a histone methyltransferase that catalyzes the methylation of lysine residue 36 on histone H3. However, although the symptomology of Sotos syndrome is well established, many aspects of NSD1 biology remain unknown. Here, we assessed the expression of Nsd1 within the mouse brain, and showed a predominantly neuronal pattern of expression for this histone-modifying factor. We also generated a mouse strain lacking one allele of Nsd1 and analyzed morphological and behavioral characteristics in these mice, showing behavioral characteristics reminiscent of some of the deficits seen in Sotos syndrome patients.

Breakthrough studies over the past decade have uncovered unique gene fusions implicated in acute lymphoblastic leukaemia (ALL). The critical gene, cytokine receptor-like factor 2 (CRLF2), is rearranged in 5-16% of B-ALL, comprising 50% of Philadelphia-like ALL and cooperates with genomic lesions in the Jak, Mapk and Ras signalling pathways. Children with Down Syndrome (DS) have a predisposition to developing CRLF2 rearranged-ALL which is observed in 60% of DS-ALL patients. These patients experience a poor survival outcome. Mutations of genes involved in epigenetic regulation are more prevalent in DS-ALL patients than non-DS ALL patients, highlighting the potential for alternative treatment strategies. DS-ALL patients also suffer greater treatment-related toxicity from current ALL treatment regimens compared to non-DS-ALL patients. An increased gene dosage of critical genes on chromosome 21 which have roles in purine synthesis and folate transport may contribute. As the genomic landscape of DS-ALL patients is different to non-DS-ALL patients, targeted therapies for individual lesions may improve outcomes. Therapeutically targeting each rearrangement with targeted or combination therapy that will perturb the transforming signalling pathways will likely improve the poor survival rates of this subset of patients.

PCDH19-Girls Clustering Epilepsy (PCDH19-GCE) is a childhood epileptic encephalopathy characterised by a spectrum of neurodevelopmental problems. PCDH19-GCE is caused by heterozygous loss-of-function mutations in the X-chromosome gene, Protocadherin 19 (PCDH19) encoding a cell-cell adhesion molecule. Intriguingly, hemizygous males are generally unaffected. As PCDH19 is subjected to random X-inactivation, heterozygous females are comprised of a mosaic of cells expressing either the normal or mutant allele, which is thought to drive pathology. Despite being the second most prevalent monogeneic cause of epilepsy, little is known about the role of PCDH19 in brain development. In this study we show that PCDH19 is highly expressed in human neural stem and progenitor cells (NSPCs) and investigate its function in vitro in these cells of both mouse and human origin. Transcriptomic analysis of mouse NSPCs lacking Pcdh19 revealed changes to genes involved in regulation of neuronal differentiation, and we subsequently show that loss of Pcdh19 causes increased NSPC neurogenesis. We reprogramed human fibroblast cells harbouring a pathogenic PCDH19 mutation into human induced pluripotent stem cells (hiPSC) and employed neural differentiation of these to extend our studies into human NSPCs. As in mouse, loss of PCDH19 function caused increased neurogenesis, and furthermore, we show this is associated with a loss of human NSPC polarity. Overall our data suggests a conserved role for PCDH19 in regulating mammalian cortical neurogenesis and has implications for the pathogenesis of PCDH19-GCE. We propose that the difference in timing or “heterochrony” of neuronal cell production originating from PCDH19 wildtype and mutant NSPCs within the same individual may lead to downstream asynchronies and abnormalities in neuronal network formation, which in-part predispose the individual to network dysfunction and epileptic activity. Refereed/Peer-reviewed

Introduction Children with Down Syndrome (DS) frequently develop hematological malignancies including acute lymphoblastic leukemia (ALL), however, the genomic basis for the predisposition to DS-ALL remains unknown. DS-ALL is associated with increased rates of relapse, chemotherapy related toxicity and poor overall survival compared to non-DS patients. The P2RY8-CRLF2 (CRLF2r) gene fusion, the result of deletion of the pseudoautosomal region 1 (PAR1) on the X/Y chromosome (chr), has been identified in 60% of DS-ALL (+21) patients and 30% of iAMP21 (intrachromosomal amplification of chr21) ALL patients compared to only 5-16% of other B-ALL patients. The high mobility group nucleosome binding protein 1, encoded by HMGN1 on chr21 may play a role in DS-ALL leukemogenesis, due to its demethylase activity associated with transcriptional activation. Methods mRNA seq was performed on the blast cells of 76 ALL patients using the Universal Plus mRNA-seq with NuQuant kit. FusionCatcher, SOAPfuse and JAFFA were used to identify fusions and featureCounts used for mRNA expression of STAR (v2.7.3a) aligned BAM files. CRISPR/Cas9 gRNAs targeting P2RY8 intron 1 or CRLF2 5' UTR created the 320 KB (PAR1) deletion. Cas9/gRNAs were transduced into Jurkat cells ± HMGN1 overexpression, activated with doxycycline and stained with TSLPR (CRLF2/IL-7Rα dimer) for single cell sort and clonal expansion. Phosphorylated proteins were measured by intracellular flow cytometry and SYBR Green RQ-PCR was used for mRNA expression. Results Significantly higher HMGN1 expression was identified in CRLF2r ALL patients, compared to the control (BCR-ABL1+ patients. Fig. 1A; p To further understand the role of HMGN1 in CRLF2r development, HMGN1 was overexpressed (1.5-fold; HMGN1H) in Jurkat Cas9 cells to represent a trisomy level of expression. gRNAs targeting P2RY8 and CRLF2 were activated in cells ± HMGN1H with undirected repair. TSLPR surface and CRLF2 mRNA expression were higher in CRLF2r + HMGN1H cells compared to CRLF2r only cells (Fig. 1B,C; p=0.034 and CRLF2r + HMGN1H cells also demonstrated significantly increased pSTAT5 (MFI: 2359 ± 1; Fig. 1D), pAKT (MFI: 2339 ± 6; Fig. 1E) and pERK (MFI: 2478 ± 48; Fig. 1F) compared to CRLF2r only cells (MFI pSTAT5: 1910 ± 10; pAKT: 1727 ± 14; pERK: 1946 ± 6; all p Conclusion The P2RY8-CRLF2 (CRLF2r) gene fusionis prevalent in DS, +21 and iAMP21 ALL patients. Here, we demonstrate that CRLF2r is associated with high expression of HMGN1 (chr21)in ALL patient cells . Using CRISPR/Cas9 in an in vitro model, we demonstrate that enforced high expression of HMGN1 alters DSB repair mechanism, favoring PAR1 deletion and the subsequent formation of the P2RY8-CRLF2 gene fusion, with attendant higher expression of STAT5 target genes. Understanding the role of HMGN1 in the disproportionate number of DS-ALL patients who are diagnosed with CRLF2r has the potential to lead to novel therapeutic interventions, in this high-risk group of patients where efficacious therapeutic options are currently poor. Figure 1 Figure 1. Disclosures Yeung: BMS: Honoraria, Research Funding; Amgen: Honoraria; Novartis: Honoraria, Research Funding; Pfizer: Honoraria. White: Novartis: Research Funding; BMS: Honoraria, Research Funding.

De novo and inherited mutations of X-chromosome cell adhesionmolecule protocadherin 19 (PCDH19) cause frequent, highly variable epilepsy, autism, cognitive decline and behavioural problems syndrome. Intriguingly, hemizygous nullmales are not affected while heterozygous females are, contradicting established X-chromosome inheritance. The disease mechanism is not known. Cellular mosaicism is the likely driver. We have identified p54nrb/NONO, amultifunctional nuclear paraspeckle protein with known roles in nuclear hormone receptor gene regulation, as a PCDH19 protein interacting partner. Using breast cancer cells we show that PCDH19-NONO complex is a positive co-regulator of ERa-mediated gene expression. Expression of mutant PCDH19 affects at least a subset of known ERα-regulated genes. These data are consistent with our findings that genes regulated by nuclear hormone receptors and those involved in themetabolism of neurosteroids in particular are dysregulated in PCDH19-epilepsy girls and affected mosaicmales. Overall we define and characterize a novelmechanism of gene regulation driven by PCDH19, which is mediated by paraspeckle constituent NONO and is ERα-dependent. This PCDH19-NONO-ERa axis is of relevance not only to PCDH19-epilepsy and its comorbidities but likely also to ERα and generally nuclear hormone receptor-associated cancers. Refereed/Peer-reviewed