Functional analysis of Collagen 17a1: A genetic modifier of junctional epidermolysis bullosa in mice.

Previous work strongly implicated Collagen 17a1 (Col17a1) as a potent genetic modifier of junctional epidermolysis bullosa (JEB) caused by a hypomorphic mutation (Lamc2jeb) in mice. The importance of the noncollagenous domain (NC4) of COLXVII was suggested by use of a congenic reduction approach that restricted the modifier effect to 2-3 neighboring amino acid changes in that domain. The current study utilizes TALEN and CRISPR/Cas9 induced amino acid replacements and in-frame indels nested to NC4 to further investigate the role of this and adjoining COLXVII domains both as modifiers and primary risk effectors. We confirm the importance of COLXVI AA 1275 S/G and 1277 N/S substitutions and utilize small nested indels to show that subtle changes in this microdomain attenuate JEB. We further show that large in-frame indels removing up to 1482 bp and 169 AA of NC6 through NC1 domains are surprisingly disease free on their own but can be very potent modifiers of Lamc2jeb/jeb JEB. Together these studies exploiting gene editing to functionally dissect the Col17a1 modifier demonstrate the importance of epistatic interactions between a primary disease-causing mutation in one gene and innocuous 'healthy' alleles in other genes.

Genetic predisposition to neuroblastoma results from a regulatory polymorphism that promotes the adrenergic cell state.

Childhood neuroblastomas exhibit plasticity between an undifferentiated neural crest-like mesenchymal cell state and a more differentiated sympathetic adrenergic cell state. These cell states are governed by autoregulatory transcriptional loops called core regulatory circuitries (CRCs), which drive the early development of sympathetic neuronal progenitors from migratory neural crest cells during embryogenesis. The adrenergic cell identity of neuroblastoma requires LMO1 as a transcriptional cofactor. Both LMO1 expression levels and the risk of developing neuroblastoma in children are associated with a single nucleotide polymorphism, G/T, that affects a GATA motif in the first intron of LMO1. Here, we showed that WT zebrafish with the GATA genotype developed adrenergic neuroblastoma, while knock-in of the protective TATA allele at this locus reduced the penetrance of MYCN-driven tumors, which were restricted to the mesenchymal cell state. Whole genome sequencing of childhood neuroblastomas demonstrated that TATA/TATA tumors also exhibited a mesenchymal cell state and were low risk at diagnosis. Thus, conversion of the regulatory GATA to a TATA allele in the first intron of LMO1 reduced the neuroblastoma-initiation rate by preventing formation of the adrenergic cell state. This mechanism was conserved over 400 million years of evolution, separating zebrafish and humans.

Genetic Predisposition to Neuroblastoma Results from a Regulatory Polymorphism that Promotes the Adrenergic Cell State.

Childhood neuroblastomas exhibit plasticity between an undifferentiated neural crest-like "mesenchymal" cell state and a more differentiated sympathetic "adrenergic" cell state. These cell states are governed by autoregulatory transcriptional loops called core regulatory circuitries (CRCs), which drive the early development of sympathetic neuronal progenitors from migratory neural crest cells during embryogenesis. The adrenergic cell identity of neuroblastoma requires LMO1 as a transcriptional co-factor. Both LMO1 expression levels and the risk of developing neuroblastoma in children are associated with a single nucleotide polymorphism G/T that affects a G ATA motif in the first intron of LMO1. Here we show that wild-type zebrafish with the G ATA genotype develop adrenergic neuroblastoma, while knock-in of the protective T ATA allele at this locus reduces the penetrance of MYCN-driven tumors, which are restricted to the mesenchymal cell state. Whole genome sequencing of childhood neuroblastomas demonstrates that T ATA/ T ATA tumors also exhibit a mesenchymal cell state and are low risk at diagnosis. Thus, conversion of the regulatory G ATA to a T ATA allele in the first intron of LMO1 reduces the neuroblastoma initiation rate by preventing formation of the adrenergic cell state, a mechanism that is conserved over 400 million years of evolution separating zebrafish and humans.

Disruption of the kringle 1 domain of prothrombin leads to late onset mortality in zebrafish.

The ability to prevent blood loss in response to injury is a conserved function of all vertebrates. Complete deficiency of the central clotting enzyme prothrombin has never been observed in humans and is incompatible with postnatal life in mice, thus limiting the ability to study its role in vivo. Zebrafish are able to tolerate severe hemostatic deficiencies that are lethal in mammals. We have generated a targeted genetic deletion in the kringle 1 domain of zebrafish prothrombin. Homozygous mutant embryos develop normally into the mid-juvenile stage but demonstrate complete mortality by 2 months of age primarily due to internal hemorrhage. Mutants are unable to form occlusive venous and arterial thrombi in response to endothelial injury, a defect that was phenocopied using direct oral anticoagulants. Human prothrombin engineered with the equivalent mutation exhibits a severe reduction in secretion, thrombin generation, and fibrinogen cleavage. Together, these data demonstrate the conserved function of thrombin in zebrafish and provide insight into the role of kringle 1 in prothrombin maturation and activity. Understanding how zebrafish are able to develop normally and survive into early adulthood without thrombin activity will provide important insight into its pleiotropic functions as well as the management of patients with bleeding disorders.

Zebrafish dscaml1 Deficiency Impairs Retinal Patterning and Oculomotor Function.

Down syndrome cell adhesion molecules (dscam and dscaml1) are essential regulators of neural circuit assembly, but their roles in vertebrate neural circuit function are still mostly unexplored. We investigated the functional consequences of dscaml1 deficiency in the larval zebrafish (sexually undifferentiated) oculomotor system, where behavior, circuit function, and neuronal activity can be precisely quantified. Genetic perturbation of dscaml1 resulted in deficits in retinal patterning and light adaptation, consistent with its known roles in mammals. Oculomotor analyses revealed specific deficits related to the dscaml1 mutation, including severe fatigue during gaze stabilization, reduced saccade amplitude and velocity in the light, greater disconjugacy, and impaired fixation. Two-photon calcium imaging of abducens neurons in control and dscaml1 mutant animals confirmed deficits in saccade-command signals (indicative of an impairment in the saccadic premotor pathway), whereas abducens activation by the pretectum-vestibular pathway was not affected. Together, we show that loss of dscaml1 resulted in impairments in specific oculomotor circuits, providing a new animal model to investigate the development of oculomotor premotor pathways and their associated human ocular disorders.SIGNIFICANCE STATEMENTDscaml1 is a neural developmental gene with unknown behavioral significance. Using the zebrafish model, this study shows that dscaml1 mutants have a host of oculomotor (eye movement) deficits. Notably, the oculomotor phenotypes in dscaml1 mutants are reminiscent of human ocular motor apraxia, a neurodevelopmental disorder characterized by reduced saccade amplitude and gaze stabilization deficits. Population-level recording of neuronal activity further revealed potential subcircuit-specific requirements for dscaml1 during oculomotor behavior. These findings underscore the importance of dscaml1 in the development of visuomotor function and characterize a new model to investigate potential circuit deficits underlying human oculomotor disorders.

Disruption of asxl1 results in myeloproliferative neoplasms in zebrafish.

Somatic loss-of-function mutations of the additional sex combs-like transcriptional regulator 1 (ASXL1) gene are common genetic abnormalities in human myeloid malignancies and induce clonal expansion of mutated hematopoietic stem cells (HSCs). To understand how ASXL1 disruption leads to myeloid cell transformation, we generated asxl1 haploinsufficient and null zebrafish lines using genome-editing technology. Here, we show that homozygous loss of asxl1 leads to apoptosis of newly formed HSCs. Apoptosis occurred via the mitochondrial apoptotic pathway mediated by upregulation of bim and bid Half of the asxl1+/- zebrafish had myeloproliferative neoplasms (MPNs) by 5 months of age. Heterozygous loss of asxl1 combined with heterozygous loss of tet2 led to a more penetrant MPN phenotype, while heterozygous loss of asxl1 combined with complete loss of tet2 led to acute myeloid leukemia (AML). These findings support the use of asxl1+/- zebrafish as a strategy to identify small-molecule drugs to suppress the growth of asxl1 mutant but not wild-type HSCs in individuals with somatically acquired inactivating mutations of ASXL1.

Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10.

Leber congenital amaurosis type 10 is a severe retinal dystrophy caused by mutations in the CEP290 gene1,2. We developed EDIT-101, a candidate genome-editing therapeutic, to remove the aberrant splice donor created by the IVS26 mutation in the CEP290 gene and restore normal CEP290 expression. Key to this therapeutic, we identified a pair of Staphylococcus aureus Cas9 guide RNAs that were highly active and specific to the human CEP290 target sequence. In vitro experiments in human cells and retinal explants demonstrated the molecular mechanism of action and nuclease specificity. Subretinal delivery of EDIT-101 in humanized CEP290 mice showed rapid and sustained CEP290 gene editing. A comparable surrogate non-human primate (NHP) vector also achieved productive editing of the NHP CEP290 gene at levels that met the target therapeutic threshold, and demonstrated the ability of CRISPR/Cas9 to edit somatic primate cells in vivo. These results support further development of EDIT-101 for LCA10 and additional CRISPR-based medicines for other inherited retinal disorders.

Efficient CRISPR/Cas9-mediated editing of trinucleotide repeat expansion in myotonic dystrophy patient-derived iPS and myogenic cells.

CRISPR/Cas9 is an attractive platform to potentially correct dominant genetic diseases by gene editing with unprecedented precision. In the current proof-of-principle study, we explored the use of CRISPR/Cas9 for gene-editing in myotonic dystrophy type-1 (DM1), an autosomal-dominant muscle disorder, by excising the CTG-repeat expansion in the 3'-untranslated-region (UTR) of the human myotonic dystrophy protein kinase (DMPK) gene in DM1 patient-specific induced pluripotent stem cells (DM1-iPSC), DM1-iPSC-derived myogenic cells and DM1 patient-specific myoblasts. To eliminate the pathogenic gain-of-function mutant DMPK transcript, we designed a dual guide RNA based strategy that excises the CTG-repeat expansion with high efficiency, as confirmed by Southern blot and single molecule real-time (SMRT) sequencing. Correction efficiencies up to 90% could be attained in DM1-iPSC as confirmed at the clonal level, following ribonucleoprotein (RNP) transfection of CRISPR/Cas9 components without the need for selective enrichment. Expanded CTG repeat excision resulted in the disappearance of ribonuclear foci, a quintessential cellular phenotype of DM1, in the corrected DM1-iPSC, DM1-iPSC-derived myogenic cells and DM1 myoblasts. Consequently, the normal intracellular localization of the muscleblind-like splicing regulator 1 (MBNL1) was restored, resulting in the normalization of splicing pattern of SERCA1. This study validates the use of CRISPR/Cas9 for gene editing of repeat expansions.

Nodal patterning without Lefty inhibitory feedback is functional but fragile.

Developmental signaling pathways often activate their own inhibitors. Such inhibitory feedback has been suggested to restrict the spatial and temporal extent of signaling or mitigate signaling fluctuations, but these models are difficult to rigorously test. Here, we determine whether the ability of the mesendoderm inducer Nodal to activate its inhibitor Lefty is required for development. We find that zebrafish lefty mutants exhibit excess Nodal signaling and increased specification of mesendoderm, resulting in embryonic lethality. Strikingly, development can be fully restored without feedback: Lethal patterning defects in lefty mutants can be rescued by ectopic expression of lefty far from its normal expression domain or by spatially and temporally uniform exposure to a Nodal inhibitor drug. While drug-treated mutants are less tolerant of mild perturbations to Nodal signaling levels than wild type embryos, they can develop into healthy adults. These results indicate that patterning without inhibitory feedback is functional but fragile.

Uromodulin p.Cys147Trp mutation drives kidney disease by activating ER stress and apoptosis.

Uromodulin-associated kidney disease (UAKD) is caused by mutations in the uromodulin (UMOD) gene that result in a misfolded form of UMOD protein, which is normally secreted by nephrons. In UAKD patients, mutant UMOD is poorly secreted and accumulates in the ER of distal kidney epithelium, but its role in disease progression is largely unknown. Here, we modeled UMOD accumulation in mice by expressing the murine equivalent of the human UMOD p.Cys148Trp point mutation (UmodC147W/+ mice). Like affected humans, these UmodC147W/+ mice developed spontaneous and progressive kidney disease with organ failure over 24 weeks. Analysis of diseased kidneys and purified UMOD-producing cells revealed early activation of the PKR-like ER kinase/activating transcription factor 4 (PERK/ATF4) ER stress pathway, innate immune mediators, and increased apoptotic signaling, including caspase-3 activation. Unexpectedly, we also detected autophagy deficiency. Human cells expressing UMOD p.Cys147Trp recapitulated the findings in UmodC147W/+ mice, and autophagy activation with mTOR inhibitors stimulated the intracellular removal of aggregated mutant UMOD. Human cells producing mutant UMOD were susceptible to TNF-α- and TRAIL-mediated apoptosis due to increased expression of the ER stress mediator tribbles-3. Blocking TNF-α in vivo with the soluble recombinant fusion protein TNFR:Fc slowed disease progression in UmodC147W/+ mice by reducing active caspase-3, thereby preventing tubule cell death and loss of epithelial function. These findings reveal a targetable mechanism for disease processes involved in UAKD.

Genome editing of factor X in zebrafish reveals unexpected tolerance of severe defects in the common pathway.

Deficiency of factor X (F10) in humans is a rare bleeding disorder with a heterogeneous phenotype and limited therapeutic options. Targeted disruption of F10 and other common pathway factors in mice results in embryonic/neonatal lethality with rapid resorption of homozygous mutants, hampering additional studies. Several of these mutants also display yolk sac vascular defects, suggesting a role for thrombin signaling in vessel development. The zebrafish is a vertebrate model that demonstrates conservation of the mammalian hemostatic and vascular systems. We have leveraged these advantages for in-depth study of the role of the coagulation cascade in the developmental regulation of hemostasis and vasculogenesis. In this article, we show that ablation of zebrafish f10 by using genome editing with transcription activator-like effector nucleases results in a major embryonic hemostatic defect. However, widespread hemorrhage and subsequent lethality does not occur until later stages, with absence of any detectable defect in vascular development. We also use f10-/- zebrafish to confirm 5 novel human F10 variants as causative mutations in affected patients, providing a rapid and reliable in vivo model for testing the severity of F10 variants. These findings as well as the prolonged survival of f10-/- mutants will enable us to expand our understanding of the molecular mechanisms of hemostasis, including a platform for screening variants of uncertain significance in patients with F10 deficiency and other coagulation disorders. Further study as to how fish tolerate what is an early lethal mutation in mammals could facilitate improvement of diagnostics and therapeutics for affected patients with bleeding disorders.

Context influences on TALE-DNA binding revealed by quantitative profiling.

Transcription activator-like effector (TALE) proteins recognize DNA using a seemingly simple DNA-binding code, which makes them attractive for use in genome engineering technologies that require precise targeting. Although this code is used successfully to design TALEs to target specific sequences, off-target binding has been observed and is difficult to predict. Here we explore TALE-DNA interactions comprehensively by quantitatively assaying the DNA-binding specificities of 21 representative TALEs to ∼5,000-20,000 unique DNA sequences per protein using custom-designed protein-binding microarrays (PBMs). We find that protein context features exert significant influences on binding. Thus, the canonical recognition code does not fully capture the complexity of TALE-DNA binding. We used the PBM data to develop a computational model, Specificity Inference For TAL-Effector Design (SIFTED), to predict the DNA-binding specificity of any TALE. We provide SIFTED as a publicly available web tool that predicts potential genomic off-target sites for improved TALE design.

Targeted Mutagenesis in Zebrafish Using CRISPR RNA-Guided Nucleases.

In recent years, the zebrafish has become a critical contributor to various areas of biomedical research, advancing our fundamental understanding of biomedicine and helping discover candidate therapeutics for human diseases. Nevertheless, to further extend the power of this important model organism requires a robust and simple-to-use genome editing platform that will enable targeted gene knockouts and introduction of specific mutations identified in human diseases into the zebrafish genome. We describe here protocols for creating insertion or deletion (indel) mutations or precise sequence modifications in zebrafish genes using customizable CRISPR-Cas9 RNA-guided nucleases (RGNs). These methods can be easily implemented in any lab and may also potentially be extended for use in other organisms.

Targeted disruption of DNMT1, DNMT3A and DNMT3B in human embryonic stem cells.

DNA methylation is a key epigenetic modification involved in regulating gene expression and maintaining genomic integrity. Here we inactivated all three catalytically active DNA methyltransferases (DNMTs) in human embryonic stem cells (ESCs) using CRISPR/Cas9 genome editing to further investigate the roles and genomic targets of these enzymes. Disruption of DNMT3A or DNMT3B individually as well as of both enzymes in tandem results in viable, pluripotent cell lines with distinct effects on the DNA methylation landscape, as assessed by whole-genome bisulfite sequencing. Surprisingly, in contrast to findings in mouse, deletion of DNMT1 resulted in rapid cell death in human ESCs. To overcome this immediate lethality, we generated a doxycycline-responsive tTA-DNMT1* rescue line and readily obtained homozygous DNMT1-mutant lines. However, doxycycline-mediated repression of exogenous DNMT1* initiates rapid, global loss of DNA methylation, followed by extensive cell death. Our data provide a comprehensive characterization of DNMT-mutant ESCs, including single-base genome-wide maps of the targets of these enzymes.

Targeted genome editing in human cells using CRISPR/Cas nucleases and truncated guide RNAs.

CRISPR RNA-guided nucleases have recently emerged as a robust genome-editing platform that functions in a wide range of organisms. To reduce off-target effects of these nucleases, we developed and validated a modified system that uses truncated guide RNAs (tru-gRNAs). The use of tru-gRNAs leads to decreases in off-target effects and does not generally compromise the on-target efficiencies of these genome-editing nucleases. In this chapter, we describe guidelines for identifying potential tru-gRNA target sites and protocols for measuring the on-target efficiencies of CRISPR RNA-guided nucleases in human cells.

Systematic screening reveals a role for BRCA1 in the response to transcription-associated DNA damage.

BRCA1 is a breast and ovarian tumor suppressor. Given its numerous incompletely understood functions and the possibility that more exist, we performed complementary systematic screens in search of new BRCA1 protein-interacting partners. New BRCA1 functions and/or a better understanding of existing ones were sought. Among the new interacting proteins identified, genetic interactions were detected between BRCA1 and four of the interactors: TONSL, SETX, TCEANC, and TCEA2. Genetic interactions were also detected between BRCA1 and certain interactors of TONSL, including both members of the FACT complex. From these results, a new BRCA1 function in the response to transcription-associated DNA damage was detected. Specifically, new roles for BRCA1 in the restart of transcription after UV damage and in preventing or repairing damage caused by stabilized R loops were identified. These roles are likely carried out together with some of the newly identified interactors. This new function may be important in BRCA1 tumor suppression, since the expression of several interactors, including some of the above-noted transcription proteins, is repeatedly aberrant in both breast and ovarian cancers.

IκB kinase ß (IKBKB) mutations in lymphomas that constitutively activate canonical nuclear factor κB (NFκB) signaling.

Somatic mutations altering lysine 171 of the IKBKB gene that encodes (IKKß), the critical activating kinase in canonical (NFκB) signaling, have been described in splenic marginal zone lymphomas and multiple myeloma. Lysine 171 forms part of a cationic pocket that interacts with the activation loop phosphate in the activated wild type kinase. We show here that K171E IKKß and K171T IKKß represent kinases that are constitutively active even in the absence of activation loop phosphorylation. Predictive modeling and biochemical studies establish why mutations in a positively charged residue in the cationic pocket of an activation loop phosphorylation-dependent kinase result in constitutive activation. Transcription activator-like effector nuclease-based knock-in mutagenesis provides evidence from a B lymphoid context that K171E IKKß contributes to lymphomagenesis.

Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing.

Monomeric CRISPR-Cas9 nucleases are widely used for targeted genome editing but can induce unwanted off-target mutations with high frequencies. Here we describe dimeric RNA-guided FokI nucleases (RFNs) that can recognize extended sequences and edit endogenous genes with high efficiencies in human cells. RFN cleavage activity depends strictly on the binding of two guide RNAs (gRNAs) to DNA with a defined spacing and orientation substantially reducing the likelihood that a suitable target site will occur more than once in the genome and therefore improving specificities relative to wild-type Cas9 monomers. RFNs guided by a single gRNA generally induce lower levels of unwanted mutations than matched monomeric Cas9 nickases. In addition, we describe a simple method for expressing multiple gRNAs bearing any 5' end nucleotide, which gives dimeric RFNs a broad targeting range. RFNs combine the ease of RNA-based targeting with the specificity enhancement inherent to dimerization and are likely to be useful in applications that require highly precise genome editing.

Broad specificity profiling of TALENs results in engineered nucleases with improved DNA-cleavage specificity.

Although transcription activator-like effector nucleases (TALENs) can be designed to cleave chosen DNA sequences, TALENs have activity against related off-target sequences. To better understand TALEN specificity, we profiled 30 unique TALENs with different target sites, array length and domain sequences for their abilities to cleave any of 10(12) potential off-target DNA sequences using in vitro selection and high-throughput sequencing. Computational analysis of the selection results predicted 76 off-target substrates in the human genome, 16 of which were accessible and modified by TALENs in human cells. The results suggest that (i) TALE repeats bind DNA relatively independently; (ii) longer TALENs are more tolerant of mismatches yet are more specific in a genomic context; and (iii) excessive DNA-binding energy can lead to reduced TALEN specificity in cells. Based on these findings, we engineered a TALEN variant that exhibits equal on-target cleavage activity but tenfold lower average off-target activity in human cells.