CRISPR-Analytics (CRISPR-A): A platform for precise analytics and simulations for gene editing.

Gene editing characterization with currently available tools does not always give precise relative proportions among the different types of gene edits present in an edited bulk of cells. We have developed CRISPR-Analytics, CRISPR-A, which is a comprehensive and versatile genome editing web application tool and a nextflow pipeline to give support to gene editing experimental design and analysis. CRISPR-A provides a robust gene editing analysis pipeline composed of data analysis tools and simulation. It achieves higher accuracy than current tools and expands the functionality. The analysis includes mock-based noise correction, spike-in calibrated amplification bias reduction, and advanced interactive graphics. This expanded robustness makes this tool ideal for analyzing highly sensitive cases such as clinical samples or experiments with low editing efficiencies. It also provides an assessment of experimental design through the simulation of gene editing results. Therefore, CRISPR-A is ideal to support multiple kinds of experiments such as double-stranded DNA break-based engineering, base editing (BE), primer editing (PE), and homology-directed repair (HDR), without the need of specifying the used experimental approach.

Evolution of CRISPR-associated endonucleases as inferred from resurrected proteins.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas9 is an effector protein that targets invading DNA and plays a major role in the prokaryotic adaptive immune system. Although Streptococcus pyogenes CRISPR-Cas9 has been widely studied and repurposed for applications including genome editing, its origin and evolution are poorly understood. Here, we investigate the evolution of Cas9 from resurrected ancient nucleases (anCas) in extinct firmicutes species that last lived 2.6 billion years before the present. We demonstrate that these ancient forms were much more flexible in their guide RNA and protospacer-adjacent motif requirements compared with modern-day Cas9 enzymes. Furthermore, anCas portrays a gradual palaeoenzymatic adaptation from nickase to double-strand break activity, exhibits high levels of activity with both single-stranded DNA and single-stranded RNA targets and is capable of editing activity in human cells. Prediction and characterization of anCas with a resurrected protein approach uncovers an evolutionary trajectory leading to functionally flexible ancient enzymes.

INSERT-seq enables high-resolution mapping of genomically integrated DNA using Nanopore sequencing.

Comprehensive characterisation of genome engineering technologies is relevant for their development and safe use in human gene therapy. Short-read based methods can overlook insertion events in repetitive regions. We develop INSERT-seq, a method that combines targeted amplification of integrated DNA, UMI-based correction of PCR bias and Oxford Nanopore long-read sequencing for robust analysis of DNA integration. The experimental pipeline improves the number of mappable insertions at repetitive regions by 4.8-7.3% and larger repeats are processed with a computational peak calling pipeline. INSERT-seq is a simple, cheap and robust method to quantitatively characterise DNA integration in diverse ex vivo and in vivo samples.

Noncanonical activation of GLI signaling in SOX2+ cells drives medulloblastoma relapse.

SRY (sex determining region Y)-box 2 (SOX2)-labeled cells play key roles in chemoresistance and tumor relapse; thus, it is critical to elucidate the mechanisms propagating them. Single-cell transcriptomic analyses of the most common malignant pediatric brain tumor, medulloblastoma (MB), revealed the existence of astrocytic Sox2+ cells expressing sonic hedgehog (SHH) signaling biomarkers. Treatment with vismodegib, an SHH inhibitor that acts on Smoothened (Smo), led to increases in astrocyte-like Sox2+ cells. Using SOX2-enriched MB cultures, we observed that SOX2+ cells required SHH signaling to propagate, and unlike in the proliferative tumor bulk, the SHH pathway was activated in these cells downstream of Smo in an MYC-dependent manner. Functionally different GLI inhibitors depleted vismodegib-resistant SOX2+ cells from MB tissues, reduced their ability to further engraft in vivo, and increased symptom-free survival. Our results emphasize the promise of therapies targeting GLI to deplete SOX2+ cells and provide stable tumor remission.

Pan-cancer pervasive upregulation of 3' UTR splicing drives tumourigenesis.

Most mammalian genes generate messenger RNAs with variable untranslated regions (UTRs) that are important post-transcriptional regulators. In cancer, shortening at 3' UTR ends via alternative polyadenylation can activate oncogenes. However, internal 3' UTR splicing remains poorly understood as splicing studies have traditionally focused on protein-coding alterations. Here we systematically map the pan-cancer landscape of 3' UTR splicing and present this in SpUR ( http://www.cbrc.kaust.edu.sa/spur/home/ ). 3' UTR splicing is widespread, upregulated in cancers, correlated with poor prognosis and more prevalent in oncogenes. We show that antisense oligonucleotide-mediated inhibition of 3' UTR splicing efficiently reduces oncogene expression and impedes tumour progression. Notably, CTNNB1 3' UTR splicing is the most consistently dysregulated event across cancers. We validate its upregulation in hepatocellular carcinoma and colon adenocarcinoma, and show that the spliced 3' UTR variant is the predominant contributor to its oncogenic functions. Overall, our study highlights the importance of 3' UTR splicing in cancer and may launch new avenues for RNA-based anti-cancer therapeutics.

MiR-138 is a potent regulator of the heterogenous MYC transcript population in cancers.

3'UTR shortening in cancer has been shown to activate oncogenes, partly through the loss of microRNA-mediated repression. This suggests that many reported microRNA-oncogene target interactions may not be present in cancer cells. One of the most well-studied oncogenes is the transcription factor MYC, which is overexpressed in more than half of all cancers. MYC overexpression is not always accompanied by underlying genetic aberrations. In this study, we demonstrate that the MYC 3'UTR is shortened in colorectal cancer (CRC). Using unbiased computational and experimental approaches, we identify and validate microRNAs that target the MYC coding region. In particular, we show that miR-138 inhibits MYC expression and suppresses tumor growth of CRC and hepatocellular carcinoma (HCC) cell lines. Critically, the intravenous administration of miR-138 significantly impedes MYC-driven tumor growth in vivo. Taken together, our results highlight the previously uncharacterized shortening of the MYC 3'UTR in cancer, and identify miR-138 as a potent regulator of the heterogenous MYC transcript population.

Find and cut-and-transfer (FiCAT) mammalian genome engineering.

While multiple technologies for small allele genome editing exist, robust technologies for targeted integration of large DNA fragments in mammalian genomes are still missing. Here we develop a gene delivery tool (FiCAT) combining the precision of a CRISPR-Cas9 (find module), and the payload transfer efficiency of an engineered piggyBac transposase (cut-and-transfer module). FiCAT combines the functionality of Cas9 DNA scanning and targeting DNA, with piggyBac donor DNA processing and transfer capacity. PiggyBac functional domains are engineered providing increased on-target integration while reducing off-target events. We demonstrate efficient delivery and programmable insertion of small and large payloads in cellulo (human (Hek293T, K-562) and mouse (C2C12)) and in vivo in mouse liver. Finally, we evolve more efficient versions of FiCAT by generating a targeted diversity of 394,000 variants and undergoing 4 rounds of evolution. In this work, we develop a precise and efficient targeted insertion of multi kilobase DNA fragments in mammalian genomes.

A novel SOCS5/miR-18/miR-25 axis promotes tumorigenesis in liver cancer.

The role of miRNAs with tumor suppressive activity in liver cancer has been well studied. However, little is known about potential oncomiRs in HCC. In our study, we conducted a systematic evaluation of candidate oncomiRs and found that upregulation of miR-18a and miR-25 in HCC was associated with poor patient survival and promoted proliferation in HCC cell lines. These two miRNAs belong to the polycistronic paralogous miR-17-92 and miR-25-106b clusters respectively. Although the members of both clusters are often upregulated in HCC, the contribution of individual miRNAs in these clusters to HCC tumorigenesis is not fully understood. We validated SOCS5 as a bona fide target of both miRNAs, and established, for the first time, the tumor suppressive role of SOCS5 in liver cancer. We further investigated the mechanism by which SOCS5 contributes to tumorigenesis, demonstrated that this SOCS5/miR-18a/miR-25 axis regulates the tumor suppressor TSC1 and downstream mTOR signaling, and highlighted the potential therapeutic use of miR-18a and miR-25 inhibition in restoring SOCS5 levels in HCC.

Cryptic Amyloidogenic Elements in the 3' UTRs of Neurofilament Genes Trigger Axonal Neuropathy.

Abnormal protein aggregation is observed in an expanding number of neurodegenerative diseases. Here, we describe a mechanism for intracellular toxic protein aggregation induced by an unusual mutation event in families affected by axonal neuropathy. These families carry distinct frameshift variants in NEFH (neurofilament heavy), leading to a loss of the terminating codon and translation of the 3' UTR into an extra 40 amino acids. In silico aggregation prediction suggested the terminal 20 residues of the altered NEFH to be amyloidogenic, which we confirmed experimentally by serial deletion analysis. The presence of this amyloidogenic motif fused to NEFH caused prominent and toxic protein aggregates in transfected cells and disrupted motor neurons in zebrafish. We identified a similar aggregation-inducing mechanism in NEFL (neurofilament light) and FUS (fused in sarcoma), in which mutations are known to cause aggregation in Charcot-Marie-Tooth disease and amyotrophic lateral sclerosis, respectively. In summary, we present a protein-aggregation-triggering mechanism that should be taken into consideration during the evaluation of stop-loss variants.

Competing endogenous RNA networks: tying the essential knots for cancer biology and therapeutics.

A recently discovered dimension of post-transcriptional gene regulation involves co-regulatory crosstalk between RNA transcripts, which compete for common pools of microRNA (miRNA) molecules. These competing endogenous RNAs (ceRNAs), or natural miRNA sponges, have an active role in regulating miRNA availability within the cell and form intertwined regulatory networks. Recent reports have implicated diverse RNA species including protein-coding messenger RNAs and non-coding RNAs as ceRNAs in human development and diseases including human cancer. In this review, we discuss the most recent discoveries that implicate natural miRNA decoys in human cancer biology, as well as exciting advances in the study of ceRNA networks and dynamics. The structure and topology of intricate genome-scale ceRNA networks can be predicted computationally, and their dynamic response to fluctuations in ceRNA and miRNA levels can be studied via mathematical modeling. Additionally, the development of new methods to quantitatively determine absolute expression levels of miRNA and ceRNA molecules have expanded the capacity to accurately study the efficiency of ceRNA crosstalk in diverse biological models. These major milestones are of critical importance to identify key components of ceRNA regulatory networks that could aid the development of new approaches to cancer diagnostics and oligonucleotide-based therapeutics.

The WNT-controlled transcriptional regulator LBH is required for mammary stem cell expansion and maintenance of the basal lineage.

The identification of multipotent mammary stem cells (MaSCs) has provided an explanation for the unique regenerative capacity of the mammary gland throughout adult life. However, it remains unclear what genes maintain MaSCs and control their specification into the two epithelial lineages: luminal and basal. LBH is a novel transcription co-factor in the WNT pathway with hitherto unknown physiological function. LBH is expressed during mammary gland development and aberrantly overexpressed in aggressive 'basal' subtype breast cancers. Here, we have explored the in vivo role of LBH in mammopoiesis. We show that in postnatal mammary epithelia, LBH is predominantly expressed in the Lin(-)CD29(high)CD24(+) basal MaSC population. Upon conditional inactivation of LBH, mice exhibit pronounced delays in mammary tissue expansion during puberty and pregnancy, accompanied by increased luminal differentiation at the expense of basal lineage specification. These defects could be traced to a severe reduction in the frequency and self-renewal/differentiation potential of basal MaSCs. Mechanistically, LBH induces expression of key epithelial stem cell transcription factor ΔNp63 to promote a basal MaSC state and repress luminal differentiation genes, mainly that encoding estrogen receptor α (Esr1/ERα). Collectively, these studies identify LBH as an essential regulator of basal MaSC expansion/maintenance, raising important implications for its potential role in breast cancer pathogenesis.

Synaptotagmin 2 mutations cause an autosomal-dominant form of lambert-eaton myasthenic syndrome and nonprogressive motor neuropathy.

Synaptotagmin 2 is a synaptic vesicle protein that functions as a calcium sensor for neurotransmission but has not been previously associated with human disease. Via whole-exome sequencing, we identified heterozygous missense mutations in the C2B calcium-binding domain of the gene encoding Synaptotagmin 2 in two multigenerational families presenting with peripheral motor neuron syndromes. An essential calcium-binding aspartate residue, Asp307Ala, was disrupted by a c.920A>C change in one family that presented with an autosomal-dominant presynaptic neuromuscular junction disorder resembling Lambert-Eaton myasthenic syndrome. A c.923C>T variant affecting an adjacent residue (p.Pro308Leu) produced a presynaptic neuromuscular junction defect and a dominant hereditary motor neuropathy in a second family. Characterization of the mutation homologous to the human c.920A>C variant in Drosophila Synaptotagmin revealed a dominant disruption of synaptic vesicle exocytosis using this transgenic model. These findings indicate that Synaptotagmin 2 regulates neurotransmitter release at human peripheral motor nerve terminals. In addition, mutations in the Synaptotagmin 2 C2B domain represent an important cause of presynaptic congenital myasthenic syndromes and link them with hereditary motor axonopathies.

Notch signaling drives stemness and tumorigenicity of esophageal adenocarcinoma.

Esophageal adenocarcinoma ranks sixth in cancer mortality in the world and its incidence has risen dramatically in the Western population over the last decades. Data presented herein strongly suggest that Notch signaling is critical for esophageal adenocarcinoma and underlies resistance to chemotherapy. We present evidence that Notch signaling drives a cancer stem cell phenotype by regulating genes that establish stemness. Using patient-derived xenograft models, we demonstrate that inhibition of Notch by gamma-secretase inhibitors (GSI) is efficacious in downsizing tumor growth. Moreover, we demonstrate that Notch activity in a patient's ultrasound-assisted endoscopic-derived biopsy might predict outcome to chemotherapy. Therefore, this study provides a proof of concept that inhibition of Notch activity will have efficacy in treating esophageal adenocarcinoma, offering a rationale to lay the foundation for a clinical trial to evaluate the efficacy of GSI in esophageal adenocarcinoma treatment.

Association between In Utero arsenic exposure, placental gene expression, and infant birth weight: a US birth cohort study.

BACKGROUND: Epidemiologic studies and animal models suggest that in utero arsenic exposure affects fetal health, with a negative association between maternal arsenic ingestion and infant birth weight often observed. However, the molecular mechanisms for this association remain elusive. In the present study, we aimed to increase our understanding of the impact of low-dose arsenic exposure on fetal health by identifying possible arsenic-associated fetal tissue biomarkers in a cohort of pregnant women exposed to arsenic at low levels. METHODS: Arsenic concentrations were determined from the urine samples of a cohort of 133 pregnant women from New Hampshire. Placental tissue samples collected from enrollees were homogenized and profiled for gene expression across a panel of candidate genes, including known arsenic regulated targets and genes involved in arsenic transport, metabolism, or disease susceptibility. Multivariable adjusted linear regression models were used to examine the relationship of candidate gene expression with arsenic exposure or with birth weight of the baby. RESULTS: Placental expression of the arsenic transporter AQP9 was positively associated with maternal urinary arsenic levels during pregnancy (coefficient estimate: 0.25; 95% confidence interval: 0.05 - 0.45). Placental expression of AQP9 related to expression of the phospholipase ENPP2 which was positively associated with infant birth weight (coefficient estimate: 0.28; 95% CI: 0.09 - 0.47). A structural equation model indicated that these genes may mediate arsenic's effect on infant birth weight (coefficient estimate: -0.009; 95% confidence interval: -0.032 - -0.001; 10,000 replications for bootstrapping). CONCLUSIONS: We identified the expression of AQP9 as a potential fetal biomarker for arsenic exposure. Further, we identified a positive association between the placental expression of phospholipase ENPP2 and infant birth weight. These findings suggest a path by which arsenic may affect birth outcomes.

Hedgehog signaling regulates bladder cancer growth and tumorigenicity.

The role of Hedgehog (HH) signaling in bladder cancer remains controversial. The gene encoding the HH receptor and negative regulator PATCHED1 (PTCH1) resides on a region of chromosome 9q, one copy of which is frequently lost in bladder cancer. Inconsistent with PTCH1 functioning as a classic tumor suppressor gene, loss-of-function mutations in the remaining copy of PTCH1 are not commonly found. Here, we provide direct evidence for a critical role of HH signaling in bladder carcinogenesis. We show that transformed human urothelial cells and many urothelial carcinoma cell lines exhibit constitutive HH signaling, which is required for their growth and tumorigenic properties. Surprisingly, rather than originating from loss of PTCH1, the constitutive HH activity observed in urothelial carcinoma cell lines was HH ligand dependent. Consistent with this finding, increased levels of HH and the HH target gene product GLI1 were found in resected human primary bladder tumors. Furthermore, on the basis of the difference in intrinsic HH dependence of urothelial carcinoma cell lines, a gene expression signature was identified that correlated with bladder cancer progression. Our findings therefore indicate that therapeutic targeting of the HH signaling pathway may be beneficial in the clinical management of bladder cancer.

Novel MLPA procedure using self-designed probes allows comprehensive analysis for CNVs of the genes involved in Hirschsprung disease.

BACKGROUND: Hirschsprung disease is characterized by the absence of intramural ganglion cells in the enteric plexuses, due to a fail during enteric nervous system formation. Hirschsprung has a complex genetic aetiology and mutations in several genes have been related to the disease. There is a clear predominance of missense/nonsense mutations in these genes whereas copy number variations (CNVs) have been seldom described, probably due to the limitations of conventional techniques usually employed for mutational analysis. In this study, we have looked for CNVs in some of the genes related to Hirschsprung (EDNRB, GFRA1, NRTN and PHOX2B) using the Multiple Ligation-dependent Probe Amplification (MLPA) approach. METHODS: CNVs screening was performed in 208 HSCR patients using a self-designed set of MLPA probes, covering the coding region of those genes. RESULTS: A deletion comprising the first 4 exons in GFRA1 gene was detected in 2 sporadic HSCR patients and in silico approaches have shown that the critical translation initiation signal in the mutant gene was abolished. In this study, we have been able to validate the reliability of this technique for CNVs screening in HSCR. CONCLUSIONS: The implemented MLPA based technique presented here allows CNV analysis of genes involved in HSCR that have not been not previously evaluated. Our results indicate that CNVs could be implicated in the pathogenesis of HSCR, although they seem to be an uncommon molecular cause of HSCR.

Involvement of SOX10 in the pathogenesis of Hirschsprung disease: report of a truncating mutation in an isolated patient.

SOX10 protein is a key transcription factor during neural crest development. Mutations in SOX10 are associated with several neurocristopathies such as Waardenburg syndrome type IV (WS4), a congenital disorder characterized by the association of hearing loss, pigmentary abnormalities, and absence of ganglion cells in the myenteric and submucosal plexus of the gastrointestinal tract, also known as aganglionic megacolon or Hirschsprung disease (HSCR). Several mutations at this locus are known to cause a high percentage of WS4 cases, but no SOX10 mutations had been ever reported associated to isolated HSCR patient. Therefore, nonsyndromic HSCR was initially thought not to be associated to mutations at this particular locus. In the present study, we describe the evaluation of the SOX10 gene in a series of 196 isolated HSCR cases, the largest patient series evaluated so far, and report a truncating c.153-155del mutation. This is the first time that a SOX10 mutation is detected in an isolated HSCR patient, which completely changes the scenario for the implications of SOX10 mutations in human disease, giving us a new tool for genetic counseling.

A new experimental approach is required in the molecular analysis of intestinal neuronal dysplasia type B patients.

Intestinal neuronal dysplasia type B (INDB) is characterized by the malformation of the parasympathetic submucous plexus of the gut. It is generally accepted that INDB has a genetic basis, and several genes produce an INDB-like phenotype in mice when disrupted, such as EDNRB. However, no mutations associated with this disease have been identified in several series analysed. In the present studu, we sought to determine whether the EDNRB/EDN3 signalling pathway plays a role in the pathogenesis of INDB in humans. Denaturing high performance liquid chromatography (dHPLC) techniques were employed to screen the EDNRB and EDN3 coding regions in 23 INDB patients. In addition, association studies were performed on these genes with single nucleotide polymorphisms strategically selected and genotyped by TaqMan technology. Although several novel variants were detected in both genes, none of these variants appeared to play a functional role in protein function or expression. Our results indicate that additional screening of other candidate genes in larger patient series is required to elucidate the molecular basis of INDB. Additionally, the systematic lack of positive results in the screening of candidate genes for INDB reported in the literature, together with our results, leads us to propose that INDB may alternatively arise as a consequence of gain of function mutations in genes related to enteric nervous system development. Therefore, the use of different molecular approaches, such as screening for genetic duplication or enhancer mutations, is recommended for future studies on the genetic basis of INDB.

New roles of EDNRB and EDN3 in the pathogenesis of Hirschsprung disease.

PURPOSE: Hirschsprung disease is characterized by the absence of intramural ganglion cells in the myenteric and submucosal plexuses within distal intestine, because of a fail in the enteric nervous system formations process. Endothelin-3-endothelin receptor B signaling pathway is known to play an essential role in this process. The aim of this study was to evaluate the implication of the EDN3 and EDNRB genes in a series of patients with Hirschsprung disease from Spain and determinate their mutational spectrum. METHODS: We performed the mutational screening of both genes in 196 patients with Hirschsprung disease using denaturing high-performance liquid chromatography technology. A case-control study using TaqMan Technology was also carried out to evaluate some common polymorphisms and haplotypes as susceptibility factors for Hirschsprung disease. RESULTS: Besides several novel mutations in both genes, we found a truncating mutation in an alternative isoform of EDNRB. Interestingly, we obtained an overrepresentation of a specific EDN3 haplotype in cases versus controls. CONCLUSIONS: Our results suggest that the isoform EDNRB Delta 3 might be playing an essential role in the formation of enteric nervous system. In addition, based on the haplotype distribution, EDN3 might be considered as a common susceptibility gene for sporadic Hirschsprung disease in a low-penetrance fashion.