Integration Locus Research Articles

Continuous integration of heading and goal directions guides steering.

Navigating animals must integrate a diverse array of sensory cues into a single locomotor decision. Insects perform intricate navigational feats using a brain region termed the central complex in which an animal's heading direction is transformed through several layers of circuitry to elicit goal-directed locomotion. These transformations occur mostly in the fan-shaped body (FB), a major locus of multi-sensory integration in the central complex. Key aspects of these sensorimotor computations have been extensively characterized by functional studies, leveraging the genetic tools available in the fruit fly. However, our understanding of how neuronal activity in the FB dictates locomotor behaviors during navigation remains enigmatic. Here, we manipulate the activity of two key neuronal populations that input into the FB-the PFN a and PFN d neurons-used to encode the direction of two complex navigational cues: wind plumes and optic flow, respectively. We find that flies presented with unidirectional optic flow steer along curved walking trajectories, but silencing PFN d neurons abolishes this curvature. We next use optogenetic activation to introduce a fictive heading signal in the PFNs to establish the causal relationship between their activity and steering behavior. Our studies reveal that the central complex guides locomotion by summing the PFN-borne directional signals and shifting movement trajectories left or right accordingly. Based on these results, we propose a model of central complex-mediated locomotion wherein the fly achieves fine-grained control of sensory-guided steering by continuously integrating its heading and goal directions over time.

Hepatitis B Virus-KMT2B Integration Drives Hepatic Oncogenic Processes in a Human Gene-edited Induced Pluripotent Stem Cells-derived Model

Background & AimsHepatitis B virus (HBV)-DNA integration into the host genome contributes to hepatocellular carcinoma (HCC) development. KMT2B is the second most frequent locus of HBV-DNA integration in HCC, however its role and function remain unclear. We aimed to clarify the impact of HBV-KMT2B integration in HCC development using a human genome-edited induced pluripotent stem cells (iPSCs) model. MethodsBased on the genetic information on HBV-KMT2B integration in HCC, we determined its complete DNA sequence and transcript variants. To exclude the effect of other oncogenic mutations, we reproduced HBV integration in healthy donor iPSCs with an intact genome and analyzed its effects using iPSC-derived hepatic progenitor cells (HPCs) and hepatocytes (iPS-Heps). ResultsThe reproduced HBV-KMT2B integration significantly upregulated the proliferation of hepatic cells. Comprehensive transcriptional and epigenetic analyses revealed enhanced expression of cell cycle-related genes in hepatic cells with HBV-KMT2B integration based on perturbation of histone 3 lysine 4 tri-methylation(H3K4me3), mimicking that in the original HCC sample. Long-read RNA-sequence detected the common KMT2B transcript variants in the HCC sample and HPCs. Overexpression of the truncated variant significantly enhanced proliferation of hepatic cells, whereas HBV-KMT2B fusion transcripts did not enhance proliferation. HBV-KMT2B-integrated HPCs exhibited replication stress and DNA damage, indicating that our model initiated the process of hepatocarcinogenesis due to abnormally promoted KMT2B function. ConclusionsOur disease model using genetically engineered iPSCs provides the first insight into both the KMT2B function in HCC development and the oncogenic processes by HBV-KMT2B integration. We clarified the novel oncogenic mechanism in HBV-related HCC due to aberrant KMT2B function.

Molecular and cytogenetic characterization of Osteospermum fruticosum lines harboring wild type pRi rol genes.

Transgenic lines engineered through wild type Rhizobium rhizogenes display an altered phenotype known as the Ri phenotype. This phenotype includes a more compact plant habit, which has proved useful to obtain more compact varieties that require less chemical growth regulation. Here, we develop a method for the molecular and cytogenetic characterization of Cape daisy (Osteospermum fruticosum Norl.) Ri lines in order to predict segregation of pRi T-DNA genes. Analysis of copy number variation (CNV) by means of digital PCR indicated large variation in the copy number of the inserted root oncogenic loci (rol) genes, ranging from 1 to more than 15 copies. In addition, up to 9 copies of the auxin biosynthesis genes (aux) were present in a single Ri line. Visualization of pRiA4 and pRi1724 rol and aux insertion in 4 Ri lines was performed through Fluorescence In Situ Hybridization. The number of rol integrated loci varied from 1 to 3 loci. In contrast, the different TR-gene copies were confined to a single locus which consistently co-localized with a TL locus, this was demonstrated for the first time. Based on CNV and FISH a single Ri line, harboring 7 pRi1724 rol gene copies dispersed over 3 integration loci, was selected for breeding. Copy number segregation in R1 progeny of 2, 3, 4 and 5 pRi1724 copies was confirmed, indicating that the evaluation of the breeding value of first generation Ri lines is possible through CNV and FISH.

Proximity ligation-based sequencing for the identification of human papillomavirus genomic integration sites in formalin-fixed paraffin embedded oropharyngeal squamous cell carcinomas.

Human papillomavirus (HPV) infections are an increasing cause of oropharyngeal squamous cell carcinomas (OPSCC). Integration of the viral genome into the host genome is suggested to affect carcinogenesis, however, the correlation with OPSCC patient prognosis is still unclear. Research on HPV integration is hampered by current integration detection technologies and their unsuitability for formalin-fixed paraffin-embedded (FFPE) tissues. This study aims to develop and validate a novel targeted proximity-ligation based sequencing method (targeted locus amplification/capture [TLA/TLC]) for HPV integration detection in cell lines and FFPE OPSCCs. For the identification of HPV integrations, TLA/TLC was applied to 7 cell lines and 27 FFPE OPSCCs. Following preprocessing steps, a polymerase chain reaction (PCR)-based HPV enrichment was performed on the cell lines and a capture-based HPV enrichment was performed on the FFPE tissues before paired-end sequencing. TLA was able to sequence up to hundreds of kb around the target, detecting exact HPV integration loci, structural variants, and chromosomal rearrangements. In all cell lines, one or more integration sites were identified, in accordance with detection of integrated papillomavirus sequences PCR data and the literature. TLC detected integrated HPV in 15/27 FFPE OPSCCs and identified simple and complex integration patterns. In general, TLA/TLC confirmed PCR data and detected additional integration sites. In conclusion TLA/TLC reliably and robustly detects HPV integration in cell lines and FFPE OPSCCs, enabling large, population-based studies on the clinical relevance of HPV integration. Furthermore, this approach might be valuable for clonality assessment of HPV-related tumors in clinical diagnostics.

Structured connectivity in the output of the cerebellar cortex

The spatial organization of a neuronal circuit is critically important for its function since the location of neurons is often associated with function. In the cerebellum, the major output of the cerebellar cortex are synapses made from Purkinje cells onto neurons in the cerebellar nuclei, yet little has been known about the spatial organization of these synapses. We explored this question using whole-cell electrophysiology and optogenetics in acute sagittal cerebellar slices to produce spatial connectivity maps of cerebellar cortical output in mice. We observed non-random connectivity where Purkinje cell inputs clustered in cerebellar transverse zones: while many nuclear neurons received inputs from a single zone, several multi-zonal connectivity motifs were also observed. Single neurons receiving input from all four zones were overrepresented in our data. These findings reveal that the output of the cerebellar cortex is spatially structured and represents a locus for multimodal integration in the cerebellum.

Host-induced RNA interference targeting the neuromotor gene FMRFamide-like peptide-14 (Mi-flp14) perturbs Meloidogyne incognita parasitic success in eggplant.

The study demonstrates the successful management of Meloidogyne incognita in eggplant using Mi-flp14 RNA interference, showing reduced nematode penetration and reproduction without off-target effects across multiple generations. Root-knot nematode, Meloidogyne incognita, causes huge yield losses worldwide. Neuromotor function in M. incognita governed by 19 neuropeptides is vital for parasitism and parasite biology. The present study establishes the utility of Mi-flp14 for managing M. incognita in eggplant in continuation of our earlier proof of concept in tobacco (US patent US2015/0361445A1). Mi-flp14 hairpin RNA construct was used for generating 19 independent transgenic eggplant events. PCR and Southern hybridization analysis confirmed transgene integration and its orientation, while RT-qPCR and Northern hybridization established the generation of dsRNA and siRNA of Mi-flp14. In vitro and in vivo bio-efficacy analysis of single-copy events against M. incognita showed reduced nematode penetration and development at various intervals that negatively impacted reproduction. Interestingly, M. incognita preferred wild-type plants over the transgenics even when unbiased equal opportunity was provided for the infection. A significant reduction in disease parameters was observed in transgenic plants viz., galls (40-48%), females (40-50%), egg masses (35-40%), eggs/egg mass (50-55%), and derived multiplication factor (60-65%) compared to wild type. A unique demonstration of perturbed expression of Mi-flp14 in partially penetrated juveniles and female nematodes established successful host-mediated RNAi both at the time of penetration even before the nematodes started withdrawing plant nutrients and later stage, respectively. The absence of off-target effects in transgenic plants was supported by the normal growth phenotype of the plants and T-DNA integration loci. Stability in the bio-efficacy against M. incognita across T1- to T4-generation transgenic plants established the utility of silencing Mi-flp14 for nematode management. This study demonstrates the significance of targeting Mi-flp14 in eggplant for nematode management, particularly to address global agricultural challenges posed by M. incognita.

Scalable protein production by Komagataella phaffii enabled by ARS plasmids and carbon source-based selection

BackgroundMost recombinant Komagataella phaffii (Pichia pastoris) strains for protein production are generated by genomic integration of expression cassettes. The clonal variability in gene copy numbers, integration loci and consequently product titers limit the aptitude for high throughput applications in drug discovery, enzyme engineering or most comparative analyses of genetic elements such as promoters or secretion signals. Circular episomal plasmids with an autonomously replicating sequence (ARS), an alternative which would alleviate some of these limitations, are inherently unstable in K. phaffii. Permanent selection pressure, mostly enabled by antibiotic resistance or auxotrophy markers, is crucial for plasmid maintenance and hardly scalable for production. The establishment and use of extrachromosomal ARS plasmids with key genes of the glycerol metabolism (glycerol kinase 1, GUT1, and triosephosphate isomerase 1, TPI1) as selection markers was investigated to obtain a system with high transformation rates that can be directly used for scalable production processes in lab scale bioreactors.ResultsIn micro-scale deep-well plate experiments, ARS plasmids employing the Ashbya gossypii TEF1 (transcription elongation factor 1) promoter to regulate transcription of the marker gene were found to deliver high transformation efficiencies and the best performances with the reporter protein (CalB, lipase B of Candida antarctica) for both, the GUT1- and TPI1-based, marker systems. The GUT1 marker-bearing strain surpassed the reference strain with integrated expression cassette by 46% upon re-evaluation in shake flask cultures regarding CalB production, while the TPI1 system was slightly less productive compared to the control. In 5 L bioreactor methanol-free fed-batch cultivations, the episomal production system employing the GUT1 marker led to 100% increased CalB activity in the culture supernatant compared to integration construct.ConclusionsFor the first time, a scalable and methanol-independent expression system for recombinant protein production for K. phaffii using episomal expression vectors was demonstrated. Expression of the GUT1 selection marker gene of the new ARS plasmids was refined by employing the TEF1 promoter of A. gossypii. Additionally, the antibiotic-free marker toolbox for K. phaffii was expanded by the TPI1 marker system, which proved to be similarly suited for the use in episomal plasmids as well as integrative expression constructs for the purpose of recombinant protein production.

Identification and functional characterisation of a locus for target site integration in Fusariumgraminearum

BackgroundFusarium Head Blight (FHB) is a destructive floral disease of different cereal crops. The Ascomycete fungus Fusariumgraminearum (Fg) is one of the main causal agents of FHB in wheat and barley. The role(s) in virulence of Fg genes include genetic studies that involve the transformation of the fungus with different expression cassettes. We have observed in several studies where Fg genes functions were characterised that integration of expression cassettes occurred randomly. Random insertion of a cassette may disrupt gene expression and/or protein functions and hence the overall conclusion of the study. Target site integration (TSI) is an approach that consists of identifying a chromosomal region where the cassette can be inserted. The identification of a suitable locus for TSI in Fg would avert the potential risks of ectopic integration.ResultsHere, we identified a highly conserved intergenic region on chromosome 1 suitable for TSI. We named this intergenic region TSI locus 1. We developed an efficient cloning vector system based on the Golden Gate method to clone different expression cassettes for use in combination with TSI locus 1. We present evidence that integrations in the TSI locus 1 affects neither fungal virulence nor fungal growth under different stress conditions. Integrations at the TSI locus 1 resulted in the expression of different gene fusions. In addition, the activities of Fg native promoters were not altered by integration into the TSI locus 1. We have developed a bespoke bioinformatic pipeline to analyse the existence of ectopic integrations, cassette truncations and tandem insertions of the cassette that may occurred during the transformation process. Finally, we established a protocol to study protein secretion in wheat coleoptiles using confocal microscopy and the TSI locus 1.ConclusionThe TSI locus 1 can be used in Fg and potentially other cereal infecting Fusarium species for diverse studies including promoter activity analysis, protein secretion, protein localisation studies and gene complementation. The bespoke bioinformatic pipeline developed in this work together with PCR amplification of the insert could be an alternative to Southern blotting, the gold standard technique used to identify ectopic integrations, cassette truncations and tandem insertions in fungal transformation.

Analysis of the transcriptional activity of model piggyBac transgenes stably integrated into different loci of the genome of CHO cells in the absence of selection pressure

CHO cells are most commonly used for the synthesis of recombinant proteins in biopharmaceutical production. When stable producer cell lines are obtained, the locus of transgene integration into the genome has a great influence on the level of its expression. Therefore, the identification of genomic loci ensuring a high level of protein production is very important. Here, we used the TRIP assay to study the influence of the local chromatin environment on the activity of transgenes in CHO cells. For this purpose, reporter constructs encoding eGFP under the control of four promoters were stably integrated into the genome of CHO cells using the piggyBac transposon. Each individual transgene contained a unique tag, a DNA barcode, and the resulting polyclonal cell population was cultured for almost a month without any selection. Next, using the high-throughput sequencing, genomic localizations of barcodes, as well as their abundances in the population and transcriptional activities were identified. In total, ~640 transgenes more or less evenly distributed across all chromosomes of CHO cells were characterized. More than half of the transgenes were completely silent. The most active transgenes were identified to be inserted in gene promoters and 5’ UTRs. Transgenes carrying Chinese hamster full-length promoter of the EF-1α gene showed the highest activity. Transgenes with a truncated version of the same promoter and with the mouse PGK gene promoter were on average 10 and 19 times less active, respectively. In total, combinations of genomic loci of CHO cells and transgene promoters that together provide different levels of transcriptional activity of the model reporter construct were described.

Distribution, inducibility, and characteristics of Latilactobacillus curvatus temperate phages.

Aim: Temperate phages are known to heavily impact the growth of their host, be it in a positive way, e.g., when beneficial genes are provided by the phage, or negatively when lysis occurs after prophage induction. This study provides an in-depth look into the distribution and variety of prophages in Latilactobacillus curvatus (L. curvatus). This species is found in a wide variety of ecological niches and is routinely used as a meat starter culture. Methods: Fourty five L. curvatus genomes were screened for prophages. The intact predicted prophages and their chromosomal integration loci were described. Six L. curvatus lysogens were analysed for phage-mediated lysis post induction via UV light and/or mitomycin C. Their lysates were analysed for phage particles via viral DNA sequencing and transmission electron microscopy. Results: Two hundred and six prophage sequences of any completeness were detected within L. curvatus genomes. The 50 as intact predicted prophages show high levels of genetic diversity on an intraspecies level with conserved regions mostly in the replication and head/tail gene clusters. Twelve chromosomal loci, mostly tRNA genes, were identified, where intact L. curvatus phages were integrated. The six analysed L. curvatus lysogens showed strain-dependent lysis in various degrees after induction, yet only four of their lysates appeared to contain fully assembled virions with the siphovirus morphotype. Conclusion: Our data demonstrate that L. curvatus is a (pro)phage-susceptible species, harbouring multiple intact prophages and remnant sequences thereof. This knowledge provides a basis to study phage-host interaction influencing microbial communities in food fermentations.

Multiomics analyses reveal pathological mechanisms of HBV infection and integration in liver cancer.

Hepatitis B virus (HBV) infection and integration are important for hepatocellular carcinoma (HCC) initiation and progression, while disease mechanisms are still largely elusive. Here, we combined bulk and single-cell sequencing technologies to tackle the disease mechanisms of HBV-related HCC. We observed high HBV mutation rate and diversity only in tumors without HBV integration. We identified human somatic risk loci for HBV integration (VIMs). Transcription factors (TFs) enriched in VIMs were involved in DNA repair and androgen receptor (AR) signaling. Aberration of AR signaling was further observed by single-cell regulon analysis in HBV-infected hepatocytes, which showed remarkable interactions between AR and the complement system that, together with the X-linked ZXDB regulon that contains albumin (ALB), probably contribute to HCC male predominance. Complement system dysregulation caused by HBV infection was further confirmed by analyses of single-cell copy numbers and cell-cell communications. Finally, HBV infection-associated immune cells presented critical defects, including TXNIP in T cells, TYROBP in NK cells, and the X-linked TIMP1 in monocytes. We further experimentally validated our findings in multiple independent patient cohorts. Collectively, our work shed light on the pathogenesis of HBV-related HCC and other liver diseases that affect billions of people worldwide.

Rethinking Brain Death-Why "Dead Enough" Is Not Good Enough: The UDDA Revision Series.

The emergence of cases of so-called "chronic brain death" seems to undermine the biophilosophical justification of brain death as true death, which was grounded in the idea that death entails the loss of integration of the organism. Severely neurologically damaged patients who can persist for years with proper support seem to be integrated organisms, and common sense suggests that they are not dead. We argue, however, that mere integration is not enough for an organism to be alive, but that living beings must be substantially self-integrating (i.e., a living organism must itself be the primary source of its integration and not an external agent such as a scientist or physician). We propose that irreversible apnea and unresponsiveness are necessary but not sufficient to judge that a human being has lost enough capacity for self-integration to be considered dead. To be declared dead, the patient must also irrevocably have lost either (1) cardiac function or (2) cerebrosomatic homeostatic control. Even if such bodies can be maintained with sufficient technological support, one may reasonably judge that the locus of integration effectively has passed from the patient to the treatment team. While organs and cells may be alive, one may justifiably declare that there is no longer a substantially autonomous, whole, living human organism. This biophilosophical conception of death implies that the notion of brain death remains viable, but that additional testing will be required to ensure that the individual is truly brain dead by virtue of having irrevocably lost not only the capacity for spontaneous respiration and conscious responsiveness but also the capacity for cerebrosomatic homeostatic control.

Differential Modulation of GABAergic and Glutamatergic Neurons in the Ventral Pallidum by GABA and Neuropeptides.

The ventral pallidum (VP) is an integral locus in the reward circuitry and a major target of GABAergic innervation of both D1-medium spiny neurons (MSNs) and D2-MSNs from the nucleus accumbens. The VP contains populations of GABAergic [VPGABA, GAD2(+), or VGluT(-)] and glutamatergic [VPGlutamate, GAD2(-), or VGluT(+)] cells that facilitate positive reinforcement and behavioral avoidance, respectively. MSN efferents to the VP exert opponent control over behavioral reinforcement with activation of D1-MSN afferents promoting and D2-MSN afferents inhibiting reward seeking. How this afferent-specific and cell type-specific control of reward seeking is integrated remains largely unknown. In addition to GABA, D1-MSNs corelease substance P to stimulate neurokinin 1 receptors (NK1Rs) and D2-MSNs corelease enkephalin to activate μ-opioid receptors (MORs) and δ-opioid receptors. These neuropeptides act in the VP to alter appetitive behavior and reward seeking. Using a combination of optogenetics and patch-clamp electrophysiology in mice, we found that GAD2(-) cells receive weaker GABA input from D1-MSN, but GAD2(+) cells receive comparable GABAergic input from both afferent types. Pharmacological activation of MORs induced an equally strong presynaptic inhibition of GABA and glutamate transmission on both cell types. Interestingly, MOR activation hyperpolarized VPGABA but not VGluT(+). NK1R activation inhibited glutamatergic transmission only on VGluT(+) cells. Our results indicate that the afferent-specific release of GABA and neuropeptides from D1-MSNs and D2-MSNs can differentially influence VP neuronal subtypes.

A Programmable CRISPR/Cas9 Toolkit Improves Lycopene Production in Bacillus subtilis.

Bacillus subtilis has been widely used and generally recognized as a safe host for the production of recombinant proteins, high-value chemicals, and pharmaceuticals. Thus, its metabolic engineering attracts significant attention. Nevertheless, the limited availability of selective markers makes this process difficult and time-consuming, especially in the case of multistep biosynthetic pathways. Here, we employ CRISPR/Cas9 technology to build an easy cloning toolkit that addresses commonly encountered obstacles in the metabolic engineering of B. subtilis, including the chromosomal integration locus, promoter, terminator, and guide RNA (gRNA) target. Six promoters were characterized, and the promoter strengths ranged from 0.9- to 23-fold that of the commonly used strong promoter P43. We characterized seven terminators in B. subtilis, and the termination efficiencies (TEs) of the seven terminators are all more than 90%. Six gRNA targets were designed upstream of the promoter and downstream of the terminator. Using a green fluorescent protein (GFP) reporter, we confirmed integration efficiency with the single-locus integration site is up to 100%. We demonstrated the applicability of this toolkit by optimizing the expression of a challenging but industrially important product, lycopene. By heterologous expression of the essential genes for lycopene synthesis on the B. subtilis genome, a total of 13 key genes involved in the lycopene biosynthetic pathway were manipulated. Moreover, our findings showed that the gene cluster ispG-idi-dxs-ispD could positively affect the production of lycopene, while the cluster dxr-ispE-ispF-ispH had a negative effect on lycopene production. Hence, our multilocus integration strategy can facilitate the pathway assembly for production of complex chemicals and pharmaceuticals in B. subtilis. IMPORTANCE We present a toolkit that allows for rapid cloning procedures and one-step subcloning to move from plasmid-based expression to stable chromosome integration and expression in a production strain in less than a week. The utility of the customized tool was demonstrated by integrating the MEP (2C-methyl-d-erythritol-4-phosphate) pathway, part of the pentose phosphate pathway (PPP), and the hetero-lycopene biosynthesis genes by stable expression in the genome. The tool could be useful to engineer B. subtilis strains through diverse recombination events and ultimately improve its potential and scope of industrial application as biological chassis.

Integration site-dependent HIV-1 promoter activity shapes host chromatin conformation.

HIV-1 integration introduces ectopic transcription factor binding sites into host chromatin. We postulate that the integrated provirus serves as an ectopic enhancer that recruits additional transcription factors to the integration locus, increases chromatin accessibility, changes 3D chromatin interactions, and enhances both retroviral and host gene expression. We used four well-characterized HIV-1-infected cell line clones having unique integration sites and low to high levels of HIV-1 expression. Using single-cell DOGMA-seq, which captured the heterogeneity of HIV-1 expression and host chromatin accessibility, we found that HIV-1 transcription correlated with HIV-1 accessibility and host chromatin accessibility. HIV-1 integration increased local host chromatin accessibility within an ∼5- to 30-kb distance. CRISPRa- and CRISPRi-mediated HIV-1 promoter activation and inhibition confirmed integration site-dependent HIV-1-driven changes of host chromatin accessibility. HIV-1 did not drive chromatin confirmation changes at the genomic level (by Hi-C) or the enhancer connectome (by H3K27ac HiChIP). Using 4C-seq to interrogate HIV-1-chromatin interactions, we found that HIV-1 interacted with host chromatin ∼100-300 kb from the integration site. By identifying chromatin regions having both increased transcription factor activity (by ATAC-seq) and HIV-1-chromatin interaction (by 4C-seq), we identified enrichment of ETS, RUNT, and ZNF-family transcription factor binding that may mediate HIV-1-host chromatin interactions. Our study has found that HIV-1 promoter activity increases host chromatin accessibility, and HIV-1 interacted with host chromatin within the existing chromatin boundaries in an integration site-dependent manner.

Rapid and precise genotyping of transgene zygosity in mice using an allele-specific method.

Precise determination of transgene zygosity is essential for use of transgenic mice in research. Because integration loci of transgenes are usually unknown due to their random insertion, assessment of transgene zygosity remains a challenge. Current zygosity genotyping methods (progeny testing, qPCR, and NGS-computational biology analysis) are time consuming, prone to error or technically challenging. Here, we developed a novel method to determine transgene zygosity requiring no knowledge of transgene insertion loci. This method applies allele-specific restriction enzyme digestion of PCR products (RE/PCR) to rapidly and reliably quantify transgene zygosity. We demonstrate the applicability of this method to three transgenic strains of mice (Atm TgC3001L, Nes-Cre, and Syn1-Cre) harboring a unique restriction enzyme site on either the transgene or its homologous sequence in the mouse genome. This method is as accurate as the gold standard of progeny testing but requires 2 d instead of a month or more. It is also exceedingly more accurate than the most commonly used approach of qPCR quantification. Our novel method represents a significant technical advance in determining transgene zygosities in mice.

An Insula-Enriched Regulator of Retinoic Acid Marks a New Intersection in the Neural Circuitry of Mouse Social Behavior

An Insula-Enriched Regulator of Retinoic Acid Marks a New Intersection in the Neural Circuitry of Mouse Social Behavior

CRISPR-Cas9 Long-Read Sequencing for Mapping Transgenes in the Mouse Genome.

Microinjected transgenes, both large and small, are known to insert randomly into the mouse genome. Traditional methods of mapping a transgene are challenging, thus complicating breeding strategies and accurate interpretation of phenotypes, particularly when a transgene disrupts critical coding or noncoding sequences. As the vast majority of transgenic mouse lines remain unmapped, we developed CRISPR-Cas9 Long-Read Sequencing (CRISPR-LRS) to ascertain transgene integration loci. This novel approach mapped a wide size range of transgenes and uncovered more complex transgene-induced host genome re-arrangements than previously appreciated. CRISPR-LRS offers a facile, informative approach to establish robust breeding practices and will enable researchers to study a gene without confounding genetic issues. Finally, CRISPR-LRS will find utility in rapidly and accurately interrogating gene/genome editing fidelity in experimental and clinical settings.

Deciphering integration loci of CHO manufacturing cell lines using long read nanopore sequencing

Despite advances in genetic characterization of Chinese hamster ovary (CHO) cell lines regarding identification of integration sites using next generation sequencing, e.g. targeted locus amplification sequencing (TLA-seq), the concatemer structure of the integrated vectors remains elusive. Here, the entire integration locus of two CHO manufacturing cell lines was reconstructed combining CRISPR/Cas9 target enrichment, nanopore sequencing and the Canu de novo assembly pipeline. An IgG producing CHO cell line integrated 3 vector copies, which were near full-length and contained all relevant vector elements such as transgenes and their promoters on each of the vector copies. In contrast, a second CHO cell line producing a bivalent bispecific antibody integrated 7 highly fragmented vector copies in different orientations leading to head-to-head and tail-to-tail fusions. The size of the vector fragments ranged from 3.0 to 11.4 kbp each carrying 1–3 transgenes. The breakpoints of the genome-vector and vector-vector junctions were validated using Sanger sequencing and Southern blotting. A comparison to TLA-seq data confirmed the genomic breakpoints, but most of the breakpoints of the vector-vector fusions were missed by TLA-seq. For the first time, the complete transgene locus of CHO manufacturing cell lines could be deciphered. Strikingly, the application of the nanopore long-read sequencing technology led to novel insights into the complexity of genomic transgene integrations of CHO manufacturing cell lines generated via random integration.

Combining manipulation of integration loci and secretory pathway on expression of an Aspergillus niger glucose oxidase gene in Trichoderma reesei

Trichoderma reesei (T. reesei) is well-known for its excellent ability to secret a large quantity of cellulase. However, unlike the endogenous proteins, little is known about the molecular mechanisms governing heterologous protein production. Herein, we focused on the integration loci and the secretory pathway, and investigated their combinatorial effects on heterologous gene expression in T. reesei using a glucose oxidase from Aspergillus niger as a model protein. Integration in the cel3c locus was more efficient than the cbh1 locus in expressing the AnGOx by increasing the transcription of AnGOx in the early stage. In addition, we discovered that interruption of the cel3c locus has an additional effect by increasing the expression of the secretory pathway component genes. Accordingly, overexpressing three secretory pathway component genes, that were snc1, sso2, and rho3, increased AnGOx expression in the cbh1 transformant but not in the cel3c transformant.