Regional random mutagenesis driven by multiple sgRNAs and diverse on-target genome editing events to identify functionally important elements in non-coding regions.

Functional regions that regulate biological phenomena are interspersed throughout eukaryotic genomes. The most definitive approach for identifying such regions is to confirm the phenotype of cells or organisms in which specific regions have been mutated or removed from the genome. This approach is invaluable for the functional analysis of genes with a defined functional element, the protein-coding sequence. By contrast, no functional analysis platforms have been established for the study of cis-elements or microRNA cluster regions consisting of multiple microRNAs with functional overlap. Whole-genome mutagenesis approaches, such as via N-ethyl-N-nitrosourea and gene trapping, have greatly contributed to elucidating the function of coding genes. These methods almost never induce deletions of genomic regions or multiple mutations within a narrow region. In other words, cis-elements and microRNA clusters cannot be effectively targeted in such a manner. Herein, we established a novel region-specific random mutagenesis method named CRISPR- and transposase-based regional mutagenesis (CTRL-mutagenesis). We demonstrate that CTRL-mutagenesis randomly induces diverse mutations within target regions in murine embryonic stem cells. Comparative analysis of mutants harbouring subtly different mutations within the same region would facilitate the further study of cis-element and microRNA clusters.

Deletion of Exoc7, but not Exoc3, in male germ cells causes severe spermatogenesis failure with spermatocyte aggregation in mice.

Vesicular trafficking is essential for the transport of intracellularly produced functional molecules to the plasma membrane and extracellular space. The exocyst complex, composed of eight different proteins, is an important functional machinery for "tethering" in vesicular trafficking. Functional studies have been conducted in laboratory mice to identify the mechanisms by which the deletion of each exocyst factor affect various biological phenomena. Interestingly, each exocyst factor-deficient mutant exhibits a different phenotype. This discrepancy may be due to the function of the exocyst factor beyond its role as a component of the exocyst complex. Male germline-specific conditional knockout (cKO) mice of the Exoc1 gene, which encodes one of the exocyst factors EXOC1 (SEC3), exhibit severe spermatogenesis defects; however, whether this abnormality also occurs in mutants lacking other exocyst factors remains unknown. In this study, we found that exocyst factor EXOC3 (SEC6) was not required for spermatogenesis, but depletion of EXOC7 (EXO70) led to severe spermatogenesis defects. In addition to being a component of the exocyst complex, EXOC1 has other functions. Notably, male germ cell-specific Exoc7 cKO and Exoc1 cKO mice exhibited phenotypic similarities, suggesting the importance of the exocyst complex for spermatogenesis. The results of this study will contribute to further understanding of spermatogenesis from the aspect of vesicular trafficking.

Implication of amino acid metabolism and cell surface integrity for the thermotolerance mechanism in the thermally adapted acetic acid bacterium Acetobacter pasteurianus TH-3.

IMPORTANCE: Acetobacter pasteurianus, an industrial vinegar-producing strain, is suffered by fermentation stress such as fermentation heat and/or high concentrations of acetic acid. By an experimental evolution approach, we have obtained a stress-tolerant strain, exhibiting significantly increased growth and acetic acid fermentation ability at higher temperatures. In this study, we report that only the three gene mutations of ones accumulated during the adaptation process, ansP, dctD, and glnD, were sufficient to reproduce the increased thermotolerance of A. pasteurianus. These mutations resulted in cell envelope modification, including increased phospholipid and lipopolysaccharide synthesis, increased respiratory activity, and cell size reduction. The phenotypic changes may cooperatively work to make the adapted cell thermotolerant by enhancing cell surface integrity, nutrient or oxygen availability, and energy generation.

The autotaxin-LPA axis promotes membrane trafficking and secretion in yolk sac visceral endoderm cells.

Autotaxin, encoded by the Enpp2 gene, is an exoenzyme that produces lysophosphatidic acid, thereby regulating many biologic functions. We previously reported that Enpp2 mRNA was abundantly expressed in yolk sac visceral endoderm (VE) cells and that Enpp2-/- mice were lethal at embryonic day 9.5 owing to angiogenic defects in the yolk sac. Enpp2-/- mice showed lysosome fragmentation in VE cells and embryonic abnormalities including allantois malformation, neural tube defects, no axial turning, and head cavity formation. However, whether the defects in endocytic vesicle formation affect membrane trafficking in VE cells remained to be directly examined. In this study, we found that pinocytosis, transcytosis, and secretion of angiogenic factors such as vascular endothelial growth factor and transforming growth factor ß1 were impaired in Enpp2-/- VE cells. Moreover, pharmacologic inhibition of membrane trafficking phenocopied the defects of Enpp2-/- mice. These findings demonstrate that Enpp2 promotes endocytosis and secretion of angiogenic factors in VE cells, thereby regulating angiogenesis/vasculogenesis and embryonic development.

Arrhythmogenic right ventricular cardiomyopathy in a Japanese patient with a homozygous founder variant of DSG2 in the East Asian population.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a hereditary cardiomyopathy that results in fatal arrhythmias and heart failure. Herein, we report a Japanese patient with ARVC whose parents were blood relatives. Genetic testing identified a homozygous rare variant, c.1592T > G (p.Phe531Cys), of DSG2 that is presumed to be a founder variant among East Asians. Genetic counseling sessions with precise risk assessment and appropriate follow-up programs were provided to the patient and family members.

Zygote Microinjection for Creating Gene Cassette Knock-in and Flox Alleles in Mice.

CRISPR-Cas technology has enabled the rapid and effortless generation of genetically modified mice. Specifically, mice and point mutant mice are readily produced by electroporation of CRISPR factors (and single-stranded oligo DNA donors) into the zygote. In contrast, gene cassette (>1 kb) knock-in and floxed mice are mainly generated by microinjection of CRISPR factors and double-stranded DNA donors into zygotes. Genome editing technologies have also increased the flexibility of genetically modified mice production. It is now possible to introduce the intended mutations in the target genomic regions in a number of beneficial inbred mouse strains. Our team has produced over 200 gene cassette knock-in mouse lines, and over 110 floxed mouse lines by zygote microinjection of CRISPR-Cas9 following requests from several countries, including Japan. Some of these genome editing used BALB/c, C3H/HeJ, and C57BL/6N inbred strains, however most used C57BL/6J. Unlike the electroporation method, genome editing by zygote microinjection in various inbred strains of mice is not that easy. However, gene cassette knock-in and floxed mice on single inbred genetic backgrounds are as critical as genetic humanized, fluorescent reporter, and conditional knockout mouse models. Therefore, this article presents the protocol for the zygote microinjection of CRISPR factors and double-stranded DNA donors in C57BL/6J mice for generating gene cassette knock-in and floxed mice. This article exclusively focuses on nuclear injection rather than cytoplasmic injection. In addition to zygote microinjection, we outline the timeline for the production process and peripheral techniques such as induction of superovulation and embryo transfer.

DAJIN enables multiplex genotyping to simultaneously validate intended and unintended target genome editing outcomes.

Genome editing can introduce designed mutations into a target genomic site. Recent research has revealed that it can also induce various unintended events such as structural variations, small indels, and substitutions at, and in some cases, away from the target site. These rearrangements may result in confounding phenotypes in biomedical research samples and cause a concern in clinical or agricultural applications. However, current genotyping methods do not allow a comprehensive analysis of diverse mutations for phasing and mosaic variant detection. Here, we developed a genotyping method with an on-target site analysis software named Determine Allele mutations and Judge Intended genotype by Nanopore sequencer (DAJIN) that can automatically identify and classify both intended and unintended diverse mutations, including point mutations, deletions, inversions, and cis double knock-in at single-nucleotide resolution. Our approach with DAJIN can handle approximately 100 samples under different editing conditions in a single run. With its high versatility, scalability, and convenience, DAJIN-assisted multiplex genotyping may become a new standard for validating genome editing outcomes.

Efficient induction of proximity-dependent labelling by biotin feeding in BMAL1-BioID knock-in mice.

Proximity-dependent biotin identification (BioID) is a useful method to identify unknown protein-protein interactions. Few reports have described genetically engineered knock-in mouse models for in vivo BioID. Thus, little is known about the proper method for biotin administration and which tissues are applicable. Here, we established a BioID knock-in mouse model of Brain and Muscle ARNT-Like 1 (BMAL1) and the BirA biotin ligase with R118G mutation (BirA*). The BMAL1-BioID mouse model was used to investigate the effect of biotin diet feeding on protein biotinylation in several tissues. The BMAL1-BirA* fusion protein-retained proper intracellular localization of BMAL1 and binding to CLOCK protein in HEK293T cells. A biotin labelling assay in mouse embryonic fibroblasts revealed the protein biotinylation activity of BMAL1-BirA* expressed in knock-in mouse cells depending on biotin supplementation. Lastly, feeding a 0.5% biotin diet for 7 days induced protein biotinylation in the brain, heart, testis and liver of BMAL1-BioID mice without adverse effects on spermatogenesis. In the kidney, the biotin diet increased biotinylated protein levels in BMAL1-BioID and control mice, suggesting the existence of endogenous biotinylation activity. These results provide valuable information to optimize the in vivo BioID procedure.

EXOC1 plays an integral role in spermatogonia pseudopod elongation and spermatocyte stable syncytium formation in mice.

The male germ cells must adopt the correct morphology at each differentiation stage for proper spermatogenesis. The spermatogonia regulates its differentiation state by its own migration. The male germ cells differentiate and mature with the formation of syncytia, failure of forming the appropriate syncytia results in the arrest at the spermatocyte stage. However, the detailed molecular mechanisms of male germ cell morphological regulation are unknown. Here, we found that EXOC1, a member of the Exocyst complex, is important for the pseudopod formation of spermatogonia and spermatocyte syncytia in mice. EXOC1 contributes to the pseudopod formation of spermatogonia by inactivating the Rho family small GTPase Rac1 and also functions in the spermatocyte syncytia with the SNARE proteins STX2 and SNAP23. Since EXOC1 is known to bind to several cell morphogenesis factors, this study is expected to be the starting point for the discovery of many morphological regulators of male germ cells.

Disruption of entire Cables2 locus leads to embryonic lethality by diminished Rps21 gene expression and enhanced p53 pathway.

In vivo function of CDK5 and Abl enzyme substrate 2 (Cables2), belonging to the Cables protein family, is unknown. Here, we found that targeted disruption of the entire Cables2 locus (Cables2d) caused growth retardation and enhanced apoptosis at the gastrulation stage and then induced embryonic lethality in mice. Comparative transcriptome analysis revealed disruption of Cables2, 50% down-regulation of Rps21 abutting on the Cables2 locus, and up-regulation of p53-target genes in Cables2d gastrulas. We further revealed the lethality phenotype in Rps21-deleted mice and unexpectedly, the exon 1-deleted Cables2 mice survived. Interestingly, chimeric mice derived from Cables2d ESCs carrying exogenous Cables2 and tetraploid wild-type embryo overcame gastrulation. These results suggest that the diminished expression of Rps21 and the completed lack of Cables2 expression are intricately involved in the embryonic lethality via the p53 pathway. This study sheds light on the importance of Cables2 locus in mouse embryonic development.

Alcohol Consumption Reduction and Clinical Outcomes of Catheter Ablation for Atrial Fibrillation.

[Figure: see text].

Characterization of a bicistronic knock-in reporter mouse model for investigating the role of CABLES2 in vivo.

Two members of the CDK5 and ABL enzyme substrate (CABLES) family, CABLES1 and CABLES2, share a highly homologous C-terminus. They interact and associate with cyclin-dependent kinase 3 (CDK3), CDK5, and c-ABL. CABLES1 mediates tumor suppression, regulates cell proliferation, and prevents protein degradation. Although Cables2 is ubiquitously expressed in adult mouse tissues at RNA level, the role of CABLES2 in vivo remains unknown. Here, we generated bicistronic Cables2 knock-in reporter mice that expressed CABLES2 tagged with 3×FLAG and 2A-mediated fluorescent reporter tdTomato. Cables2-3×FLAG-2A-tdTomato (Cables2Tom) mice confirmed the expression of Cables2 in various mouse tissues. Interestingly, high intensity of tdTomato fluorescence was observed in the brain, testis and ovary, especially in the corpus luteum. Furthermore, immunoprecipitation analysis using the brain and testis in Cables2Tom/Tom revealed interaction of CABLES2 with CDK5. Collectively, our new Cables2 knock-in reporter model will enable the comprehensive analysis of in vivo CABLES2 function.

Efficient production of large deletion and gene fragment knock-in mice mediated by genome editing with Cas9-mouse Cdt1 in mouse zygotes.

Genetically modified mouse models are essential for in vivo investigation of gene function and human disease research. Targeted mutations can be introduced into mouse embryos using genome editing technology such as CRISPR-Cas. Although mice with small indel mutations can be produced, the production of mice carrying large deletions or gene fragment knock-in alleles remains inefficient. We introduced the nuclear localisation property of Cdt1 protein into the CRISPR-Cas system for efficient production of genetically engineered mice. Mouse Cdt1-connected Cas9 (Cas9-mC) was present in the nucleus of HEK293T cells and mouse embryos. Cas9-mC induced a bi-allelic full deletion of Dmd, GC-rich fragment knock-in, and floxed allele knock-in with high efficiency compared to standard Cas9. These results indicate that Cas9-mC is a useful tool for producing mouse models carrying targeted mutations.

Generation of B6-Ddx4em1(CreERT2)Utr , a novel CreERT2 knock-in line, for germ cell lineage by CRISPR/Cas9.

Germ cell development is essential for maintaining reproduction in animals. In postpubertal females, oogenesis is a highly complicated event for producing fertilizable oocytes. It starts when dormant primordial oocytes undergo activation to become growing oocytes. In postpubertal males, spermatogenesis is a differentiation process for producing sperm from spermatogonial stem cells. To obtain full understanding of the molecular mechanisms underlying germ cell development, the Cre/loxP system has been widely applied for conditional knock-out mouse studies. In this study, we established a novel knock-in mouse line, B6-Ddx4 em1(CreERT2)Utr , which expresses CreERT2 recombinase under the control of the endogenous DEAD-box helicase 4 (Ddx4) gene promoter. Ddx4 was specifically expressed in both female and male germ cell lineages. We mated the CreERT2 mice with R26GRR mice, expressing enhanced green fluorescent protein (EGFP) and tDsRed before and after Cre recombination. We found tDsRed signals in the testes and ovaries of tamoxifen-treated B6-Ddx4 em1(CreERT2)Utr ::R26GRR mice, but not in untreated mice. Immunostaining of their ovaries clearly showed that Cre recombination occurred in all oocytes at every follicle stage. We also found 100% Cre recombination efficiency in male germ cells via the progeny test. In summary, our results indicate that B6-Ddx4 em1(CreERT2)Utr is beneficial for studying female and male germ cell development.

Generation of bicistronic reporter knockin mice for visualizing germ layers.

Knockout mouse models are commonly used in developmental biology to investigate the functions of specific genes, and the knowledge obtained in such models has yielded insights into the molecular mechanisms underlying developmental processes. Gastrulation is the most dynamic process in embryogenesis during which differentiation into three germ layers occurs. However, the functions of genes involved in gastrulation are not completely understood. One major reason for this is the technical difficulty of embryo analysis to understand germ layer location. We have generated three reporter mouse strains in which the germ layers are distinguished by different fluorescent reporters. Using CRISPR/Cas9 genome editing in mouse zygotes, the fluorescent reporter genes, EGFP, tdTomato, and TagBFP including 2A peptide sequences were knocked into the appropriate sites before the stop codon of the Sox17 (endoderm marker), Otx2 (ectoderm marker), and T (mesoderm marker) genes, respectively. Founder mice were successfully generated in the Sox17-2A-EGFP, Otx2-2A-tdTomato, and T-2A-TagBFP knockin reporter strains. Further, homozygous knockin mice of all strains appeared morphologically normal and were fertile. On stereomicroscopic analysis, fluorescent signals were detected in a germ layer-specific manner from heterozygous embryos at embryonic day (E) 6.5-8.5 in all strains, and were immunohistochemically demonstrated to match their respective germ layer-specific marker protein at E7.5. Taken together, these observations suggest that the Sox17-2A-EGFP, Otx2-2A-tdTomato, and T-2A-TagBFP knockin reporter mice may be useful for comprehensive analysis of gene function in germ layer formation.

Incidence, Clinical Characteristics, and Long-term Outcome of the Dilated Phase of Hypertrophic Cardiomyopathy.

Some patients with hypertrophic cardiomyopathy (HCM) develop systolic dysfunction, called the dilated phase of HCM (d-HCM), which is associated with increased morbidity and mortality. We conducted a retrospective study using an HCM database to clarify the incidence, clinical characteristics, and long-term outcomes of d-HCM. We analyzed an HCM cohort consisting of 434 patients (273 with apical HCM and 161 with non-apical HCM; 18 had obstructive HCM, 16 had dilated HCM, and 127 had other HCM) diagnosed by echocardiography in our hospital between 1991 and 2010. The follow-up period was 8.4 ± 6.7 years. The mean age at final follow-up was 67 ± 14 years, and 304 patients (70%) were men. The mean age of the 16 d-HCM patients at the initial visit was 45 ± 17 years, the age at final follow-up was 59 ± 18 years, and 13 were men. Thirteen d-HCM patients developed atrial fibrillation and six patients developed ischemic stroke. Twelve d-HCM patients were implanted with cardiac devices: one pacemaker, nine implantable cardioverter-defibrillators, and two cardiac resynchronization therapy with defibrillator. Five patients died of progressive heart failure at the age of 61 ± 23 years. The age at the initial visit and final follow-up were lower and the NYHA class, brain natriuretic peptide levels, and left ventricular function at initial evaluation were worse in the d-HCM group. Univariate analysis demonstrated that a lower age at the initial visit was associated with d-HCM (hazard ratio 0.955/1 year increase; 95% CI 0.920-0.991, P = 0.015). In our HCM cohort, the incidence of d-HCM was 4%. A high prevalence of atrial fibrillation and cerebral infarction and poor prognosis were noted in this group, despite patients undergoing medication and device implantation.

Asymptomatic Cerebral Infarction During Catheter Ablation for Atrial Fibrillation: Comparing Uninterrupted Rivaroxaban and Warfarin (ASCERTAIN).

OBJECTIVES: This randomized study compared uninterrupted rivaroxaban therapy with warfarin therapy as prophylaxis against catheter ablation (CA)-induced asymptomatic cerebral infarction (ACI) and identified the risk factors of rivaroxaban. BACKGROUND: The reported incidence of ACI during CA for atrial fibrillation (AF) remains at 10% to 30%, and periprocedural oral anticoagulation could affect this incidence. METHODS: Patients with nonvalvular AF undergoing radiofrequency CA were randomly assigned to receive either uninterrupted rivaroxaban or warfarin as periprocedural anticoagulation therapy. CA was performed after at least 1 month of adequate anticoagulation. Cerebral magnetic resonance imaging (MRI) was performed within 2 weeks before and 1 day after CA to detect ACI. RESULTS: A total 132 patients were enrolled; 127 (median: 60.0 years of age; 83.5% males; 64.6% incidence of paroxysmal AF) complied with the study protocol and were analyzed; 64 patients received rivaroxaban, and 63 patients received warfarin. The rates of CA-induced ACI in the rivaroxaban group (15.6% [10 of 64 patients]) were similar to those in the warfarin group (15.9% [10 of 63 patients]; p = 1.000). No thromboembolic events developed; no differences in major or nonmajor bleeding rates were observed between the 2 drug groups (3.1% vs. 1.6%, respectively, or 18.8% vs. 19.0%, respectively). Multiple regression analysis indicated that the presence of deep and subcortical white matter hyperintensity (p = 0.002; odds ratio [OR]: 5.323) and the frequency of cardioversions (p = 0.016; OR: 1.250) were associated with the incidence of ACI. CONCLUSIONS: No notable differences were found between the incidence of CA-induced ACI in the rivaroxaban group and that in the warfarin group in this randomized study.

Important cardiac transcription factor genes are accompanied by bidirectional long non-coding RNAs.

BACKGROUND: Heart development is a relatively fragile process in which many transcription factor genes show dose-sensitive characteristics such as haploinsufficiency and lower penetrance. Despite efforts to unravel the genetic mechanism for overcoming the fragility under normal conditions, our understanding still remains in its infancy. Recent studies on the regulatory mechanisms governing gene expression in mammals have revealed that long non-coding RNAs (lncRNAs) are important modulators at the transcriptional and translational levels. Based on the hypothesis that lncRNAs also play important roles in mouse heart development, we attempted to comprehensively identify lncRNAs by comparing the embryonic and adult mouse heart and brain. RESULTS: We have identified spliced lncRNAs that are expressed during development and found that lncRNAs that are expressed in the heart but not in the brain are located close to genes that are important for heart development. Furthermore, we found that many important cardiac transcription factor genes are located in close proximity to lncRNAs. Importantly, many of the lncRNAs are divergently transcribed from the promoter of these genes. Since the lncRNA divergently transcribed from Tbx5 is highly evolutionarily conserved, we focused on and analyzed the transcript. We found that this lncRNA exhibits a different expression pattern than that of Tbx5, and knockdown of this lncRNA leads to embryonic lethality. CONCLUSION: These results suggest that spliced lncRNAs, particularly bidirectional lncRNAs, are essential regulators of mouse heart development, potentially through the regulation of neighboring transcription factor genes.

Simple generation of hairless mice for in vivo imaging.

The in vivo imaging of mice makes it possible to analyze disease progress non-invasively through reporter gene expression. As the removal of hair improves the accuracy of in vivo imaging, gene-modified mice with a reporter gene are often crossed with Hos:HR-1 mutant mice homozygous for the spontaneous Hrhr mutation that exhibit a hair loss phenotype. However, it is time consuming to produce mice carrying both the reporter gene and mutant Hrhr gene by mating. In addition, there is a risk that genetic background of the gene-modified mice would be altered by mating. To resolve these issues, we established a simple method to generate hairless mice maintaining the original genetic background by CRISPR technology. First, we constructed the pX330 vector, which targets exon 3 of Hr. This DNA vector (5 ng/µl) was microinjected into the pronuclei of C57BL/6J mice. Induced Hr gene mutations were found in many founders (76.1%) and these mutations were heritable. Next, we performed in vivo imaging using these gene-modified hairless mice. As expected, luminescent objects in their body were detected by in vivo imaging. This study clearly showed that hairless mice could be simply generated by the CRISPR/Cas9 system, and this method may be useful for in vivo imaging studies with various gene-modified mice.

A cost-utility analysis for catheter ablation of atrial fibrillation in combination with warfarin and dabigatran based on the CHADS2 score in Japan.

BACKGROUND: We aimed to clarify the cost-effectiveness of an expensive combination therapy for atrial fibrillation (AF) using both catheter ablation and dabigatran compared with warfarin at each CHADS2 score for patients in Japan. METHODS: A Markov model was constructed to analyze costs and quality-adjusted life years associated with AF therapeutic options with a time horizon of 10 years. The target population was 60-year-old patients with paroxysmal AF. The indication for anticoagulation was determined according to the Japanese guideline. Anticoagulation-related data were derived from the RE-LY study and the AF recurrence rate was set at 2.7% per month during the first 12 months and at 0.40% per month afterwards. Stroke risk was determined according to AF recurrence, anticoagulation, and CHADS2 score. The risks for stroke recurrence and stroke death were also considered. Costs were calculated from the healthcare payer's perspective, and only direct medical costs were included. RESULTS: Warfarin was the most preferred option for patients with a CHADS2 score of 0 from a health economics aspect. Ablation under warfarin was preferred for a CHADS2 score of 1-3, while ablation under dabigatran was preferred for a CHADS2 score ≥4. The quality of life score for AF had the largest impact on the incremental cost-effectiveness ratios in the analysis between the anticoagulation arm and the anticoagulation+ablation arm for a CHADS2 score of 2. Within the range of the Japanese willingness-to-pay threshold (¥5,000,000), the ablation+warfarin arm became the best option with its probability of 81.7% for a CHADS2 score of 2; the dabigatran+ablation arm was the most preferred option with its probability of 56.1% for a CHADS2 score of 4. CONCLUSIONS: Ablation under dabigatran therapy is an expensive therapeutic option, but it might benefit patients with a low quality of life and a high CHADS2 score.