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SpCas9 and AsCas12a are widely utilized as genome-editing tools in human cells. However, their relatively large size poses a limitation for delivery by cargo-size-limited adeno-associated virus (AAV) vectors. The type V-F Cas12f from Acidibacillus sulfuroxidans is exceptionally compact (422 amino acids) and has been harnessed as a compact genome-editing tool. Here, we developed an approach, combining deep mutational scanning and structure-informed design, to successfully generate two AsCas12f activity-enhanced (enAsCas12f) variants. Remarkably, the enAsCas12f variants exhibited genome-editing activities in human cells comparable with those of SpCas9 and AsCas12a. The cryoelectron microscopy (cryo-EM) structures revealed that the mutations stabilize the dimer formation and reinforce interactions with nucleic acids to enhance their DNA cleavage activities. Moreover, enAsCas12f packaged with partner genes in an all-in-one AAV vector exhibited efficient knock-in/knock-out activities and transcriptional activation in mice. Taken together, enAsCas12f variants could offer a minimal genome-editing platform for in vivo gene therapy.
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Sistemas CRISPR-Cas , Edição de Genes , Animais , Humanos , Camundongos , Microscopia Crioeletrônica , Mutação , Terapia GenéticaRESUMO
Autophagy degrades unnecessary proteins or damaged organelles to maintain cellular function. Therefore, autophagy has a preventive role against various diseases including hepatic disorders, neurodegenerative diseases, and cancer. Although autophagy in germ cells or Sertoli cells is known to be required for spermatogenesis and male fertility, it remains poorly understood how autophagy participates in spermatogenesis. We found that systemic knockout mice of Rubicon, a negative regulator of autophagy, exhibited a substantial reduction in testicular weight, spermatogenesis, and male fertility, associated with upregulation of autophagy. Rubicon-null mice also had lower levels of mRNAs of Sertoli cell-related genes in testis. Importantly, Rubicon knockout in Sertoli cells, but not in germ cells, caused a defect in spermatogenesis and germline stem cell maintenance in mice, indicating a critical role of Rubicon in Sertoli cells. In mechanistic terms, genetic loss of Rubicon promoted autophagic degradation of GATA4, a transcription factor that is essential for Sertoli cell function. Furthermore, androgen antagonists caused a significant decrease in the levels of Rubicon and GATA4 in testis, accompanied by elevated autophagy. Collectively, we propose that Rubicon promotes Sertoli cell function by preventing autophagic degradation of GATA4, and that this mechanism could be regulated by androgens.
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Fator de Transcrição GATA4/metabolismo , Técnicas de Inativação de Genes/métodos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Células de Sertoli/fisiologia , Animais , Autofagia , Linhagem Celular , Fertilidade , Humanos , Masculino , Camundongos , Proteólise , Células de Sertoli/citologia , Análise de Célula Única , Espermatogênese , Testículo/crescimento & desenvolvimento , Testículo/metabolismoRESUMO
Periodically repeating DNA and protein elements are involved in various important biological events including genomic evolution, gene regulation, protein complex formation, and immunity. Notably, the currently used genome editing tools such as ZFNs, TALENs, and CRISPRs are also all associated with periodically repeating biomolecules of natural organisms. Despite the biological importance of periodically repeating sequences and the expectation that new genome editing modules could be discovered from such periodical repeats, no software that globally detects such structured elements in large genomic resources in a high-throughput and unsupervised manner has been developed. We developed new software, SPADE (Search for Patterned DNA Elements), that exhaustively explores periodic DNA and protein repeats from large-scale genomic datasets based on k-mer periodicity evaluation. With a simple constraint, sequence periodicity, SPADE captured reported genome-editing-associated sequences and other protein families involving repeating domains such as tetratricopeptide, ankyrin and WD40 repeats with better performance than the other software designed for limited sets of repetitive biomolecular sequences, suggesting the high potential of this software to contribute to the discovery of new biological events and new genome editing modules.
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DNA/química , Genômica/métodos , Sequências Repetitivas de Aminoácidos , Sequências Repetitivas de Ácido Nucleico , Software , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Humanos , Efetores Semelhantes a Ativadores de Transcrição/química , Nucleases de Dedos de Zinco/químicaRESUMO
The RNA-guided DNA endonuclease Cas9 cleaves double-stranded DNA targets complementary to an RNA guide, and is widely used as a powerful genome-editing tool. Here, we report the crystal structure of Brevibacillus laterosporus Cas9 (BlCas9, also known as BlatCas9), in complex with a guide RNA and its target DNA at 2.4-Å resolution. The structure reveals that the BlCas9 guide RNA adopts an unexpected architecture containing a triple-helix, which is specifically recognized by BlCas9, and that BlCas9 recognizes a unique N4CNDN protospacer adjacent motif through base-specific interactions on both the target and non-target DNA strands. Based on the structure, we rationally engineered a BlCas9 variant that exhibits enhanced genome- and base-editing activities with an expanded target scope in human cells. This approach may further improve the performance of the enhanced BlCas9 variant to generate useful genome-editing tools that require only a single C PAM nucleotide and can be packaged into a single AAV vector for in vivo gene therapy.
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Brevibacillus , Proteína 9 Associada à CRISPR , Edição de Genes , Brevibacillus/genética , Brevibacillus/metabolismo , Brevibacillus/enzimologia , Edição de Genes/métodos , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Proteína 9 Associada à CRISPR/química , RNA Guia de Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas/metabolismo , Humanos , Sistemas CRISPR-Cas , Engenharia de Proteínas/métodosRESUMO
Male fertility decreases with aging, with spermatogenic decline being one of its causes. Altered testis environment is suggested as a cause of the phenotype; however, the associated mechanisms remain unclear. Herein, we investigated the age-related changes in testicular somatic cells on spermatogenic activity. The number and proliferation of spermatogonia significantly reduced with aging in mice. Interestingly, senescence-associated ß-galactosidase-positive cells appeared in testicular endothelial cell (EC) populations, but not in germ cell populations, with aging. Transcriptome analysis of ECs indicated that senescence occurred in the ECs of aged mice. Furthermore, the support capacity of ECs for spermatogonial proliferation significantly decreased with aging; however, the senolytic-induced removal of senescent cells from aged ECs restored their supporting capacity to a comparable level as that of young ECs. Our results suggest that the accumulation of senescent ECs in the testis is a potential factor contributing to the age-related decline in spermatogenic activity.
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This work introduces a perspective-corrected video see-through mixed-reality head-mounted display with edge-preserving occlusion and low-latency capabilities. To realize the consistent spatial and temporal composition of a captured real world containing virtual objects, we perform three essential tasks: 1) to reconstruct captured images so as to match the user's view; 2) to occlude virtual objects with nearer real objects, to provide users with correct depth cues; and 3) to reproject the virtual and captured scenes to be matched and to keep up with users' head motions. Captured image reconstruction and occlusion-mask generation require dense and accurate depth maps. However, estimating these maps is computationally difficult, which results in longer latencies. To obtain an acceptable balance between spatial consistency and low latency, we rapidly generated depth maps by focusing on edge smoothness and disocclusion (instead of fully accurate maps), to shorten the processing time. Our algorithm refines edges via a hybrid method involving infrared masks and color-guided filters, and it fills disocclusions using temporally cached depth maps. Our system combines these algorithms in a two-phase temporal warping architecture based upon synchronized camera pairs and displays. The first phase of warping is to reduce registration errors between the virtual and captured scenes. The second is to present virtual and captured scenes that correspond with the user's head motion. We implemented these methods on our wearable prototype and performed end-to-end measurements of its accuracy and latency. We achieved an acceptable latency due to head motion (less than 4 ms) and spatial accuracy (less than 0.1° in size and less than 0.3° in position) in our test environment. We anticipate that this work will help improve the realism of mixed reality systems.
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DNA constructs and their annotated sequence maps have been rapidly accumulating with the advancement of DNA cloning, synthesis, and assembly methods. Such resources have also been utilized in designing and building new DNA materials. However, as commonly seen in the life sciences, no framework exists to describe reproducible DNA construction processes. Furthermore, the use of previously developed DNA materials and building protocols is usually not appropriately credited. Here, we report a framework QUEEN (framework to generate quinable and efficiently editable nucleotide sequence resources) to resolve these issues and accelerate the building of DNA. QUEEN enables the flexible design of new DNA by using existing DNA material resource files and recording its construction process in an output file (GenBank file format). A GenBank file generated by QUEEN can regenerate the process code such that it perfectly clones itself and bequeaths the same process code to its successive GenBank files, recycling its partial DNA resources. QUEEN-generated GenBank files are compatible with existing DNA repository services and software. We propose QUEEN as a solution to start significantly advancing the material and protocol sharing of DNA resources.
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DNA , Software , DNA/genética , Bases de Dados de Ácidos NucleicosRESUMO
The RNA-guided DNA endonuclease Cas9 is a versatile genome-editing tool. However, the molecular weight of the commonly used Streptococcus pyogenes Cas9 is relatively large. Consequently, its gene cannot be efficiently packaged into an adeno-associated virus vector, thereby limiting its applications for therapeutic genome editing. Here, we biochemically characterized the compact Cas9 from Campylobacter jejuni (CjCas9) and found that CjCas9 has a previously unrecognized preference for the N3VRYAC protospacer adjacent motif. We thus rationally engineered a CjCas9 variant (enCjCas9), which exhibits enhanced cleavage activity and a broader targeting range both in vitro and in human cells, as compared with CjCas9. Furthermore, a nickase version of enCjCas9, but not CjCas9, fused with a cytosine deaminase mediated C-to-T conversions in human cells. Overall, our findings expand the CRISPR-Cas toolbox for therapeutic genome engineering.
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Sistemas CRISPR-Cas , Campylobacter jejuni , Campylobacter jejuni/genética , Edição de Genes , HumanosRESUMO
Phylogeny estimation (the reconstruction of evolutionary trees) has recently been applied to CRISPR-based cell lineage tracing, allowing the developmental history of an individual tissue or organism to be inferred from a large number of mutated sequences in somatic cells. However, current computational methods are not able to construct phylogenetic trees from extremely large numbers of input sequences. Here, we present a deep distributed computing framework to comprehensively trace accurate large lineages (FRACTAL) that substantially enhances the scalability of current lineage estimation software tools. FRACTAL first reconstructs only an upstream lineage of the input sequences and recursively iterates the same produce for its downstream lineages using independent computing nodes. We demonstrate the utility of FRACTAL by reconstructing lineages from >235 million simulated sequences and from >16 million cells from a simulated experiment with a CRISPR system that accumulates mutations during cell proliferation. We also successfully applied FRACTAL to evolutionary tree reconstructions and to an experiment using error-prone PCR (EP-PCR) for large-scale sequence diversification.
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Algoritmos , Software , Linhagem da Célula/genética , Mutação , FilogeniaRESUMO
Cell behavior is controlled by complex gene regulatory networks. Although studies have uncovered diverse roles of individual genes, it has been challenging to record or control sequential genetic events in living cells. In this study, we designed two cellular chain reaction systems that enable sequential sgRNA activation in mammalian cells using a nickase Cas9 tethering of a cytosine nucleotide deaminase (nCas9-CDA). In these systems, thymidine (T)-to-cytosine (C) substitutions in the scaffold region of the sgRNA or the TATA box-containing loxP sequence (TATAloxP) are corrected by the nCas9-CDA, leading to activation of the next sgRNA. These reactions can occur multiple times, resulting in cellular chain reactions. As a proof of concept, we established a chain reaction by repairing sgRNA scaffold mutations in 293 T cells. Importantly, the results obtained in yeast or in vitro did not match those obtained in mammalian cells, suggesting that in vivo chain reactions need to be optimized in appropriate cellular contexts. Our system may lay the foundation for building cellular chain reaction systems that have a broad utility in the future biomedical research.
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Sistemas CRISPR-Cas , Reparo do DNA , Edição de Genes , Mutação , RNA Guia de Cinetoplastídeos/genética , Citosina Desaminase/genética , Citosina Desaminase/metabolismo , Células HEK293 , Humanos , TATA Box/genética , Timidina/genéticaRESUMO
Bacillus sp. strain KH172YL63 is a Gram-positive bacterium isolated from the deep-sea floor surface sediment at 3,308 m below sea level in the Nankai Trough in Japan. Here, we report the complete genome sequence of Bacillus sp. strain KH172YL63, which has a genome size of 4,251,700 bp and a G+C content of 44.8%.
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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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We describe base editors that combine both cytosine and adenine base-editing functions. A codon-optimized fusion of the cytosine deaminase PmCDA1, the adenosine deaminase TadA and a Cas9 nickase (Target-ACEmax) showed a high median simultaneous C-to-T and A-to-G editing activity at 47 genomic targets. On-target as well as DNA and RNA off-target activities of Target-ACEmax were similar to those of existing single-function base editors.
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Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , DNA/genética , Edição de Genes , Adenina/metabolismo , Adenosina Desaminase/genética , Citosina/metabolismo , Desoxirribonuclease I/genética , Genoma Humano/genética , Glicoproteínas/genética , Guanina/metabolismo , Células HEK293 , Humanos , Mutação/genética , Proteínas Nucleares/genética , RNA/genéticaRESUMO
Mammalian development involves continuous dynamic processes in which cells propagate, differentiate, orchestrate, and decease to produce high-order functions. Although accurate cell lineage information can provide a strong foundation to understand such complex processes, the cell lineages involved in development of the whole mammalian body remain largely unclear, except for in early embryogenesis, which is observable under a microscope. With CRISPR genome editing, the concept of 'evolving DNA barcodes' has rapidly emerged for large-scale, high-resolution cell lineage tracing, where cell-embedded DNA barcodes continuously accumulate random mutations that are inherited from mother to daughter cells. Similar to evolutionary tree reconstruction using species' DNA sequences, cell lineages can be reconstructed using shared mutation patterns in the DNA barcodes identified using massively parallel sequencing. The dramatic developments of single-cell and imaging technologies have enabled analyses of the molecular and spatial architecture of heterogeneous cells. The evolving DNA barcodes can also consolidate this information on a reconstructed cell lineage tree and accelerate our understanding of multicellular organisms.
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Linhagem da Célula , Código de Barras de DNA Taxonômico , Animais , Biomarcadores , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Evolução Molecular , Humanos , Mutação , Análise de Célula ÚnicaRESUMO
While current omics and single cell technologies have enabled measurements of high-resolution molecular snapshots of cells at a large scale, these technologies all require destruction of samples and prevent us from analyzing dynamic changes in molecular profiles, phenotypes, and behaviors of individual cells in a complex system. One possible direction to overcome this issue is the development of a cell-embedded 'event recorder' system, whereby molecular and phenotypic information of a cell(s) can be obtained at the time of observation with their past event information stored in 'heritable polymers' of the same cell. This concept has been demonstrated by many synthetic cellular circuits that monitor and transmit a certain set of environmental and intracellular signals into DNA, and have now been further accelerated by recent CRISPR-related technologies. Notably, the discovery of the RT-Cas1-Cas2 system, which acquires sequences of cellular transcripts into a specific host genomic region, has enabled recording of a broader range of molecular profile histories in the DNA tapes of cells, to understand the dynamics of complex biological processes that cannot be addressed by current technologies.
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Células , DNA/genética , Animais , Sistemas CRISPR-Cas , Comunicação Celular , Humanos , Recombinação Genética , TranscriptomaRESUMO
Psychrobacter sp. strain KH172YL61 is a Gram-negative bacterium isolated from deep-sea sediment in the Nankai Trough in Japan. Here, we report the complete genome sequence of this strain, which has a genome size of 3.19 Mb, with a G+C content of 44.0%.
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The RNA-guided endonuclease Cas9 cleaves its target DNA and is a powerful genome-editing tool. However, the widely used Streptococcus pyogenes Cas9 enzyme (SpCas9) requires an NGG protospacer adjacent motif (PAM) for target recognition, thereby restricting the targetable genomic loci. Here, we report a rationally engineered SpCas9 variant (SpCas9-NG) that can recognize relaxed NG PAMs. The crystal structure revealed that the loss of the base-specific interaction with the third nucleobase is compensated by newly introduced non-base-specific interactions, thereby enabling the NG PAM recognition. We showed that SpCas9-NG induces indels at endogenous target sites bearing NG PAMs in human cells. Furthermore, we found that the fusion of SpCas9-NG and the activation-induced cytidine deaminase (AID) mediates the C-to-T conversion at target sites with NG PAMs in human cells.
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Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sistemas CRISPR-Cas , Endonucleases/química , Endonucleases/genética , Edição de Genes , Proteína 9 Associada à CRISPR , Cristalografia por Raios X , Células HEK293 , Humanos , Engenharia de ProteínasRESUMO
Sarcocystis fayeri (S. fayeri) is a newly identified causative agent of foodborne disease that is associated with the consumption of raw horse meat. The testing methods prescribed by the Ministry of Health, Labour and Welfare of Japan are time consuming and require the use of expensive equipment and a high level of technical expertise. Accordingly, these methods are not suitable for use in the routine sanitary control setting to prevent outbreaks of foodborne disease. In order to solve these problems, we have developed a new, rapid and simple testing method using LAMP, which takes only 1 hour to perform and which does not involve the use of any expensive equipment or expert techniques. For the validation of this method, an inter-laboratory study was performed among 5 institutes using 10 samples infected with various concentrations of S. fayeri. The results of the inter-laboratory study demonstrated that our LAMP method could detect S. fayeri at concentrations greater than 10(4) copies/g. Thus, this new method could be useful in screening for S. fayeri as a routine sanitary control procedure.
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Análise de Alimentos , Carne/parasitologia , Sarcocystis/classificação , Sarcocystis/genética , Animais , Inocuidade dos Alimentos , Doenças Transmitidas por Alimentos/parasitologia , Doenças Transmitidas por Alimentos/prevenção & controle , Cavalos , Humanos , Técnicas de Amplificação de Ácido Nucleico , Reprodutibilidade dos TestesRESUMO
The conditions of one step RT-PCR method for detection of virus RNA in field-collected mosquitoes, and preservation period of infected mosquitoes for one step RT-PCR were examined. We compared several virus RNA extraction methods with artificially contaminated mosquito pools with dengue virus (DV), Japanese encephalitis virus (JEV), and yellow fever virus (YFV) with a known amount of plaque forming unit (PFU) to establish the condition of one step RT-PCR. In this study, most effective RNA extraction method was ISOGEN-LS extraction combined with supernatant of centrifuged mosquito homogenates. Detection limit of one step RT-PCR using flavivirus universal primer in ten mosquitoes/tube (pool) was 10 PFU of DV, JEV and YFV, 1 PFU of each viruses using species-specific primer respectively, in one hundred mosquitoes/tube, 100 PFU/tube using universal primer pairs, 10 PFU/tube using species-specific primer pairs respectively. Dengue virus infected single mosquito was mixed with 99 un-infected mosquitoes, and tested by one step RT-PCR. We could detect single infected mosquito in pools containing 99 un-infected mosquitoes. Aedes aegypti and Aedes albopictus were inoculated intrathoracically with a mouse-adapted strain of dengue-1 virus and were kept up to 30 days at different temperature. Then examined by one step RT-PCR to determine the appropriate mosquito handling method and the condition of transportation. Positive result was obtained up to 30 days after the mosquito died naturally. These results suggested that we could detect flavivirus RNA tested not only from live mosquitoes but also dead mosquitoes as well, and could apply one step RT-PCR as a rapid, specific, and highly sensitive tool for flavivirus surveillance.
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Culicidae/genética , Flavivirus/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , AnimaisRESUMO
A sum-frequency-generation system for differential absorption lidar measurement of atmospheric nitrogen dioxide in the lower troposphere was developed. The system uses a combination of a pair of KD*P crystals and a tunable dye laser with LDS 765 dye pumped by the second harmonic of a Nd:YAG laser to generate lambdaon and lambdaoff alternatively. Compared with the conventional system that uses Coumarin 445 dye pumped by the third harmonic, the output energy and long-term stability were improved. By use of this system, atmospheric NO2 concentrations of approximately 10-50 ppb were measured, with an instrumental error of approximately 7 ppb.