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CRISPR/Cas-mediated knock-in of DNA sequences enables precise genome engineering for research and therapeutic applications. However, designing effective guide RNAs (gRNAs) and homology-directed repair (HDR) donors remains a bottleneck. Here, we present protoSpaceJAM, an open-source algorithm to automate and optimize gRNA and HDR donor design for CRISPR/Cas insertional knock-in experiments, currently supporting SpCas9, SpCas9-VQR and enAsCas12a Cas enzymes. protoSpaceJAM utilizes biological rules to rank gRNAs based on specificity, distance to insertion site, and position relative to regulatory regions. protoSpaceJAM can introduce 'recoding' mutations (silent mutations and mutations in non-coding sequences) in HDR donors to prevent re-cutting and increase knock-in efficiency. Users can customize parameters and design double-stranded or single-stranded donors. We validated protoSpaceJAM's design rules by demonstrating increased knock-in efficiency with recoding mutations and optimal strand selection for single-stranded donors. An additional module enables the design of genotyping primers for deep sequencing of edited alleles. Overall, protoSpaceJAM streamlines and optimizes CRISPR knock-in experimental design in a flexible and modular manner to benefit diverse research and therapeutic applications. protoSpaceJAM is available open-source as an interactive web tool at protospacejam.czbiohub.org or as a standalone Python package at github.com/czbiohub-sf/protoSpaceJAM.
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BACKGROUND: Genome streamlining, the process by which genomes become smaller and encode fewer genes over time, is a common phenomenon among pathogenic bacteria. This reduction is driven by selection for minimized energy expenditure in a nutrient-rich environment. As pathogens evolve to become more reliant on the host, metabolic genes and resulting capabilities are lost in favor of siphoning metabolites from the host. Characterizing genome streamlining, gene loss, and metabolic pathway degradation can be useful in assessing pathogen dependency on host metabolism and identifying potential targets for host-directed therapeutics. RESULTS: PoMeLo (Predictor of Metabolic Loss) is a novel evolutionary genomics-guided computational approach for identifying metabolic gaps in the genomes of pathogenic bacteria. PoMeLo leverages a centralized public database of high-quality genomes and annotations and allows the user to compare an unlimited number of genomes across individual genes and pathways. PoMeLo runs locally using user-friendly prompts in a matter of minutes and generates tabular and visual outputs for users to compare predicted metabolic capacity between groups of bacteria and individual species. Each pathway is assigned a Predicted Metabolic Loss (PML) score to assess the magnitude of genome streamlining. Optionally, PoMeLo places the results in an evolutionary context by including phylogenetic relationships in visual outputs. It can also initially compute phylogenetically-weighted mean genome sizes to identify genome streamlining events. Here, we describe PoMeLo and demonstrate its use in identifying metabolic gaps in genomes of pathogenic Treponema species. CONCLUSIONS: PoMeLo represents an advance over existing methods for identifying metabolic gaps in genomic data, allowing comparison across large numbers of genomes and placing the resulting data in a phylogenetic context. PoMeLo is freely available for academic and non-academic use at https://github.com/czbiohub-sf/pomelo .
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Genoma , Genómica , Filogenia , Genómica/métodos , Evolución Biológica , Bacterias/genética , Programas InformáticosRESUMEN
The sequencing of antibody repertoires of B-cells at increasing coverage and depth has led to the identification of vast numbers of immunoglobulin heavy and light chains. However, the size and complexity of these Adaptive Immune Receptor Repertoire sequencing (AIRR-seq) datasets makes it difficult to perform exploratory analyses. To aid in data exploration, we have developed AIRRscape, an R Shiny-based interactive web browser application that enables B-cell receptor (BCR) and antibody feature discovery through comparisons among multiple repertoires. Using AIRR-seq data as input, AIRRscape starts by aggregating and sorting repertoires into interactive and explorable bins of germline V-gene, germline J-gene, and CDR3 length, providing a high-level view of the entire repertoire. Interesting subsets of repertoires can be quickly identified and selected, and then network topologies of CDR3 motifs can be generated for further exploration. Here we demonstrate AIRRscape using patient BCR repertoires and sequences of published monoclonal antibodies to investigate patterns of humoral immunity to three viral pathogens: SARS-CoV-2, HIV-1, and DENV (dengue virus). AIRRscape reveals convergent antibody sequences among datasets for all three pathogens, although HIV-1 antibody datasets display limited convergence and idiosyncratic responses. We have made AIRRscape available as a web-based Shiny application, along with code on GitHub to encourage its open development and use by immuno-informaticians, virologists, immunologists, vaccine developers, and other scientists that are interested in exploring and comparing multiple immune receptor repertoires.
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Formación de Anticuerpos , COVID-19 , Anticuerpos Monoclonales , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Receptores de Antígenos de Linfocitos B/genética , SARS-CoV-2/genéticaRESUMEN
Targeted PCR amplification and high-throughput sequencing (amplicon sequencing) of 16S rRNA gene fragments is widely used to profile microbial communities. New long-read sequencing technologies can sequence the entire 16S rRNA gene, but higher error rates have limited their attractiveness when accuracy is important. Here we present a high-throughput amplicon sequencing methodology based on PacBio circular consensus sequencing and the DADA2 sample inference method that measures the full-length 16S rRNA gene with single-nucleotide resolution and a near-zero error rate. In two artificial communities of known composition, our method recovered the full complement of full-length 16S sequence variants from expected community members without residual errors. The measured abundances of intra-genomic sequence variants were in the integral ratios expected from the genuine allelic variants within a genome. The full-length 16S gene sequences recovered by our approach allowed Escherichia coli strains to be correctly classified to the O157:H7 and K12 sub-species clades. In human fecal samples, our method showed strong technical replication and was able to recover the full complement of 16S rRNA alleles in several E. coli strains. There are likely many applications beyond microbial profiling for which high-throughput amplicon sequencing of complete genes with single-nucleotide resolution will be of use.
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Secuenciación de Nucleótidos de Alto Rendimiento , Microbiota/genética , Filogenia , ARN Ribosómico 16S/genética , Escherichia coli K12/clasificación , Escherichia coli K12/genética , Escherichia coli O157/clasificación , Escherichia coli O157/genética , Heces/microbiología , Humanos , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Nucleótido Simple/genética , Análisis de Secuencia de ADNRESUMEN
The experimental induction of RNA silencing in plants often involves expression of transgenes encoding inverted repeat (IR) sequences to produce abundant dsRNAs that are processed into small RNAs (sRNAs). These sRNAs are key mediators of post-transcriptional gene silencing (PTGS) and determine its specificity. Despite its application in agriculture and broad utility in plant research, the mechanism of IR-PTGS is incompletely understood. We generated four sets of 60 Arabidopsis plants, each containing IR transgenes expressing different configurations of uidA and CHALCONE Synthase (At-CHS) gene fragments. Levels of PTGS were found to depend on the orientation and position of the fragment in the IR construct. Deep sequencing and mapping of sRNAs to corresponding transgene-derived and endogenous transcripts identified distinctive patterns of differential sRNA accumulation that revealed similarities among sRNAs associated with IR-PTGS and endogenous sRNAs linked to uncapped mRNA decay. Detailed analyses of poly-A cleavage products from At-CHS mRNA confirmed this hypothesis. We also found unexpected associations between sRNA accumulation and the presence of predicted open reading frames in the trigger sequence. In addition, strong IR-PTGS affected the prevalence of endogenous sRNAs, which has implications for the use of PTGS for experimental or applied purposes.
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Silenciador del Gen , Interferencia de ARN , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Aciltransferasas/genética , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Secuenciación de Nucleótidos de Alto Rendimiento , Secuencias Invertidas Repetidas/genética , Plantas Modificadas Genéticamente/genética , Estabilidad del ARN/genética , ARN Bicatenario/genética , Transducción de SeñalRESUMEN
Two independent temporal-spatial clusters of hospital-onset Rhizopus infections were evaluated using whole-genome sequencing (WGS). Phylogenetic analysis confirmed that isolates within each cluster were unrelated despite epidemiological suspicion of outbreaks. The ITS1 region alone was insufficient for accurate analysis. WGS has utility for rapid rule-out of suspected nosocomial Rhizopus outbreaks.
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Genoma Bacteriano , Rhizopus , Humanos , Rhizopus/genética , Filogenia , Hospitales , Brotes de EnfermedadesRESUMEN
Lettuce downy mildew caused by Bremia lactucae is the most important disease of lettuce globally. This oomycete is highly variable and rapidly overcomes resistance genes and fungicides. The use of multiple read types results in a high-quality, near-chromosome-scale, consensus assembly. Flow cytometry plus resequencing of 30 field isolates, 37 sexual offspring, and 19 asexual derivatives from single multinucleate sporangia demonstrates a high incidence of heterokaryosis in B. lactucae. Heterokaryosis has phenotypic consequences on fitness that may include an increased sporulation rate and qualitative differences in virulence. Therefore, selection should be considered as acting on a population of nuclei within coenocytic mycelia. This provides evolutionary flexibility to the pathogen enabling rapid adaptation to different repertoires of host resistance genes and other challenges. The advantages of asexual persistence of heterokaryons may have been one of the drivers of selection that resulted in the loss of uninucleate zoospores in multiple downy mildews.
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Núcleo Celular/genética , Interacciones Huésped-Patógeno/genética , Lactuca/microbiología , Oomicetos/genética , Enfermedades de las Plantas/microbiología , Núcleo Celular/efectos de los fármacos , Resistencia a la Enfermedad/genética , Fungicidas Industriales/farmacología , Genómica , Lactuca/genética , Oomicetos/citología , Oomicetos/patogenicidad , Selección Genética/efectos de los fármacos , Transcriptoma/efectos de los fármacos , Transcriptoma/genética , Virulencia/genéticaRESUMEN
BACKGROUND: The epidemiology of mortality and morbidity from carbon monoxide poisoning in Canada has received little attention. Our objective was to evaluate trends in mortality and hospital admission rates for unintentional nonfire-related carbon monoxide poisoning across Canada. METHODS: Age- and sex-standardized mortality (1981-2009) and hospital admission (1995-2010) rates by age group, sex and site of carbon monoxide exposure were calculated for each province and for all of Canada. We quantified the long-term trends by calculating the average annual percent change. Multivariable Poisson regression was used to estimate incidence rate ratios (IRRs) of carbon monoxide poisoning across age groups, sex and month of occurrence. RESULTS: In Canada, there were 1808 unintentional nonfire-related carbon monoxide poisoning deaths between 1981 and 2009 and 1984 admissions to hospital between 1995 and 2010. Average annual decreases of 3.46% (95% confidence interval [CI] -4.59% to -2.31%) and 5.83% (95% CI -7.79% to -3.83%) were observed for mortality and hospital admission rates, respectively. Mortality (IRR 5.31, 95% CI 4.57 to 6.17) and hospital admission (IRR 2.77, 95% CI 2.51 to 3.03) rates were elevated in males compared with females. Decreased trends in the rates were observed for all sites of carbon monoxide exposure, but the magnitude of this decrease was lowest in residential environments. Deaths and admissions to hospital were most frequent from September to April, with peaks in December and January. INTERPRETATION: Mortality and hospital admission rates for unintentional nonfire-related carbon monoxide poisoning in Canada have declined steadily. Continued efforts should focus on reducing carbon monoxide poisoning during the cooler months and in residential environments.