Computational pipeline for designing guide RNAs for mismatch-CRISPRi.
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ABSTRACT:
SUBMITTER: van Gestel J
PROVIDER: S-EPMC8121773 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:CRISPR-mediated base editors have opened unique avenues for scar-free genome-wide mutagenesis. Here, we describe a comprehensive computational workflow called beditor that can be broadly adapted for designing guide RNA libraries with a range of CRISPR-mediated base editors, Protospacer Adjacent Motif (PAM) recognition sequences, and genomes of many species. Additionally, to assist users in selecting the best sets of guide RNAs for their experiments, a priori estimates of editing efficiency, called beditor scores, are calculated. These beditor scores are intended to select guide RNAs that conform to requirements for optimal base editing: the editable base falls within maximum activity window of the CRISPR-mediated base editor and produces nonconfounding mutational effects with minimal predicted off-target effects. We demonstrate the utility of the software by designing guide RNAs for base editing to model or correct thousands of clinically important human disease mutations.
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Project description:BackgroundAs complete and accurate genome sequences are becoming easier to obtain, more researchers wish to get one or more of them to support their research endeavors. Reliable and well-documented sequence assembly workflows find use in reference or pangenome projects.ResultsWe describe modifications to the TRITEX genome assembly workflow motivated by the rise of fast and easy long-read contig assembly of inbred plant genomes and the routine deployment of the toolchains in pangenome projects. New features include the use as surrogates of or complements to dense genetic maps and the introduction of user-editable tables to make the curation of contig placements easier and more intuitive.ConclusionEven maximally contiguous sequence assemblies of the telomere-to-telomere sort, and to a yet greater extent, the fragmented kind require validation, correction, and comparison to reference standards. As pangenomics is burgeoning, these tasks are bound to become more widespread and TRITEX is one tool to get them done. This technical guide is supported by a step-by-step computational tutorial accessible under https://tritexassembly.bitbucket.io/ . The TRITEX source code is hosted under this URL: https://bitbucket.org/tritexassembly .
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Project description:Microbes naturally coexist in complex, multistrain communities. However, extracting individual microbes from and specifically manipulating the composition of these consortia remain challenging. The sequence-specific nature of CRISPR guide RNAs can be leveraged to accurately differentiate microorganisms and facilitate the creation of tools that can achieve these tasks. We developed a computational program, ssCRISPR, which designs strain-specific CRISPR guide RNA sequences with user-specified target strains, protected strains, and guide RNA properties. We experimentally verify the accuracy of the strain specificity predictions in both Escherichia coli and Pseudomonas spp. and show that up to three nucleotide mismatches are often required to ensure perfect specificity. To demonstrate the functionality of ssCRISPR, we apply computationally designed CRISPR-Cas9 guide RNAs to two applications: the purification of specific microbes through one- and two-plasmid transformation workflows and the targeted removal of specific microbes using DNA-loaded liposomes. For strain purification, we utilize gRNAs designed to target and kill all microbes in a consortium except the specific microbe to be isolated. For strain elimination, we utilize gRNAs designed to target only the unwanted microbe while protecting all other strains in the community. ssCRISPR will be of use in diverse microbiota engineering applications.
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Project description:Hypoxia is associated with several diseases, including cancer. Cells that are deprived of adequate oxygen supply trigger transcriptional and post-transcriptional responses, which control cellular pathways such as angiogenesis, proliferation, and metabolic adaptation. Circular RNAs (circRNAs) are a novel class of mainly non-coding RNAs, which have been implicated in multiple cancers and attract increasing attention as potential biomarkers. Here, we characterize the circRNA signatures of three different cancer cell lines from cervical (HeLa), breast (MCF-7), and lung (A549) cancer under hypoxia. In order to reliably detect circRNAs, we integrate available tools with custom approaches for quantification and statistical analysis. Using this consolidated computational pipeline, we identify ~12000 circRNAs in the three cancer cell lines. Their molecular characteristics point to an involvement of complementary RNA sequences as well as trans-acting factors in circRNA biogenesis, such as the RNA-binding protein HNRNPC. Notably, we detect a number of circRNAs that are more abundant than their linear counterparts. In addition, 64 circRNAs significantly change in abundance upon hypoxia, in most cases in a cell type-specific manner. In summary, we present a comparative circRNA profiling in human cancer cell lines, which promises novel insights into the biogenesis and function of circRNAs under hypoxic stress.
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Project description:Long noncoding RNAs (lncRNAs) are versatile RNA molecules recently identified as key regulators of gene expression in response to environmental stress. Our primary focus in this study was to develop a robust computational pipeline for identifying structurally identical lncRNAs across replicates from publicly available bulk RNA-seq datasets. In order to demonstrate the effectiveness of the pipeline, we utilized the transcriptome of the thermophilic fungus Thermothelomyces thermophilus and assessed the expression pattern of lncRNAs in conjunction with Heat Shock Proteins (HSP), a well-known protein family critical for the cell's response to high temperatures. Our findings demonstrate that the identification of structurally identical transcripts among replicates in this thermophilic fungus ensures the reliability and accuracy of RNA studies, contributing to the validity of biological interpretations. Furthermore, the majority of lncRNAs exhibited a distinct expression pattern compared to HSPs. Our study contributes to advancing the understanding of the biological mechanisms comprising lncRNAs in thermophilic fungi.
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Project description:The availability of genomes across the tree of life is highly biased toward vertebrates, pathogens, human disease models, and organisms with relatively small and simple genomes. Recent progress in genomics has enabled the de novo decoding of the genome of virtually any organism, greatly expanding its potential for understanding the biology and evolution of the full spectrum of biodiversity. The increasing diversity of sequencing technologies, assays, and de novo assembly algorithms have augmented the complexity of de novo genome sequencing projects in non-model organisms. To reduce the costs and challenges in de novo genome sequencing projects and streamline their experimental design and analysis, we developed iWGS (in silico Whole Genome Sequencer and Analyzer), an automated pipeline for guiding the choice of appropriate sequencing strategy and assembly protocols. iWGS seamlessly integrates the four key steps of a de novo genome sequencing project: data generation (through simulation), data quality control, de novo assembly, and assembly evaluation and validation. The last three steps can also be applied to the analysis of real data. iWGS is designed to enable the user to have great flexibility in testing the range of experimental designs available for genome sequencing projects, and supports all major sequencing technologies and popular assembly tools. Three case studies illustrate how iWGS can guide the design of de novo genome sequencing projects and evaluate the performance of a wide variety of user-specified sequencing strategies and assembly protocols on genomes of differing architectures. iWGS, along with a detailed documentation, is freely available at https://github.com/zhouxiaofan1983/iWGS.
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Project description:In eukaryotes, box H/ACA small nucleolar RNAs (snoRNAs) guide sites of pseudouridine (Psi) formation in rRNA. These snoRNAs reside in RNP complexes containing the putative Psi synthase, Cbf5p. In this study we have identified Cbf5p-associated RNAs in Euglena gracilis, an early diverging eukaryote, by immunoprecipitating Cbf5p-containing complexes from cellular extracts. We characterized one box H/ACA-like RNA which, however, does not appear to guide Psi formation in rRNA. We also identified four single Psi-guide box AGA RNAs. We determined target sites for these putative Psi-guide RNAs and confirmed that the predicted Psi modifications do, in fact, occur at these positions in Euglena rRNA. The Cbf5p-associated snoRNAs appear to be encoded by multicopy genes, some of which are clustered in the genome together with methylation-guide snoRNA genes. These modification-guide snoRNAs and snoRNA genes are the first ones to be reported in euglenid protists, the evolutionary sister group to the kinetoplastid protozoa. Unexpectedly, we also found and have partially characterized a selenocysteine tRNA homolog in the anti-Cbf5p-immunoprecipitated sample.
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