RESUMO
Regulatory regions harbor multiple transcription factor (TF) recognition sites; however, the contribution of individual sites to regulatory function remains challenging to define. We describe an approach that exploits the error-prone nature of genome editing-induced double-strand break repair to map functional elements within regulatory DNA at nucleotide resolution. We demonstrate the approach on a human erythroid enhancer, revealing single TF recognition sites that gate the majority of downstream regulatory function.
Assuntos
Proteínas de Transporte/genética , Pegada de DNA/métodos , Genômica/métodos , Proteínas Nucleares/genética , Sequências Reguladoras de Ácido Nucleico , Sequência de Bases , Sítios de Ligação , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Elementos Facilitadores Genéticos , Eritrócitos/fisiologia , Eritropoese , Genoma Humano , Humanos , Mutação , Proteínas Repressoras , Fatores de Transcrição/metabolismoRESUMO
Nucleobase editors represent an emerging technology that enables precise single-base edits to the genomes of eukaryotic cells. Most nucleobase editors use deaminase domains that act upon single-stranded DNA and require RNA-guided proteins such as Cas9 to unwind the DNA prior to editing. However, the most recent class of base editors utilizes a deaminase domain, DddAtox, that can act upon double-stranded DNA. Here, we target DddAtox fragments and a FokI-based nickase to the human CIITA gene by fusing these domains to arrays of engineered zinc fingers (ZFs). We also identify a broad variety of Toxin-Derived Deaminases (TDDs) orthologous to DddAtox that allow us to fine-tune properties such as targeting density and specificity. TDD-derived ZF base editors enable up to 73% base editing in T cells with good cell viability and favorable specificity.
Assuntos
Citidina Desaminase , Edição de Genes , Humanos , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , DNA/metabolismo , Dedos de Zinco , Citidina/genética , Sistemas CRISPR-CasRESUMO
UNLABELLED: Interactive examination of RNA multiple alignments for covariant mutations is a useful step in non-coding RNA sequence analysis. We present three parallel implementations of an RNA visualization metaphor: Colorstock, a command-line script using ANSI terminal color; SScolor, a Perl script that generates static HTML pages; and Ratón, an AJAX web application generating dynamic HTML. Each tool can be used to color RNA alignments by secondary structure and to visually highlight compensatory mutations in stems. AVAILABILITY: All source code is freely available under the GPL. The source code can be downloaded and a prototype of Ratón can be accessed at http://biowiki.org/RnaAlignmentViewers.
Assuntos
Biologia Computacional/métodos , Alinhamento de Sequência/métodos , Análise de Sequência de RNA/métodos , Software , Sequência de Bases , Dados de Sequência Molecular , RNA Viral/genéticaRESUMO
Engineered nucleases have gained broad appeal for their ability to mediate highly efficient genome editing. However the specificity of these reagents remains a concern, especially for therapeutic applications, given the potential mutagenic consequences of off-target cleavage. Here we have developed an approach for improving the specificity of zinc finger nucleases (ZFNs) that engineers the FokI catalytic domain with the aim of slowing cleavage, which should selectively reduce activity at low-affinity off-target sites. For three ZFN pairs, we engineered single-residue substitutions in the FokI domain that preserved full on-target activity but showed a reduction in off-target indels of up to 3,000-fold. By combining this approach with substitutions that reduced the affinity of zinc fingers, we developed ZFNs specific for the TRAC locus that mediated 98% knockout in T cells with no detectable off-target activity at an assay background of ~0.01%. We anticipate that this approach, and the FokI variants we report, will enable routine generation of nucleases for gene editing with no detectable off-target activity.
Assuntos
Clivagem do DNA , Edição de Genes/métodos , Linfócitos T , Sequência de Bases , DNA/genética , DNA/metabolismo , Citometria de Fluxo , Células-Tronco Hematopoéticas , Humanos , Células K562 , Domínios Proteicos , RNA MensageiroRESUMO
BACKGROUND: Recent years have seen the emergence of genome annotation methods based on the phylo-grammar, a probabilistic model combining continuous-time Markov chains and stochastic grammars. Previously, phylo-grammars have required considerable effort to implement, limiting their adoption by computational biologists. RESULTS: We have developed an open source software tool, xrate, for working with reversible, irreversible or parametric substitution models combined with stochastic context-free grammars. xrate efficiently estimates maximum-likelihood parameters and phylogenetic trees using a novel "phylo-EM" algorithm that we describe. The grammar is specified in an external configuration file, allowing users to design new grammars, estimate rate parameters from training data and annotate multiple sequence alignments without the need to recompile code from source. We have used xrate to measure codon substitution rates and predict protein and RNA secondary structures. CONCLUSION: Our results demonstrate that xrate estimates biologically meaningful rates and makes predictions whose accuracy is comparable to that of more specialized tools.
Assuntos
Algoritmos , Inteligência Artificial , Mapeamento Cromossômico/métodos , Alinhamento de Sequência/métodos , Análise de Sequência de DNA/métodos , Software , Sequência Conservada , FilogeniaRESUMO
Linking phenotypic with genotypic diversity has become a major requirement for basic and applied genome-centric biological research. To meet this need, a comprehensive database backend for efficiently storing, querying and analyzing large experimental data sets is necessary. Chado, a generic, modular, community-based database schema is widely used in the biological community to store information associated with genome sequence data. To meet the need to also accommodate large-scale phenotyping and genotyping projects, a new Chado module called Natural Diversity has been developed. The module strictly adheres to the Chado remit of being generic and ontology driven. The flexibility of the new module is demonstrated in its capacity to store any type of experiment that either uses or generates specimens or stock organisms. Experiments may be grouped or structured hierarchically, whereas any kind of biological entity can be stored as the observed unit, from a specimen to be used in genotyping or phenotyping experiments, to a group of species collected in the field that will undergo further lab analysis. We describe details of the Natural Diversity module, including the design approach, the relational schema and use cases implemented in several databases.
Assuntos
Biodiversidade , Biologia Computacional/métodos , Sistemas de Gerenciamento de Base de Dados , Bases de Dados Factuais , Animais , Genótipo , Internet , Fenótipo , PlantasRESUMO
We performed benchmarks of phylogenetic grammar-based ncRNA gene prediction, experimenting with eight different models of structural evolution and two different programs for genome alignment. We evaluated our models using alignments of twelve Drosophila genomes. We find that ncRNA prediction performance can vary greatly between different gene predictors and subfamilies of ncRNA gene. Our estimates for false positive rates are based on simulations which preserve local islands of conservation; using these simulations, we predict a higher rate of false positives than previous computational ncRNA screens have reported. Using one of the tested prediction grammars, we provide an updated set of ncRNA predictions for D. melanogaster and compare them to previously-published predictions and experimental data. Many of our predictions show correlations with protein-coding genes. We found significant depletion of intergenic predictions near the 3' end of coding regions and furthermore depletion of predictions in the first intron of protein-coding genes. Some of our predictions are colocated with larger putative unannotated genes: for example, 17 of our predictions showing homology to the RFAM family snoR28 appear in a tandem array on the X chromosome; the 4.5 Kbp spanned by the predicted tandem array is contained within a FlyBase-annotated cDNA.