Showing papers by "Eivind Valen published in 2016"
TL;DR: This major update of CHOPCHOP introduces tools for the next generation of CRISPR advances, including Cpf1 and Cas9 nickases, and provides support for custom length sgRNAs and evaluates the sequence composition of the whole sgRNA and its surrounding region using models compiled from multiple large-scale studies.
Abstract: In just 3 years CRISPR genome editing has transformed biology, and its popularity and potency continue to grow. New CRISPR effectors and rules for locating optimum targets continue to be reported, highlighting the need for computational CRISPR targeting tools to compile these rules and facilitate target selection and design. CHOPCHOP is one of the most widely used web tools for CRISPR- and TALEN-based genome editing. Its overarching principle is to provide an intuitive and powerful tool that can serve both novice and experienced users. In this major update we introduce tools for the next generation of CRISPR advances, including Cpf1 and Cas9 nickases. We support a number of new features that improve the targeting power, usability and efficiency of CHOPCHOP. To increase targeting range and specificity we provide support for custom length sgRNAs, and we evaluate the sequence composition of the whole sgRNA and its surrounding region using models compiled from multiple large-scale studies. These and other new features, coupled with an updated interface for increased usability and support for a continually growing list of organisms, maintain CHOPCHOP as one of the leading tools for CRISPR genome editing. CHOPCHOP v2 can be found at http://chopchop.cbu.uib.no.
748 citations
TL;DR: It is shown that both genomic context and internal gRNA interactions can interfere with Cas9-mediated cleavage and illuminate previously uncharacterized features of Cas9–gRNA complex formation.
Abstract: The CRISPR/Cas system uses guide RNAs (gRNAs) to direct sequence-specific DNA cleavage. Not every gRNA elicits cleavage and the mechanisms that govern gRNA activity have not been resolved. Low activity could result from either failure to form a functional Cas9-gRNA complex or inability to recognize targets in vivo. Here we show that both phenomena influence Cas9 activity by comparing mutagenesis rates in zebrafish embryos with in vitro cleavage assays. In vivo, our results suggest that genomic factors such as CTCF inhibit mutagenesis. Comparing near-identical gRNA sequences with different in vitro activities reveals that internal gRNA interactions reduce cleavage. Even though gRNAs containing these structures do not yield cleavage-competent complexes, they can compete with active gRNAs for binding to Cas9. These results reveal that both genomic context and internal gRNA interactions can interfere with Cas9-mediated cleavage and illuminate previously uncharacterized features of Cas9-gRNA complex formation.
121 citations
TL;DR: A model capable of predicting translation initiation sites and demonstrating its high accuracy using N-terminal proteomics is built and 61 novel genes previously undiscovered in the genome are identified.
Abstract: While methods for annotation of genes are increasingly reliable the exact identification of the translation initiation site remains a challenging problem. Since the N-termini of proteins often contain regulatory and targeting information developing a robust method for start site identification is crucial. Ribosome profiling reads show distinct patterns of read length distributions around translation initiation sites. These patterns are typically lost in standard ribosome profiling analysis pipelines, when reads from footprints are adjusted to determine the specific codon being translated. Using these unique signatures we build a model capable of predicting translation initiation sites and demonstrate its high accuracy using N-terminal proteomics. Applying this to prokaryotic samples, we re-annotate translation initiation sites and provide evidence of N-terminal truncations and elongations of annotated coding sequences. These re-annotations are supported by the presence of Shine-Dalgarno sequences, structural and sequence based features and N-terminal peptides. Finally, our model identifies 61 novel genes previously undiscovered in the genome.
1 citations