In silico design and analysis of targeted genome editing with CRISPR

CRISPR/Cas systems have become a tool of choice for targeted genome engineering in recent years. Scientists around the world want to accelerate their research with the use of CRISPR/Cas systems, but are being slowed down by the need to understand the technology and computational steps needed for design and analysis. However, bioinformatics tools for the design and analysis of CRISPR experiments are being created to aid those scientists. For the design of CRISPR targeted genome editing experiments, CHOPCHOP has become one of the most cited and most used tools. After the initial publication of CHOPCHOP, our understanding of the CRISPR system underwent a scientific evolution. I therefore updated CHOPCHOP to accommodate the latest discoveries, such as designs for nickase and isoform targeting, machine learning algorithms for efficiency scoring and repair profile prediction, in addition to many others. On the other spectrum of genome engineering with CRISPR, there is a need for analysis of the data and validation of mutants. For the analysis of the CRISPR targeted genome editing experiments, I have created ampliCan, an R package that with the use of ‘editing aware’ alignment and automated normalization, performs precise estimation of editing efficiencies for thousands of CRISPR experiments. I have benchmarked ampliCan to display its strengths at handling a variety of editing indels, filtering out contaminant reads and performing HDR editing estimates. Both of these tools were developed with the idea that biologists without a deep understanding of CRISPR should be able to use them, and at the same time seasoned experts can adjust the settings for their purposes. I hope that these tools will facilitate adaptation of CRISPR systems for targeted genome editing and indirectly allow for great discoveries in the future

Deep conservation of ribosome stall sites across RNA processing genes

The rate of translation can vary depending on the mRNA template. During the elongation phase the ribosome can transiently pause or permanently stall. A pause can provide the nascent protein with the time to fold or be transported, while stalling can serve as quality control and trigger degradation of aberrant mRNA and peptide. Ribosome profiling has allowed for the genome-wide detection of such pauses and stalls, but due to library-specific biases, these predictions are often unreliable. Here, we take advantage of the deep conservation of protein synthesis machinery, hypothesizing that similar conservation could exist for functionally important locations of ribosome slowdown, here collectively called stall sites. We analyze multiple ribosome profiling datasets from phylogenetically diverse eukaryotes: yeast, fruit fly, zebrafish, mouse and human to identify conserved stall sites. We find thousands of stall sites across multiple species, with the enrichment of proline, glycine and negatively charged amino acids around conserved stalling. Many of the sites are found in RNA processing genes, suggesting that stalling might have a conserved role in RNA metabolism. In summary, our results provide a rich resource for the study of conserved stalling and indicate possible roles of stalling in gene regulation

CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing

The CRISPR–Cas system is a powerful genome editing tool that functions in a diverse array of organisms and cell types. The technology was initially developed to induce targeted mutations in DNA, but CRISPR–Cas has now been adapted to target nucleic acids for a range of purposes. CHOPCHOP is a web tool for identifying CRISPR–Cas single guide RNA (sgRNA) targets. In this major update of CHOPCHOP, we expand our toolbox beyond knockouts. We introduce functionality for targeting RNA with Cas13, which includes support for alternative transcript isoforms and RNA accessibility predictions. We incorporate new DNA targeting modes, including CRISPR activation/repression, targeted enrichment of loci for long-read sequencing, and prediction of Cas9 repair outcomes. Finally, we expand our results page visualization to reveal alternative isoforms and downstream ATG sites, which will aid users in avoiding the expression of truncated proteins. The CHOPCHOP web tool now supports over 200 genomes and we have released a command-line script for running larger jobs and handling unsupported genomes. CHOPCHOP v3 can be found at https://chopchop.cbu.uib.nopublishedVersio

TailFindR: Alignment-free poly(A) length measurement for Oxford Nanopore RNA and DNA sequencing

Polyadenylation at the 3′-end is a major regulator of messenger RNA and its length is known to affect nuclear export, stability, and translation, among others. Only recently have strategies emerged that allow for genome-wide poly(A) length assessment. These methods identify genes connected to poly(A) tail measurements indirectly by short-read alignment to genetic 3′-ends. Concurrently, Oxford Nanopore Technologies (ONT) established full-length isoform-specific RNA sequencing containing the entire poly(A) tail. However, assessing poly(A) length through base-calling has so far not been possible due to the inability to resolve long homopolymeric stretches in ONT sequencing. Here we present tailfindr, an R package to estimate poly(A) tail length on ONT long-read sequencing data. tailfindr operates on unaligned, base-called data. It measures poly(A) tail length from both native RNA and DNA sequencing, which makes poly(A) tail studies by full-length cDNA approaches possible for the first time. We assess tailfindr’s performance across different poly(A) lengths, demonstrating that tailfindr is a versatile tool providing poly(A) tail estimates across a wide range of sequencing conditions.publishedVersio

Rapid genome editing by CRISPR-Cas9-POLD3 fusion

Precision CRISPR gene editing relies on the cellular homology-directed DNA repair (HDR) to introduce custom DNA sequences to target sites. The HDR editing efficiency varies between cell types and genomic sites, and the sources of this variation are incompletely understood. Here, we have studied the effect of 450 DNA repair protein-Cas9 fusions on CRISPR genome editing outcomes. We find the majority of fusions to improve precision genome editing only modestly in a locus- and cell-type specific manner. We identify Cas9-POLD3 fusion that enhances editing by speeding up the initiation of DNA repair. We conclude that while DNA repair protein fusions to Cas9 can improve HDR CRISPR editing, most need to be optimized to the cell type and genomic site, highlighting the diversity of factors contributing to locus-specific genome editing outcomes.Peer reviewe

In silico design and analysis of targeted genome editing with CRISPR

CRISPR/Cas systems have become a tool of choice for targeted genome engineering in recent years. Scientists around the world want to accelerate their research with the use of CRISPR/Cas systems, but are being slowed down by the need to understand the technology and computational steps needed for design and analysis. However, bioinformatics tools for the design and analysis of CRISPR experiments are being created to aid those scientists. For the design of CRISPR targeted genome editing experiments, CHOPCHOP has become one of the most cited and most used tools. After the initial publication of CHOPCHOP, our understanding of the CRISPR system underwent a scientific evolution. I therefore updated CHOPCHOP to accommodate the latest discoveries, such as designs for nickase and isoform targeting, machine learning algorithms for efficiency scoring and repair profile prediction, in addition to many others. On the other spectrum of genome engineering with CRISPR, there is a need for analysis of the data and validation of mutants. For the analysis of the CRISPR targeted genome editing experiments, I have created ampliCan, an R package that with the use of ‘editing aware’ alignment and automated normalization, performs precise estimation of editing efficiencies for thousands of CRISPR experiments. I have benchmarked ampliCan to display its strengths at handling a variety of editing indels, filtering out contaminant reads and performing HDR editing estimates. Both of these tools were developed with the idea that biologists without a deep understanding of CRISPR should be able to use them, and at the same time seasoned experts can adjust the settings for their purposes. I hope that these tools will facilitate adaptation of CRISPR systems for targeted genome editing and indirectly allow for great discoveries in the future

CRISPR Genome Editing Made Easy Through the CHOPCHOP Website

The design of optimal guide RNA (gRNA) sequences for CRISPR systems is challenged by the need to achieve highly efficient editing at the desired location (on-target editing) with minimal editing at unintended locations (off-target editing). Although laboratory validation should ideally be used to detect off-target activity, computational predictions are almost always preferred in practice due to their speed and low cost. Several studies have therefore explored gRNA-DNA interactions in order to understand how CRISPR complexes select their genomic targets. CHOPCHOP (https://chopchop.cbu.uib.no/) leverages these developments to build a user-friendly web interface that helps users design optimal gRNAs. CHOPCHOP supports a wide range of CRISPR applications, including gene knock-out, sequence knock-in, and RNA knock-down. Furthermore, CHOPCHOP offers visualization that enables an informed choice of gRNAs and supports experimental validation. In these protocols, we describe the best practices for gRNA design using CHOPCHOP

CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering

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.n

CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering

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

Deep conservation of ribosome stall sites across RNA processing genes

The rate of translation can vary depending on the mRNA template. During the elongation phase the ribosome can transiently pause or permanently stall. A pause can provide the nascent protein with the time to fold or be transported, while stalling can serve as quality control and trigger degradation of aberrant mRNA and peptide. Ribosome profiling has allowed for the genome-wide detection of such pauses and stalls, but due to library-specific biases, these predictions are often unreliable. Here, we take advantage of the deep conservation of protein synthesis machinery, hypothesizing that similar conservation could exist for functionally important locations of ribosome slowdown, here collectively called stall sites. We analyze multiple ribosome profiling datasets from phylogenetically diverse eukaryotes: yeast, fruit fly, zebrafish, mouse and human to identify conserved stall sites. We find thousands of stall sites across multiple species, with the enrichment of proline, glycine and negatively charged amino acids around conserved stalling. Many of the sites are found in RNA processing genes, suggesting that stalling might have a conserved role in RNA metabolism. In summary, our results provide a rich resource for the study of conserved stalling and indicate possible roles of stalling in gene regulation