Eivind Valen

• Labun, Kornel; Rio, Oline Eriksen; Dahal-Koirala, Shiva; Komisarczuk, Anna Zofia; Valen, Eivind & Haapaniemi, Emma Maria (2026). SNIPSNP: precision design of CRISPR/Cas9 knock-in reagents for variant correction and disease modeling. Nucleic Acids Research (NAR). ISSN 0305-1048. 54(W1). doi: 10.1093/nar/gkag409. Fulltekst i vitenarkiv Vis sammendrag

  Abstract We present SNIPSNP (crisprtools.org/snipsnp), a comprehensive bioinformatics pipeline for designing experiments for CRISPR-induced homology-directed repair (HDR). The tool addresses the critical challenge of Cas9 re-cleavage by simplifying the selection of “blocking” silent variants that are effective at inhibiting RNP binding upon donor-templated editing. SNIPSNP handles complex edits, including indels, and uses multi-objective optimization to balance editing efficiency with biological safety. From user-defined wild-type and desired HDR alleles, the pipeline identifies candidate guides, annotating them with integrated efficiency scores and genome-wide off-target assessments. Uniquely, SNIPSNP evaluates guide binding against the post-edit genome to determine whether the therapeutic variant alone disrupts repeated Cas9 recognition. When necessary, it introduces synonymous blocking variants, prioritizing PAM and seed regions to minimize re-cleavage probability and editing of the wild-type (WT) allele when editing heterozygous variants. All candidate modifications undergo safety profiling and prioritization of known benign variants from dbSNP. We experimentally validated SNIPSNP and benchmarked it on pathogenic inborn error of immunity variants in primary patient T-cells. Across loci, SNIPSNP-designed templates outperform standard “correction-only” strategies, demonstrating enhanced precision editing, and reduced re-cleavage, establishing SNIPSNP as a robust platform for genome editing and disease modeling.

• Auxillos, Jamie; Stigliani, Arnaud; Vaagensø, Christian Skov; Garland, William; Niazi, Adnan Muhammad & Valen, Eivind [Vis alle 8 forfattere av denne artikkelen] (2025). True length of diverse capped RNA sequencing (TLDR-seq): 5′–3′-end sequencing of capped RNAs regardless of 3′-end status. Nucleic Acids Research (NAR). ISSN 0305-1048. 53(6). doi: 10.1093/nar/gkaf240. Fulltekst i vitenarkiv Vis sammendrag

  Analysis of transcript function is greatly aided by knowledge of the full-length RNA sequence. New long-read sequencing enabled by Oxford Nanopore and PacBio devices have the potential to provide full-length transcript information; however, standard methods still lack the ability to capture true RNA 5′ ends and select for polyadenylated (pA+) transcripts only. Here, we present a method that, by utilizing cap trapping and 3′-end adapter ligation, sequences transcripts between their exact 5′ and 3′ ends regardless of polyadenylation status and without the need for ribosomal RNA depletion, with the ability to characterize polyadenylation length of RNAs, if any. The method shows high reproducibility, can faithfully detect 5′ ends, 3′ ends and splice junctions, and produces gene-expression estimates that are highly correlated to those of short-read sequencing techniques. We also demonstrate that the method can detect and sequence full-length nonadenylated (pA−) RNAs, including long noncoding RNAs, promoter upstream transcripts, and enhancer RNAs, and present cases where pA+ and pA− RNAs show preferences for different but closely located transcription start sites. Our method is therefore useful for the characterization of diverse capped RNA species and analysis of relationships between transcription initiation, termination, and RNA processing.

• Chothani, Sonia; Ruiz-Orera, Jorge; Tierney, Jack A. S.; Clauwaert, Jim; Deutsch, Eric W & Alba, M. Mar [Vis alle 46 forfattere av denne artikkelen] (2025). An expanded reference catalog of translated open reading frames for biomedical research. bioRxiv. ISSN 2692-8205. doi: 10.1101/2025.07.03.662928. Fulltekst i vitenarkiv Vis sammendrag

  Non-canonical (i.e., unannotated) open reading frames (ncORFs) have until recently been omitted from reference genome annotations, despite evidence of their translation, limiting their incorporation into biomedical research. To address this, in 2022, we initiated the TransCODE consortium and built the first community-driven consensus catalog of human ncORFs, which was openly distributed to the research community via Ensembl-GENCODE. While this catalog represented a starting point for reference ncORF annotation, major technical and scientific issues remained. In particular, this initial catalogue had no standardized framework to judge the evidence of translation for individual ncORFs. Here, we present an expanded and refined catalog of the human reference annotation of ncORFs. By incorporating more datasets and by lifting constraints on ORF length and start-codon, we define a comprehensive set of 28,359 ncORFs that is nearly four times the size of the previous catalog. Furthermore, to aid users who wish to work with ncORFs with the strongest and most reproducible signals of translation, we utilized a data-driven framework (i.e. translation signature scores) to assess the accumulated evidence for any individual ncORF. Using this approach, we derive a subset of 7,888 ncORFs with translation evidence on par with canonical protein-coding genes, which we refer to as the Primary set. This set can serve as a reliable reference for downstream analyses and validation, with a particular emphasis on high quality. Overall, this update reflects continual community-driven efforts to make ncORFs accessible and actionable to the broader research public and further iterations of the catalog will continue to expand and refine this resource.

• Begik, Oguzhan; Pryszcz, Leszek P.; Niazi, Adnan Muhammad; Valen, Eivind & Novoa, Eva Maria (2025). Nano3P-seq: charting the coding and noncoding transcriptome at single-molecule resolution. Nature Protocols. ISSN 1754-2189. 20(12), s. 3607–3628. doi: 10.1038/s41596-025-01205-0. Fulltekst i vitenarkiv Vis sammendrag

  RNA polyadenylation is crucial for RNA maturation, stability and function, with poly(A) tail lengths significantly influencing mRNA translation, efficiency and decay. Here, we provide a step-by-step protocol to perform Nanopore 3′ end-capture sequencing (Nano3P-seq), a nanopore-based cDNA sequencing method to simultaneously capture RNA abundances and tail-composition and tail-length estimates at single-molecule resolution. Taking advantage of a template switching–based protocol, Nano3P-seq can sequence any RNA-derived molecule from its 3′ end, regardless of its polyadenylation status, without the need for PCR amplification or RNA adapter ligation. We provide an updated Nano3P-seq protocol that is compatible with R10.4 flow cells, as well as compatible software for poly(A) tail length and content prediction, which we term ‘PolyTailor’. We demonstrate that PolyTailor provides accurate estimates of transcript abundances and tail lengths and composition, while capturing both coding and noncoding RNA biotypes, including mRNAs, small nucleolar RNAs and ribosomal RNAs. Nano3P-seq can be applied to RNA samples prepared by using different methods (e.g., poly(A)-selected, ribodepleted or total RNA) and can be completed in 1 day. The protocol requires experience in molecular biology techniques and nanopore sequencing library preparation and basic knowledge of Linux Bash syntax and R programming. This protocol makes Nano3P-seq accessible and easy to implement by future users aiming to study the tail dynamics and heterogeneity of both coding and noncoding transcriptomes in a comprehensive and reproducible manner.

• Kute, Preeti; Pauzin, Francois Philippe; Labun, Kornel; Bramham, Clive Raymond Evjen & Valen, Eivind (2025). Transcriptome-wide mapping of small ribosomal subunits elucidates scanning mechanisms of translation initiation in the mammalian brain. Communications Biology. 8(1). doi: 10.1038/s42003-025-08804-3. Fulltekst i vitenarkiv Vis sammendrag

  Abstract Neuronal protein synthesis is highly compartmentalised and regulated, with key roles for translation initiation and elongation factors. Ribosome profiling, the most widely used transcriptome-wide method for measuring translation, captures translation elongation, but not the initiation phase involving small ribosomal subunit (SSU) scanning. Here, we adapted ribosome complex profiling (RCP-seq) for mouse dentate gyrus and cerebral cortex, to characterize translation initiation. In both tissues, SSUs accumulate near the start codon on synaptically localised RNAs, and this ‘poised’ SSU configuration correlates with enhanced translational efficiency. Upstream open reading frames (uORFs) are associated with less poised SSUs, potentially by disassociating the SSUs. We further find that neuron-specific transcripts recruit more ribosomes and are more efficiently translated than glia-specific transcripts. For neuronal transcripts, monosome-preferring mRNAs show less SSU occupancy relative to polysome-preferring mRNAs, suggesting reduced recruitment of ribosomes. In summary, RCP-seq elucidates translation initiation dynamics and cell-type- and transcript-specific regulation in the mammalian brain.

• Engelfriet, Melanie Lianne; Guo, Yanwu; Arnold, Andreas; Valen, Eivind & Ciosk, Rafal (2025). Reprograming gene expression in ‘hibernating’ C. elegans involves the IRE-1/XBP-1 pathway. eLIFE. 13. doi: 10.7554/elife.101186.3. Fulltekst i vitenarkiv Vis sammendrag

  In nature, many animals respond to cold by entering hibernation, while in clinical settings, controlled cooling is used in transplantation and emergency medicine. However, the molecular mechanisms that enable cells to survive severe cold are still not fully understood. One key aspect of cold adaptation is the global downregulation of protein synthesis. Studying it in the nematode Caenorhabditis elegans , we find that the translation of most mRNAs continues in the cold, albeit at a slower rate, and propose that cold-specific gene expression is regulated primarily at the transcription level. Supporting this idea, we found that the transcription of certain cold-induced genes is linked to the activation of unfolded protein response (UPR) through the conserved IRE-1/XBP-1 signaling pathway. Our findings suggest that this pathway is triggered by cold-induced perturbations in proteins and lipids within the endoplasmic reticulum, and that its activation is beneficial for cold survival.

• Labun, Kornel; Rio, Oline Eriksen; Tjeldnes, Håkon; Swirski, Michał; Komisarczuk, Anna Zofia & Haapaniemi, Emma Maria [Vis alle 7 forfattere av denne artikkelen] (2025). CHOPOFF: symbolic alignments enable fast and sensitive CRISPR off-target detection. bioRxiv - the preprint server for biology. doi: 10.1101/2025.01.06.603201. Fulltekst i vitenarkiv Vis sammendrag

  ABSTRACT CRISPR/Cas systems offer powerful tools for genome editing, but their therapeutic application is hampered by the risk of unintended off-target mutations. Many molecular methods have been established to detect off-target editing, however, their sensitivity depends on first identifying potential sites using in silico methods. However, these in silico prediction methods are challenged by a trade-off between speed and sensitivity, and can fail to comprehensively detect all edited off-target sites. Here, we demonstrate that ignoring bulges has led to missing editing at off-target sites in previous studies and that continuing this practice can lead to inflated claims of fidelity. As a solution, we introduce the concept of symbolic alignments to efficiently identify off-targets without sacrificing sensitivity. We further present specialized data structures that enable rapid, alignment-free probabilistic ranking of guide RNAs based on their predicted off-target burden. Implemented in the tool CHOPOFF, these innovations accommodate mismatches, bulges (insertions/deletions), and incorporate genomic sequence variants for personalized off-target assessment. Benchmarking demonstrates that CHOPOFF significantly outperforms state-of-the-art tools in both prediction accuracy and computational speed. Availability CHOPOFF command line available at https://github.com/JokingHero/CHOPOFF.jl CHOPOFF web server available at https://crisprtools.org/chopoff

• Mamia, Katariina Aino Inkeri; Kolbeinsdottir, Solrun; Labun, Kornel; Li, Zhuokun; Komisarczuk, Anna Zofia & Keskitalo, Salla [Vis alle 32 forfattere av denne artikkelen] (2025). Precision T cell correction platform for Inborn Errors of Immunity. Molecular Therapy. ISSN 1525-0016. 33(11), s. 5624–5643. doi: 10.1016/j.ymthe.2025.08.018. Fulltekst i vitenarkiv Vis sammendrag

  CRISPR/Cas9 gene editing is a promising tool to correct pathogenic variants for autologous cell therapies targeting Inborn errors of immunity (IEI). Current strategies, such as gene knockout or cDNA knock-in, address many single-gene defects but can disrupt gene expression, highlighting the need for precise correction platforms. While transplanting corrected autologous hematopoietic stem cells is a curative approach, it is unsuitable for patients with advanced disease, inflammation or acute infections. As correcting T cells is an alternative therapeutic strategy for lymphoid IEIs, we present an efficient T cell single nucleotide variant (SNV) correction platform based on homology-directed repair (HDR). By using STAT1 gain-of-function (STAT1-GOF), Cartilage Hair Hypoplasia (CHH), Deficiency of ADA2 (DADA2), and Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) as IEI models, we demonstrate that our platform achieves up to 80% correction, with resultant functional correction of the disease phenotype in the selected models. Furthermore, we performed safety profiling using GUIDE-seq, single-cell RNA sequencing, long-read genome sequencing, and proteomics analysis and detected no genomic, transcriptomic, or proteomic aberrations. This study establishes HDR-based SNV editing as a portable method for developing clinical autologous T cell therapies and represents a promising step toward a broad-spectrum gene correction platform for treating diverse monogenic immune disorders.

• Tierney, Jack A.S.; Świrski, Michał I.; Tjeldnes, Håkon; Kiran, Anmol M.; Carancini, Gionmattia & Kiniry, Stephen J. [Vis alle 10 forfattere av denne artikkelen] (2025). RiboSeq.Org: an integrated suite of resources for ribosome profiling data analysis and visualization. Nucleic Acids Research (NAR). ISSN 0305-1048. 53(1), s. D268–D274. doi: 10.1093/nar/gkae1020. Fulltekst i vitenarkiv
• Deutsch, Eric W; Kok, Leron W; Mudge, Jonathan M; Ruiz-Orera, Jorge; Fierro-Monti, Ivo & Sun, Zhi [Vis alle 43 forfattere av denne artikkelen] (2024). High-quality peptide evidence for annotating non-canonical open reading frames as human proteins. bioRxiv. ISSN 2692-8205. doi: 10.1101/2024.09.09.612016. Fulltekst i vitenarkiv Vis sammendrag

  Abstract A major scientific drive is to characterize the protein-coding genome as it provides the primary basis for the study of human health. But the fundamental question remains: what has been missed in prior genomic analyses? Over the past decade, the translation of non-canonical open reading frames (ncORFs) has been observed across human cell types and disease states, with major implications for proteomics, genomics, and clinical science. However, the impact of ncORFs has been limited by the absence of a large-scale understanding of their contribution to the human proteome. Here, we report the collaborative efforts of stakeholders in proteomics, immunopeptidomics, Ribo-seq ORF discovery, and gene annotation, to produce a consensus landscape of protein-level evidence for ncORFs. We show that at least 25% of a set of 7,264 ncORFs give rise to translated gene products, yielding over 3,000 peptides in a pan-proteome analysis encompassing 3.8 billion mass spectra from 95,520 experiments. With these data, we developed an annotation framework for ncORFs and created public tools for researchers through GENCODE and PeptideAtlas. This work will provide a platform to advance ncORF-derived proteins in biomedical discovery and, beyond humans, diverse animals and plants where ncORFs are similarly observed.

• Sivertsen, Audun; Mortensen, Nicolay; Solem, Unni; Valen, Eivind; Bullita, Marie Francoise & Wensaas, Knut-Arne [Vis alle 10 forfattere av denne artikkelen] (2024). Comprehensive contact tracing during an outbreak of alpha-variant SARS-CoV-2 in a rural community reveals less viral genomic diversity and higher household secondary attack rates than expected. mSphere. 9(8). doi: 10.1128/msphere.00114-24. Fulltekst i vitenarkiv Vis sammendrag

  Sequencing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes throughout the COVID-19 pandemic has generated a wealth of data on viral evolution across populations, but only a few studies have so far explored SARS-CoV-2 evolution across large connected transmission networks. Here, we couple data from SARS-CoV-2 sequencing with contact tracing data from an outbreak with a single origin in a rural Norwegian community where samples from all exposed persons were collected prospectively. A total of 134 nasopharyngeal samples were positive by PCR. Among the 121 retrievable genomes, 81 were identical to the genome of the introductor, thus demonstrating that genomics beyond clustering genotypically similar viral genomes to confirm relatedness offers limited additional value to manual contact tracing. In the cases where mutations were discovered, five small genetic clusters were identified. We observed a household secondary attack rate of 77%, with 92% of household members infected among households with secondary transmission, suggesting that SARS-CoV-2 introduction into large families is likely to affect all household members.

• Kelley, Leanne H.; Caldas, Ian V.; Sullenberger, Matthew T.; Yongblah, Kevin E.; Niazi, Adnan Muhammad & Iyer, Anoop [Vis alle 11 forfattere av denne artikkelen] (2024). Poly(U) polymerase activity in Caenorhabditis elegans regulates abundance and tailing of sRNA and mRNA. Genetics. ISSN 0016-6731. 228(2). doi: 10.1093/genetics/iyae120. Fulltekst i vitenarkiv Vis sammendrag

  Terminal nucleotidyltransferases add nucleotides to the 3′ end of RNA to modify their stability and function. In Caenorhabditis elegans, the terminal uridyltransferases/poly(U) polymerases PUP-1 (aka CID-1, CDE-1), PUP-2, and PUP-3 affect germline identity, survival, and development. Here, we identify small RNA (sRNA) and mRNA targets of these PUPs and of a fourth predicted poly(U) polymerase, F43E2.1/PUP-4. Using genetic and RNA sequencing approaches, we identify RNA targets of each PUP and the U-tail frequency and length of those targets. At the whole organism level, PUP-1 is responsible for most sRNA U-tailing, and other PUPs contribute to modifying discrete subsets of sRNAs. Moreover, the expression of PUP-2, PUP-3, and especially PUP-4 limits uridylation on some sRNAs. The relationship between uridylation status and sRNA abundance suggests that U-tailing can have a negative or positive effect on abundance depending on context. sRNAs modified by PUP activity primarily target mRNAs that are ubiquitously expressed or most highly expressed in the germline. mRNA data obtained with a Nanopore-based method reveal that the addition of U-tails to nonadenylated mRNA is substantially reduced in the absence of PUP-3. Overall, this work identifies PUP RNA targets, defines the effect of uridylation loss on RNA abundance, and reveals the complexity of PUP regulation in C. elegans development.

• Tierney, Jack A.S.; Sẃirski, Michał; Tjeldnes, Håkon; Mudge, Jonathan M.; Kufel, Joanna & Whiffin, Nicola [Vis alle 8 forfattere av denne artikkelen] (2024). Ribosome decision graphs for the representation of eukaryotic RNA translation complexity. Genome Research. ISSN 1088-9051. 34(4), s. 530–538. doi: 10.1101/gr.278810.123. Fulltekst i vitenarkiv
• Svalastoga, Pernille; Kaci, Alba; Molnes, Janne; Solheim, Marie Holm; Johansson, Bente Berg & Krogvold, Lars [Vis alle 16 forfattere av denne artikkelen] (2023). Characterisation of HNF1A variants in paediatric diabetes in Norway using functional and clinical investigations to unmask phenotype and monogenic diabetes. Diabetologia. ISSN 0012-186X. 66, s. 2226–2237. doi: 10.1007/s00125-023-06012-4. Fulltekst i vitenarkiv Vis sammendrag

  Aims/hypothesis Correctly diagnosing MODY is important, as individuals with this diagnosis can discontinue insulin injections; however, many people are misdiagnosed. We aimed to develop a robust approach for determining the pathogenicity of variants of uncertain significance in hepatocyte nuclear factor-1 alpha (HNF1A)-MODY and to obtain an accurate estimate of the prevalence of HNF1A-MODY in paediatric cases of diabetes. Methods We extended our previous screening of the Norwegian Childhood Diabetes Registry by 830 additional samples and comprehensively genotyped HNF1A variants in autoantibody-negative participants using next-generation sequencing. Carriers of pathogenic variants were treated by local healthcare providers, and participants with novel likely pathogenic variants and variants of uncertain significance were enrolled in an investigator-initiated, non-randomised, open-label pilot study (ClinicalTrials.gov registration no. NCT04239586). To identify variants associated with HNF1A-MODY, we functionally characterised their pathogenicity and assessed the carriers’ phenotype and treatment response to sulfonylurea. Results In total, 615 autoantibody-negative participants among 4712 cases of paediatric diabetes underwent genetic sequencing, revealing 19 with HNF1A variants. We identified nine carriers with novel variants classified as variants of uncertain significance or likely to be pathogenic, while the remaining ten participants carried five pathogenic variants previously reported. Of the nine carriers with novel variants, six responded favourably to sulfonylurea. Functional investigations revealed their variants to be dysfunctional and demonstrated a correlation with the resulting phenotype, providing evidence for reclassifying these variants as pathogenic. Conclusions/interpretation Based on this robust classification, we estimate that the prevalence of HNF1A-MODY is 0.3% in paediatric diabetes. Clinical phenotyping is challenging and functional investigations provide a strong complementary line of evidence. We demonstrate here that combining clinical phenotyping with functional protein studies provides a powerful tool to obtain a precise diagnosis of HNF1A-MODY.

• Bizuayehu, Teshome Tilahun; Labun, Kornel; Jakubec, Martin; Jefimov, Kirill; Niazi, Adnan Muhammad & Valen, Eivind Dale (2022). Long-read single-molecule RNA structure sequencing using nanopore. Nucleic Acids Research (NAR). ISSN 0305-1048. 50(20). doi: 10.1093/nar/gkac775. Fulltekst i vitenarkiv Vis sammendrag

  RNA molecules can form secondary and tertiary structures that can regulate their localization and function. Using enzymatic or chemical probing together with high-throughput sequencing, secondary structure can be mapped across the entire transcriptome. However, a limiting factor is that only population averages can be obtained since each read is an independent measurement. Although long-read sequencing has recently been used to determine RNA structure, these methods still used aggregate signals across the strands to detect structure. Averaging across the population also means that only limited information about structural heterogeneity across molecules or dependencies within each molecule can be obtained. Here, we present Single-Molecule Structure sequencing (SMS-seq) that combines structural probing with native RNA sequencing to provide non-amplified, structural profiles of individual molecules with novel analysis methods. Our new approach using mutual information enabled single molecule structural interrogation. Each RNA is probed at numerous bases enabling the discovery of dependencies and heterogeneity of structural features. We also show that SMS-seq can capture tertiary interactions, dynamics of riboswitch ligand binding, and mRNA structural features.

• Kute, Preeti; Soukarieh, Omar; Tjeldnes, Håkon; Trégouët, David-Alexandre & Valen, Eivind Dale (2022). Small Open Reading Frames, How to Find Them and Determine Their Function. Frontiers in Genetics. 12. doi: 10.3389/fgene.2021.796060. Fulltekst i vitenarkiv Vis sammendrag

  Advances in genomics and molecular biology have revealed an abundance of small open reading frames (sORFs) across all types of transcripts. While these sORFs are often assumed to be non-functional, many have been implicated in physiological functions and a significant number of sORFs have been described in human diseases. Thus, sORFs may represent a hidden repository of functional elements that could serve as therapeutic targets. Unlike protein-coding genes, it is not necessarily the encoded peptide of an sORF that enacts its function, sometimes simply the act of translating an sORF might have a regulatory role. Indeed, the most studied sORFs are located in the 5′UTRs of coding transcripts and can have a regulatory impact on the translation of the downstream protein-coding sequence. However, sORFs have also been abundantly identified in non-coding RNAs including lncRNAs, circular RNAs and ribosomal RNAs suggesting that sORFs may be diverse in function. Of the many different experimental methods used to discover sORFs, the most commonly used are ribosome profiling and mass spectrometry. These can confirm interactions between transcripts and ribosomes and the production of a peptide, respectively. Extensions to ribosome profiling, which also capture scanning ribosomes, have further made it possible to see how sORFs impact the translation initiation of mRNAs. While high-throughput techniques have made the identification of sORFs less difficult, defining their function, if any, is typically more challenging. Together, the abundance and potential function of many of these sORFs argues for the necessity of including sORFs in gene annotations and systematically characterizing these to understand their potential functional roles. In this review, we will focus on the high-throughput methods used in the detection and characterization of sORFs and discuss techniques for validation and functional characterization.

• Begik, Oguzhan; Diensthuber, Gregor; Liu, Huanle; Delgado-Tejedor, Anna; Kontur, Cassandra & Niazi, Adnan Muhammad [Vis alle 11 forfattere av denne artikkelen] (2022). Nano3P-seq: transcriptome-wide analysis of gene expression and tail dynamics using end-capture nanopore cDNA sequencing. Nature Methods. ISSN 1548-7091. 20. doi: 10.1038/s41592-022-01714-w. Fulltekst i vitenarkiv Vis sammendrag

  RNA polyadenylation plays a central role in RNA maturation, fate, and stability. In response to developmental cues, polyA tail lengths can vary, affecting the translation efficiency and stability of mRNAs. Here we develop Nanopore 3′ end-capture sequencing (Nano3P-seq), a method that relies on nanopore cDNA sequencing to simultaneously quantify RNA abundance, tail composition, and tail length dynamics at per-read resolution. By employing a template-switching-based sequencing protocol, Nano3P-seq can sequence RNA molecule from its 3′ end, regardless of its polyadenylation status, without the need for PCR amplification or ligation of RNA adapters. We demonstrate that Nano3P-seq provides quantitative estimates of RNA abundance and tail lengths, and captures a wide diversity of RNA biotypes. We find that, in addition to mRNA and long non-coding RNA, polyA tails can be identified in 16S mitochondrial ribosomal RNA in both mouse and zebrafish models. Moreover, we show that mRNA tail lengths are dynamically regulated during vertebrate embryogenesis at an isoform-specific level, correlating with mRNA decay. Finally, we demonstrate the ability of Nano3P-seq in capturing non-A bases within polyA tails of various lengths, and reveal their distribution during vertebrate embryogenesis. Overall, Nano3P-seq is a simple and robust method for accurately estimating transcript levels, tail lengths, and tail composition heterogeneity in individual reads, with minimal library preparation biases, both in the coding and non-coding transcriptome.

• Grapotte, Mathys; Saraswat, Manu; Bessière, Chloé; Menichelli, Christophe; Ramilowski, Jordan A. & Severin, Jessica [Vis alle 490 forfattere av denne artikkelen] (2021). Discovery of widespread transcription initiation at microsatellites predictable by sequence-based deep neural network. Nature Communications. 12(1). doi: 10.1038/s41467-021-23143-7. Fulltekst i vitenarkiv Vis sammendrag

  Abstract Using the Cap Analysis of Gene Expression (CAGE) technology, the FANTOM5 consortium provided one of the most comprehensive maps of transcription start sites (TSSs) in several species. Strikingly, ~72% of them could not be assigned to a specific gene and initiate at unconventional regions, outside promoters or enhancers. Here, we probe these unassigned TSSs and show that, in all species studied, a significant fraction of CAGE peaks initiate at microsatellites, also called short tandem repeats (STRs). To confirm this transcription, we develop Cap Trap RNA-seq, a technology which combines cap trapping and long read MinION sequencing. We train sequence-based deep learning models able to predict CAGE signal at STRs with high accuracy. These models unveil the importance of STR surrounding sequences not only to distinguish STR classes, but also to predict the level of transcription initiation. Importantly, genetic variants linked to human diseases are preferentially found at STRs with high transcription initiation level, supporting the biological and clinical relevance of transcription initiation at STRs. Together, our results extend the repertoire of non-coding transcription associated with DNA tandem repeats and complexify STR polymorphism.

• Niazi, Adnan Muhammad; Krause, Maximilian & Valen, Eivind Dale (2021). Transcript Isoform-Specific Estimation of Poly(A) Tail Length by Nanopore Sequencing of Native RNA. Methods in molecular biology. ISSN 1064-3745. 2284, s. 543–567. doi: 10.1007/978-1-0716-1307-8_30. Fulltekst i vitenarkiv
• Tjeldnes, Håkon; Labun, Kornel; Cleuren, Yamila Nicole Torres; Chyzynska, Katarzyna; Swirski, Michal & Valen, Eivind Dale (2021). ORFik: a comprehensive R toolkit for the analysis of translation. BMC Bioinformatics. 22, s. 1–16. doi: 10.1186/s12859-021-04254-w. Fulltekst i vitenarkiv Vis sammendrag

  Background With the rapid growth in the use of high-throughput methods for characterizing translation and the continued expansion of multi-omics, there is a need for back-end functions and streamlined tools for processing, analyzing, and characterizing data produced by these assays. Results Here, we introduce ORFik, a user-friendly R/Bioconductor API and toolbox for studying translation and its regulation. It extends GenomicRanges from the genome to the transcriptome and implements a framework that integrates data from several sources. ORFik streamlines the steps to process, analyze, and visualize the different steps of translation with a particular focus on initiation and elongation. It accepts high-throughput sequencing data from ribosome profiling to quantify ribosome elongation or RCP-seq/TCP-seq to also quantify ribosome scanning. In addition, ORFik can use CAGE data to accurately determine 5′UTRs and RNA-seq for determining translation relative to RNA abundance. ORFik supports and calculates over 30 different translation-related features and metrics from the literature and can annotate translated regions such as proteins or upstream open reading frames (uORFs). As a use-case, we demonstrate using ORFik to rapidly annotate the dynamics of 5′ UTRs across different tissues, detect their uORFs, and characterize their scanning and translation in the downstream protein-coding regions. Conclusion In summary, ORFik introduces hundreds of tested, documented and optimized methods. ORFik is designed to be easily customizable, enabling users to create complete workflows from raw data to publication-ready figures for several types of sequencing data. Finally, by improving speed and scope of many core Bioconductor functions, ORFik offers enhancement benefiting the entire Bioconductor environment.

• Labun, Kornel; Krause, Maximilian; Cleuren, Yamila Nicole Torres & Valen, Eivind Dale (2021). CRISPR Genome Editing Made Easy Through the CHOPCHOP Website. Current Protocols in Bioinformatics. ISSN 1934-3396. 1. doi: 10.1002/cpz1.46. Fulltekst i vitenarkiv Vis sammendrag

  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.

• Chyzynska, Katarzyna; Labun, Kornel; Jones, Carl Matthew; Grellscheid, Sushma Nagaraja & Valen, Eivind Dale (2021). Deep conservation of ribosome stall sites across RNA processing genes. NAR Genomics and Bioinformatics. 3(2), s. 1–13. doi: 10.1093/nargab/lqab038. Fulltekst i vitenarkiv Vis sammendrag

  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.

• Reint, Ganna; Li, Zhuokun; Labun, Kornel; Keskitalo, Salla; Soppa, Inkeri & Mamia, Katariina Aino Inkeri [Vis alle 19 forfattere av denne artikkelen] (2021). Rapid genome editing by CRISPR-Cas9-POLD3 fusion. eLIFE. 11. doi: 10.7554/eLife.75415. Fulltekst i vitenarkiv Vis sammendrag

  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.

• Svalastoga, Pernille; Sulen, Åsta Nordsveen; Fehn, Jarle Røneid; Aukland, Stein Magnus; Irgens, Henrik Underthun & Sirnes, Eivind [Vis alle 10 forfattere av denne artikkelen] (2020). Intellectual disability in KATP channel neonatal diabetes. Diabetes Care. ISSN 0149-5992. 43(3), s. 526–533. doi: 10.2337/dc19-1013. Fulltekst i vitenarkiv
• Poduval, Deepak; Berge, Elisabet Ognedal; Sichmanovà, Zuzana; Valen, Eivind; Iversen, Gjertrud Titlestad & Minsaas, Laura [Vis alle 8 forfattere av denne artikkelen] (2020). Assessment of tumor suppressor promoter methylation in healthy individuals. Clinical Epigenetics. 12:131, s. 1–15. doi: 10.1186/s13148-020-00920-7. Fulltekst i vitenarkiv Vis sammendrag

  Background The number of tumor suppressor genes for which germline mutations have been linked to cancer risk is steadily increasing. However, while recent reports have linked constitutional normal tissue promoter methylation of BRCA1 and MLH1 to ovarian and colon cancer risk, the role of epigenetic alterations as cancer risk factors remains largely unknown, presenting an important area for future research. Currently, we lack fast and sensitive methods for assessment of promoter methylation status across known tumor suppressor genes. Results In this paper, we present a novel NGS-based approach assessing promoter methylation status across a large panel of defined tumor suppressor genes to base-pair resolution. The method omits the limitations related to commonly used array-approaches. Our panel includes 565 target regions covering the promoters of 283 defined tumor suppressors, selected by pre-specified criteria, and was applied for rapid targeted methylation-specific NGS. The feasibility of the method was assessed by analyzing normal tissue DNA (white blood cells, WBC) samples from 34 healthy postmenopausal women and by performing preliminary assessment of the methylation landscape of tumor suppressors in these individuals. The mean target coverage was 189.6x providing a sensitivity of 0.53%, sufficient for promoter methylation assessment of low-level methylated genes like BRCA1. Within this limited test-set, we detected 206 regions located in the promoters of 149 genes to be differentially methylated (hyper- or hypo-) at > 99% confidence level. Seven target regions in gene promoters (CIITA, RASSF1, CHN1, PDCD1LG2, GSTP1, XPA, and ZNF668) were found to be hyper-methylated in a minority of individuals, with a > 20 percent point difference in mean methylation across the region between individuals. In an exploratory hierarchical clustering analysis, we found that the individuals analyzed may be grouped into two main groups based on their WBC methylation profile across the 283 tumor suppressor gene promoters. Conclusions Methylation-specific NGS of our tumor suppressor panel, with detailed assessment of differential methylation in healthy individuals, presents a feasible method for identification of novel epigenetic risk factors for cancer.

• Giess, Adam; Cleuren, Yamila Nicole Torres; Tjeldnes, Håkon; Krause, Maximilian; Bizuayehu, Teshome Tilahun & Hiensch, Senna [Vis alle 9 forfattere av denne artikkelen] (2020). Profiling of Small Ribosomal Subunits Reveals Modes and Regulation of Translation Initiation. Cell reports. 31(3). doi: 10.1016/j.celrep.2020.107534. Fulltekst i vitenarkiv Vis sammendrag

  Translation initiation is often attributed as the rate-determining step of eukaryotic protein synthesis and key to gene expression control. Despite this centrality, the series of steps involved in this process is poorly understood. Here, we capture the transcriptome-wide occupancy of ribosomes across all stages of translation initiation, enabling us to characterize the transcriptome-wide dynamics of ribosome recruitment to mRNAs, scanning across 5′ UTRs and stop codon recognition, in a higher eukaryote. We provide mechanistic evidence for ribosomes attaching to the mRNA by threading the mRNA through the small subunit. Moreover, we identify features that regulate the recruitment and processivity of scanning ribosomes and redefine optimal initiation contexts. Our approach enables deconvoluting translation initiation into separate stages and identifying regulators at each step.

• Pálfy, Máté; Schulze, Gunnar; Valen, Eivind & Vastenhouw, Nadine L. (2020). Chromatin accessibility established by Pou5f3, Sox19b and Nanog primes genes for activity during zebrafish genome activation. PLoS Genetics. ISSN 1553-7390. 16(1), s. 1–25. doi: 10.1371/journal.pgen.1008546. Fulltekst i vitenarkiv Vis sammendrag

  In many organisms, early embryonic development is driven by maternally provided factors until the controlled onset of transcription during zygotic genome activation. The regulation of chromatin accessibility and its relationship to gene activity during this transition remain poorly understood. Here, we generated chromatin accessibility maps with ATAC-seq from genome activation until the onset of lineage specification. During this period, chromatin accessibility increases at regulatory elements. This increase is independent of RNA polymerase II-mediated transcription, with the exception of the hypertranscribed miR-430 locus. Instead, accessibility often precedes the transcription of associated genes. Loss of the maternal transcription factors Pou5f3, Sox19b, and Nanog, which are known to be required for zebrafish genome activation, results in decreased accessibility at regulatory elements. Importantly, the accessibility of regulatory regions, especially when established by Pou5f3, Sox19b and Nanog, is predictive for future transcription. Our results show that the maternally provided transcription factors Pou5f3, Sox19b, and Nanog open up chromatin and prime genes for activity during zygotic genome activation in zebrafish.

• Danks, Gemma Barbara; Galbiati, Heloisa; Raasholm, Martina; Cleuren, Yamila Nicole Torres; Valen, Eivind & Navratilova, Pavla [Vis alle 7 forfattere av denne artikkelen] (2019). Trans-splicing of mRNAs links gene transcription to translational control regulated by mTOR. BMC Genomics. 20(1). doi: 10.1186/s12864-019-6277-x. Fulltekst i vitenarkiv Vis sammendrag

  Background In phylogenetically diverse organisms, the 5′ ends of a subset of mRNAs are trans-spliced with a spliced leader (SL) RNA. The functions of SL trans-splicing, however, remain largely enigmatic. Results We quantified translation genome-wide in the marine chordate, Oikopleura dioica, under inhibition of mTOR, a central growth regulator. Translation of trans-spliced TOP mRNAs was suppressed, consistent with a role of the SL sequence in nutrient-dependent translational control of growth-related mRNAs. Under crowded, nutrient-limiting conditions, O. dioica continued to filter-feed, but arrested growth until favorable conditions returned. Upon release from unfavorable conditions, initial recovery was independent of nutrient-responsive, trans-spliced genes, suggesting animal density sensing as a first trigger for resumption of development. Conclusion Our results are consistent with a proposed role of trans-splicing in the coordinated translational down-regulation of nutrient-responsive genes under growth-limiting conditions.

• Krause, Maximilian; Niazi, Adnan Muhammad; Labun, Kornel; Cleuren, Yamila Nicole Torres; Müller, Florian Sebastian Alexander & Valen, Eivind (2019). tailfindr: Alignment-free poly(A) length measurement for Oxford Nanopore RNA and DNA sequencing. RNA: A publication of the RNA Society. ISSN 1355-8382. 25(10), s. 1229–1241. doi: 10.1261/rna.071332.119. Fulltekst i vitenarkiv Vis sammendrag

  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.

• Labun, Kornel; Montague, Tessa G.; Krause, Maximilian; Cleuren, Yamila Nicole Torres; Tjeldnes, Håkon & Valen, Eivind (2019). CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing. Nucleic Acids Research (NAR). ISSN 0305-1048. 47(W1), s. W171–W174. doi: 10.1093/nar/gkz365. Fulltekst i vitenarkiv Vis sammendrag

  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.no

• Labun, Kornel; Guo, Xiaoge; Chavez, Alejandro; Church, George; Gagnon, James A. & Valen, Eivind (2019). Accurate analysis of genuine CRISPR editing events with ampliCan. Genome Research. ISSN 1088-9051. 29(5), s. 843–847. doi: 10.1101/gr.244293.118. Fulltekst i vitenarkiv Vis sammendrag

  We present ampliCan, an analysis tool for genome editing that unites highly precise quantification and visualization of genuine genome editing events. ampliCan features nuclease-optimized alignments, filtering of experimental artifacts, event-specific normalization, and off-target read detection and quantifies insertions, deletions, HDR repair, as well as targeted base editing. It is scalable to thousands of amplicon sequencing–based experiments from any genome editing experiment, including CRISPR. It enables automated integration of controls and accounts for biases at every step of the analysis. We benchmarked ampliCan on both real and simulated data sets against other leading tools, demonstrating that it outperformed all in the face of common confounding factors.

• Birkeland, Åsmund; Chyzynska, Katarzyna & Valen, Eivind (2018). Shoelaces: An interactive tool for ribosome profiling processing and visualization. BMC Genomics. 19. doi: 10.1186/s12864-018-4912-6. Fulltekst i vitenarkiv Vis sammendrag

  Background The emergence of ribosome profiling to map actively translating ribosomes has laid the foundation for a diverse range of studies on translational regulation. The data obtained with different variations of this assay is typically manually processed, which has created a need for tools that would streamline and standardize processing steps. Results We present Shoelaces, a toolkit for ribosome profiling experiments automating read selection and filtering to obtain genuine translating footprints. Based on periodicity, favoring enrichment over the coding regions, it determines the read lengths corresponding to bona fide ribosome protected fragments. The specific codon under translation (P-site) is determined by automatic offset calculations resulting in sub-codon resolution. Shoelaces provides both a user-friendly graphical interface for interactive visualisation in a genome browser-like fashion and a command line interface for integration into automated pipelines. We process 79 libraries and show that studies typically discard excessive amounts of quality data in their manual analysis pipelines. Conclusions Shoelaces streamlines ribosome profiling analysis offering automation of the processing, a range of interactive visualization features and export of the data into standard formats. Shoelaces stores all processing steps performed in an XML file that can be used by other groups to exactly reproduce the processing of a given study. We therefore anticipate that Shoelaces can aid researchers by automating what is typically performed manually and contribute to the overall reproducibility of studies. The tool is freely distributed as a Python package, with additional instructions, tutorial and demo datasets available at https://bitbucket.org/valenlab/shoelaces.

• Giess, Adam; Jonckheere, Veronique; Ndah, Elvis; Chyzynska, Katarzyna; Damme, Petra Van & Valen, Eivind (2017). Ribosome signatures aid bacterial translation initiation site identification. BMC Biology. 15:76, s. 1–14. doi: 10.1186/s12915-017-0416-0. Fulltekst i vitenarkiv Vis sammendrag

  Background While methods for annotation of genes are increasingly reliable, the exact identification of translation initiation sites 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. Results Utilising these signatures in combination with nucleotide sequence information, we build a model capable of predicting translation initiation sites and demonstrate its high accuracy using N-terminal proteomics. Applying this to prokaryotic translatomes, we re-annotate translation initiation sites and provide evidence of N-terminal truncations and extensions of previously annotated coding sequences. These re-annotations are supported by the presence of structural and sequence-based features next to N-terminal peptide evidence. Finally, our model identifies 61 novel genes previously undiscovered in the Salmonella enterica genome. Conclusions Signatures within ribosome profiling read length distributions can be used in combination with nucleotide sequence information to provide accurate genome-wide identification of translation initiation sites.

• Ndah, Elvis; Jonckheere, Veronique; Giess, Adam; Valen, Eivind; Menschaert, Gerben & Damme, Petra Van (2017). REPARATION: ribosome profiling assisted (re-) annotation of bacterial genomes. Nucleic Acids Research (NAR). ISSN 0305-1048. 45(20). doi: 10.1093/nar/gkx758. Fulltekst i vitenarkiv Vis sammendrag

  Prokaryotic genome annotation is highly dependent on automated methods, as manual curation cannot keep up with the exponential growth of sequenced genomes. Current automated methods depend heavily on sequence context and often underestimate the complexity of the proteome. We developed REPARATION (RibosomeE Profiling Assisted (Re-)AnnotaTION), a de novo algorithm that takes advantage of experimental protein translation evidence from ribosome profiling (Ribo-seq) to delineate translated open reading frames (ORFs) in bacteria, independent of genome annotation. REPARATION evaluates all possible ORFs in the genome and estimates minimum thresholds based on a growth curve model to screen for spurious ORFs. We applied REPARATION to three annotated bacterial species to obtain a more comprehensive mapping of their translation landscape in support of experimental data. In all cases, we identified hundreds of novel (small) ORFs including variants of previously annotated ORFs. Our predictions were supported by matching mass spectrometry (MS) proteomics data, sequence composition and conservation analysis. REPARATION is unique in that it makes use of experimental translation evidence to perform de novo ORF delineation in bacterial genomes irrespective of the sequence context of the reading frame.

• Valen, Eivind; Labun, Kornel; Montague, Tessa G.; Gagnon, James A. & Thyme, Summer B. (2016). CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering. Nucleic Acids Research (NAR). ISSN 0305-1048. 44. doi: 10.1093/nar/gkw398. Fulltekst i vitenarkiv Vis sammendrag

  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.

• Thyme, Summer B.; Akhmetova, Laila; Montague, Tessa G.; Valen, Eivind & Schier, Alexander F. (2016). Internal guide RNA interactions interfere with Cas9-mediated cleavage. Nature Communications. 7:11750. doi: 10.1038/ncomms11750. Fulltekst i vitenarkiv Vis sammendrag

  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.

• Merkle, Florian T.; Neuhausser, Werner M.; Santos, David; Valen, Eivind; Gagnon, James A. & Maas, Kristi [Vis alle 9 forfattere av denne artikkelen] (2015). Efficient CRISPR-Cas9-mediated generation of knockin human pluripotent stem cells lacking undesired mutations at the targeted locus. Cell reports. 11(6), s. 875–883. doi: 10.1016/j.celrep.2015.04.007. Fulltekst i vitenarkiv Vis sammendrag

  The CRISPR-Cas9 system has the potential to revolutionize genome editing in human pluripotent stem cells (hPSCs), but its advantages and pitfalls are still poorly understood. We systematically tested the ability of CRISPR-Cas9 to mediate reporter gene knockin at 16 distinct genomic sites in hPSCs. We observed efficient gene targeting but found that targeted clones carried an unexpectedly high frequency of insertion and deletion (indel) mutations at both alleles of the targeted gene. These indels were induced by Cas9 nuclease, as well as Cas9-D10A single or dual nickases, and often disrupted gene function. To overcome this problem, we designed strategies to physically destroy or separate CRISPR target sites at the targeted allele and developed a bioinformatic pipeline to identify and eliminate clones harboring deleterious indels at the other allele. This two-pronged approach enables the reliable generation of knockin hPSC reporter cell lines free of unwanted mutations at the targeted locus.

• Andersson, Robin; Andersen, Peter Refsing; Valen, Eivind; Core, Leighton J.; Bornholdt, Jette & Boyd, Mette [Vis alle 8 forfattere av denne artikkelen] (2014). Nuclear stability and transcriptional directionality separate functionally distinct RNA species. Nature Communications. 5:5336. doi: 10.1038/ncomms6336. Fulltekst i vitenarkiv Vis sammendrag

  Mammalian genomes are pervasively transcribed, yielding a complex transcriptome with high variability in composition and cellular abundance. Although recent efforts have identified thousands of new long non-coding (lnc) RNAs and demonstrated a complex transcriptional repertoire produced by protein-coding (pc) genes, limited progress has been made in distinguishing functional RNA from spurious transcription events. This is partly due to present RNA classification, which is typically based on technical rather than biochemical criteria. Here we devise a strategy to systematically categorize human RNAs by their sensitivity to the ribonucleolytic RNA exosome complex and by the nature of their transcription initiation. These measures are surprisingly effective at correctly classifying annotated transcripts, including lncRNAs of known function. The approach also identifies uncharacterized stable lncRNAs, hidden among a vast majority of unstable transcripts. The predictive power of the approach promises to streamline the functional analysis of known and novel RNAs.

Se alle arbeider i NVA

• Rio, Oline Eriksen; Labun, Kornel; Dahal-Koirala, Shiva; Komisarczuk, Anna Zofia; Valen, Eivind & Haapaniemi, Emma Maria (2026). SNIPSNP: precision design of CRISPR reagents. Fulltekst i vitenarkiv
• Świrski, Michał I.; Tierney, Jack A. S.; Albà, M. Mar; Andreev, Dmitry E.; Aspden, Julie L. & Atkins, John F. [Vis alle 170 forfattere av denne artikkelen] (2025). Translon: a single term for translated regions. Nature Methods. ISSN 1548-7091. 22(10), s. 2002–2006. doi: 10.1038/s41592-025-02810-3. Fulltekst i vitenarkiv
• Haugen, Frida Loe; Mamia, Katariina Aino Inkeri; Rio, Oline Eriksen; Sollano, Anniken Solveig Matheson; Li, Zhuokun & Labun, Kornel [Vis alle 20 forfattere av denne artikkelen] (2025). CRISPR/Cas9-based pipeline to introduce custom edits in autologous T cells. Fulltekst i vitenarkiv
• Haugen, Frida Loe; Mamia, Katariina Aino Inkeri; Rio, Oline Eriksen; Sollano, Anniken Solveig Matheson; Li, Zhuokun & Labun, Kornel [Vis alle 20 forfattere av denne artikkelen] (2025). CRISPR-Cas9 pipeline for custom editing in autologous T cells. doi: https:/event.fourwaves.com/crisprmed25/abstracts/b5911e8e-de8e-4ee4-b8e2-9f08cc2f31d8. Fulltekst i vitenarkiv Vis sammendrag

  CRISPR/Cas9 gene editing technology is a promising tool for correcting pathogenic variants in Inborn Errors of Immunity (IEIs). Current IEI correction strategies rely on cDNA knock-in or gene knockout, which can disrupt gene expression and need extensive optimization. To address this, we developed a safe, efficient CRISPR/Cas9-based T cell editing pipeline using HDR for SNV correction. We observe a rapid and selective expansion of CD3+ T cells from patient peripheral blood following an 8-day cytokine stimulation. To enhance HDR outcomes, we explored multiple strategies and developed an optimized T cell pipeline capable of achieving up to 80% HDR. We found no pre-malignant off-target effects or karyotypic, transcriptomic, or proteomic abnormalities in patient T cells analysed with GUIDE-seq, scRNA-seq, long-read WGS, and proteomics. We observed functional correction of the disease phenotype in patient T cells demonstrated on transcriptomic level by single cell RNA sequencing, on proteomic level by Mass Spectrometry and in cell culture-based assays. So far, we have implemented the pipeline and corrected 12 unique SNV mutations causing 8 diverse IEIs. This platform is designed to streamline CRISPR tool discovery/optimization and can be further developed into a GMP-compatible, scaled-up process for salvage therapy in T cell-related IEIs.

• Mamia, Katariina Aino Inkeri; Kolbeinsdottir, Solrun; Li, Zhuokun; Reint, Ganna; Komisarczuk, Anna Zofia & Haugen, Frida Høsøien [Vis alle 21 forfattere av denne artikkelen] (2024). CRISPR/Cas9 T cell editing pipeline for monogenic primary immunodeficiency disorders. Fulltekst i vitenarkiv
• Mamia, Katariina Aino Inkeri; Kolbeinsdottir, Solrun; Li, Zhuokun; Reint, Ganna; Komisarczuk, Anna Zofia & Haugen, Frida Høsøien [Vis alle 21 forfattere av denne artikkelen] (2024). CRISPR/Cas9 T cell editing pipeline for monogenic primary immunodeficiency disorders. Fulltekst i vitenarkiv
• Mamia, Katariina Aino Inkeri; Kolbeinsdottir, Solrun; Li, Zhuokun; Reint, Ganna; Komisarczuk, Anna Zofia & Haugen, Frida Høsøien [Vis alle 21 forfattere av denne artikkelen] (2024). CRISPR/Cas9 T cell editing pipeline for monogenic primary immunodeficiency disorders. Fulltekst i vitenarkiv
• Reint, Ganna; Li, Zhuokun; Labun, Kornel; Keskitalo, Salla; Soppa, Inkeri & Mamia, Katariina Aino Inkeri [Vis alle 19 forfattere av denne artikkelen] (2022). Rapid genome editing by CRISPR-Cas9-POLD3 fusion. Fulltekst i vitenarkiv
• Grapotte, Mathys; Saraswat, Manu; Bessière, Chloé; Menichelli, Christophe; Ramilowski, Jordan A. & Severin, Jessica [Vis alle 489 forfattere av denne artikkelen] (2022). Author Correction: Discovery of widespread transcription initiation at microsatellites predictable by sequence-based deep neural network (Nature Communications, (2021), 12, 1, (3297), 10.1038/s41467-021-23143-7). Nature Communications. 13(1). doi: 10.1038/s41467-022-28758-y. Fulltekst i vitenarkiv
• Mamia, Katariina Aino Inkeri; Saarela, Janna Saija; Li, Zhuokun; Reint, Ganna; Fatkhutdinov, Nail & Haugen, Frida Høsøien [Vis alle 13 forfattere av denne artikkelen] (2022). CRISPR-Cas9 T cell editing pipeline for Finnish founder diseases. Fulltekst i vitenarkiv
• Valen, Eivind (2022). Nye CRISPR-metoder og mer presis presisjonsmedisin. Tidsskrift for Den norske legeforening (Tidsskriftet). ISSN 0029-2001. 142(17). doi: 10.4045/tidsskr.22.0689. Fulltekst i vitenarkiv
• Holt, Eric; Halberg, Nils Henrik & Valen, Eivind (2024). The pancreas remembers: epigenetic memory of obesity as subtle, coordinated changes in promoter accessibility. Universitetet i Bergen. Fulltekst i vitenarkiv Vis sammendrag

  Background: Epigenetic memory involves heritable gene expression changes without DNA sequence alterations, impacting cellular functions long after the initial triggers have ceased. The impact of obesity on pancreatic function has been well documented, but epigenetic memory in this context remains unclear. Objective: To explore epigenetic memory of obesity in the pancreas. Methods: Pancreata from mice with three dietary histories (obese, post-obese, lean) were analyzed using RNA sequencing (RNA-seq), chromatin immunoprecipitation followed by sequencing (ChIP-seq) for histone H3 lysine 27 acetylation (H3K27ac), and assay for transposase-accessible chromatin using sequencing (ATAC-seq), in which Tn5 transposase binding sites were used to assess chromatin accessibility. Results of differential gene expression, promoter accessibility, and histone acetylation analyses were compared. Gene set enrichment analysis (GSEA) was performed based on these results, and the identified epigenetic memory (EM) gene sets and their core genes were characterized. Results: Few individual genes were differential (𝑃adj < 0.05) in H3K27ac and Tn5 across all groups and in RNA-seq between post-obese and lean mice. GSEA revealed 240 EM gene sets, enriched in both obese and post-obese compared to lean, predominantly in Tn5. EM gene sets included 36 related to lipid metabolism, 29 to cellular respiration, and others related to RNA processing, nucleotide metabolism, translation, protein processing, mitosis, and chromatin. EM core genes had high promoter counts and showed subtle yet consistent and widespread, highly correlated changes in Tn5 and H3K27ac but not in RNA. EM core genes were upregulated in obese compared to lean in both Tn5 and H3K27ac; in post-obese, they were downregulated compared to obese but upregulated compared to lean in Tn5, while H3K27ac showed reversed trends. Conclusion: Epigenetic memory of obesity in the pancreas manifests as subtle, coordinated changes in promoter accessibility, not continuous gene expression changes. Histone acetylation, contrary to its general characterization as the upstream regulator of chromatin accessibility, does not act as such in this context. Bivalent chromatin may instead mediate the latent memory. The findings need replication, and further studies could investigate bivalency, DNA methylation, nucleosome occupancy, promoter-enhancer interaction, and temporal changes post-intervention.

• Giess, Adam & Valen, Eivind (2019). Understanding translational landscapes through the footprints of ribosomes. Universitetet i Bergen. ISSN 9788230835142. Fulltekst i vitenarkiv

Se alle arbeider i NVA