The accurate structural modeling of B- and T-cell receptors is fundamental to gain a detailed insight in the mechanisms underlying immunity and in developing new drugs and therapies. The LYRA (LYmphocyte Receptor Automated modeling) web server (http://www.cbs.dtu.dk/services/LYRA/) implements a complete and automated method for building of B- and T-cell receptor structural models starting from their amino acid sequence alone. The webserver is freely available and easy to use for non-specialists. Upon submission, LYRA automatically generates alignments using ad hoc profiles, predicts the structural class of each hypervariable loop, selects the best templates in an automatic fashion, and provides within minutes a complete 3D model that can be downloaded or inspected online. Experienced users can manually select or exclude template structures according to case specific information. LYRA is based on the canonical structure method, that in the last 30 years has been successfully used to generate antibody models of high accuracy, and in our benchmarks this approach proves to achieve similarly good results on TCR modeling, with a benchmarked average RMSD accuracy of 1.29 and 1.48 Å for B- and T-cell receptors, respectively. To the best of our knowledge, LYRA is the first automated server for the prediction of TCR structure.

Recombineering and multiplex automated genome engineering (MAGE) offer the possibility to rapidly modify multiple genomic or plasmid sites at high efficiencies. This enables efficient creation of genetic variants including both single mutants with specifically targeted modifications as well as combinatorial cell libraries. Manual design of oligonucleotides for these approaches can be tedious, time-consuming, and may not be practical for larger projects targeting many genomic sites. At present, the change from a desired phenotype (e.g. altered expression of a specific protein) to a designed MAGE oligo, which confers the corresponding genetic change, is performed manually. To address these challenges, we have developed the MAGE Oligo Design Tool (MODEST). This web-based tool allows designing of MAGE oligos for (i) tuning translation rates by modifying the ribosomal binding site, (ii) generating translational gene knockouts and (iii) introducing other coding or non-coding mutations, including amino acid substitutions, insertions, deletions and point mutations. The tool automatically designs oligos based on desired genotypic or phenotypic changes defined by the user, which can be used for high efficiency recombineering and MAGE. MODEST is available for free and is open to all users at http://modest.biosustain.dtu.dk.

Diffuse large B‐cell lymphoma (DLBCL) is characterized by extensive genetic heterogeneity, and this results in unpredictable responses to the current treatment, R‐CHOP, which consists of a cancer drug combination supplemented with the humanized CD20‐targeting monoclonal antibody rituximab. Despite improvements in the patient response rate through rituximab addition to the treatment plan, up to 40% of DLBCL patients end in a relapsed or refractory state due to inherent or acquired resistance to the regimen. Here, we employ a lentiviral genome‐wide clustered regularly interspaced short palindromic repeats library screening approach to identify genes involved in facilitating the rituximab response in cancerous B cells. Along with the CD20‐encoding MS4A1 gene, we identify genes related to B‐cell receptor (BCR) signaling as mediators of the intracellular signaling response to rituximab. More specifically, the B‐cell linker protein (BLNK) and Bruton's tyrosine kinase (BTK) genes stand out as pivotal genes in facilitating direct rituximab‐induced apoptosis through mechanisms that occur alongside complement‐dependent cytotoxicity (CDC). Our findings demonstrate that rituximab triggers BCR signaling in a BLNK‐ and BTK‐dependent manner and support the existing notion that intertwined CD20 and BCR signaling pathways in germinal center B‐cell‐like‐subtype DLBCL lead to programmed cell death.

Prime editing (PE) is a novel CRISPR-derived genome editing technique facilitating precision editing without double-stranded DNA breaks. PE, mediated by a Cas9-reverse transcriptase fusion protein, is based on dual-functioning prime editing guide RNAs (pegRNAs), serving both as guide molecules and as templates carrying the desired edits. Due to such diverse functions, manual pegRNA design is a subject to error and not suited for large-scale setups. Here, we present pegIT, a user-friendly web tool for rapid pegRNA design for numerous user-defined edits, including large-scale setups. pegIT is freely available at https://pegit.giehmlab.dk.